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269552005	pes2o/s2orc	v3-fos-license	An In-depth Analysis of Multisensory Reaction Time Disparities between Yogic and Non-Yogic Practitioners Background. Auditory and visual reaction time refers to the duration between the introduction of a sound or light and the onset of a response. It is an important aspect of human behaviour, influencing performance in various activities ranging from daily tasks to sports and emergency situations. Objectives. The purpose of this study was to investigate reaction time (auditory and visual) as a component of skill-related fitness between male college students practicing yoga and those who do not. Materials and methods. A total of forty male college-going students (N = 40) were randomly selected to participate in this study. Out of the total, twenty participants (n = 20) were dedicated yoga practitioners actively involved in district and state-level yoga competitions. The remaining twenty individuals (n = 20) followed a sedentary lifestyle and were not involved in yoga practices. The subjects ʼ ages ranged from 17 to 25 years, encompassing a cohort of young adults. The data pertaining to visual reaction time (VRT) and auditory reaction time (ART) was collected using an audio-visual reaction timer. Each participant performed the task thrice, and the mean score was used to indicate the experimental reaction time in milliseconds (ms). Descriptive statistics and the independent samples t-test were subsequently conducted to evaluate the significance level, with a predetermined threshold set at p < 0.05. Results. The results showed that the comparison between VRT and ART of yoga and non-yoga practitioners revealed that for yoga practitioners, t (38) = 2.91, and p < 0.006, whereas for non-yoga practitioners, t (38) = 3.55, and p < 0.001. Regarding VRT between yogic and non-yogic practitioners, t (38) = 1.99, and p > 0.054, and for ART between yogic and non-yogic practitioners, t (38) = 2.12, and p < 0.041. Conclusions . The findings suggest that both yogic and non-yogic practitioners demonstrated significantly lower level of ART compared to VRT. Further results indicate that hand speed in terms of VRT is nearly identical between yogic and non-yogic practitioners, but the ART of yogic practitioners was observed to be faster than that of non-yogic practitioners. Yoga has been associated with various physical and mental health benefits, and studies suggest that it may have a positive impact on reaction times. Introduction Reaction time refers to the period between the presentation of a stimulus and the initiation of a response. In various fields such as psychology, sports training, and physiology, reaction time is measured to assess how quickly an individual can respond to a specific stimulus.It is a crucial parameter in understanding cognitive and motor skills, as well as the efficiency of sensory and neural processes.The significance of reaction time in performance is paramount, acting as a dependable gauge of an individual's sensorimotor coordination and overall attentiveness (Balakrishnan et al., 2014).This assessment of how quickly information is processed in the center and how well-coordinated movements are in the periphery relies on the nervous system's ability to detect stimuli.Neurons send signals to the brain and spinal cord before directing actions to the hands and fingers through motor neurons to execute a suitable response.Reaction time indicates how quickly an organism responds to particular stimuli and is affected by factors like age, gender, handedness, visual concentration, practice, fatigue, fasting, breathing rhythms, personality traits, physical activity, and cognitive capabilities (Jain et al., 2015).In the context of events, reaction time pertains to the ability to swiftly and effectively respond to stimuli such as sound or light, demonstrating quick and suitable posture and control (Reza et al., 2018;Singh & Singh, 2024).Auditory and visual reaction time, vital in this context, denotes the time interval from the introduction of a sound or light stimulus to the commencement of the corresponding response. Reaction time is a key component in the skill-related aspects of physical fitness, distinct from agility but holding unique significance within the spectrum of physical fitness components.It plays a significant role in various sporting scenarios, with experienced players in soccer demonstrating quicker reaction times compared to their less-experienced counterparts (Theofilou et al., 2022).In sports like basketball, and tennis, rapid reaction times are crucial for adapting promptly to opponents' movements or shots (Bourgase, 2009;Debrousse, 2023).In cricket, for instance, a swift reaction to an edge results in a skillful slip catch.Reaction time is particularly indicative of sprinting ability, where a quick response to the starting signal can significantly impact the outcome of a close race (Jackson, 2017).Athletes dedicate years to training, aiming to enhance reaction time as milliseconds can be the game-changer between victory and missed opportunities.Improving reaction time can lead to heightened on-court efficiency, swifter responses to stimuli, and a decreased likelihood of injuries (Debrousse, 2023).In various activities, spanning from everyday tasks to engagements in sports and emergency situations, optimal performance is associated with improved reaction time in specific situations. Dutch physiologist Franciscus C. Donders led the way in measuring reaction time in a lab, challenging the prior belief before 1865 that mental processes were too rapid to be measured.Donders (1868), found it intriguing to quantify the duration of fundamental mental processes.The emphasis on precise measurement of reaction time laid the foundation for contemporary psychological experiments, influencing research methodologies to this day (Vinupradha, 2016).The length of cognitive processes is frequently measured in experimental psychology using reaction time (Yadav et al., 2013).Within the realm of cognitive psychology, reaction time serves as a metric for assessing the duration it takes an individual to process information.It has been demonstrated that pharmacological and physiological variables affect reaction time (Mohan et al., 1984;Malathi et al., 1990).According to Annett (1981), the left hemisphere governs motor function in the right hand, while the right hemisphere regulates motor function in the left hand.The mind serves as the motivating factor for actions, as it commands and directs the movements of the limbs in the human body (Rahman & Islam, 2021a).Despite this, the majority of hand patterns are still right-handed, accounting for around 90% of the human population (Scharoun & Bryden, 2014).Researcher has looked into three different kinds of reaction time: simple, recognition, and choice reaction time (Luce, 2008).For assessing routine reaction time in humans, the practicality of utilizing visual, auditory, and tactile neural pathways has been identified (Godlove et al., 2014;Jain et al., 2015). The holistic approach of yogic practices, as defined by Patanjali, integrates the mastery of the mind and the regulation of stress in individuals (Sonwane & Mishra, 2016).This ancient discipline covers a range of techniques like asana, pranayama, and meditation, all aimed at syncing up your mind and body for optimal harmony (Bagya et al., 2018;Kuppusamy et al., 2017).Through practices like meditation, individuals cultivate mindfulness and awareness, reducing distractions and enhancing their ability to perceive stimuli promptly.Regular participation in yoga leads to improvements in mental well-being, central nervous system processing, and sensorimotor performance (Kuppusamy et al., 2020).The mindful movement, breath control, and mental concentration in yoga positively influence reaction time and overall skill-related fitness.Research shows that yoga enhances sensory-motor conduction velocity, information processing, and concentration, resulting in decreased visual and auditory reaction times (Madanmohan et al., 1992;Madanmohan et al., 2012;Andreou, 2017).The practice of yoga reduces mental fatiguability and increases performance quotient by improving arousal and concentration (Andreou, 2017).Pranayama techniques contribute to heightened concentration and alertness, crucial for faster reactions, as seen in Bhramari pranayama improving cognitive function (Kuppusamy et al., 2020).Yoga poses not only improve coordination and balance but also body awareness and flexibility.It can increase a player's mental and physical relaxation, promote the development of body awareness (Rahman & Islam, 2020), and provide quick responses to external stimuli.Mountain Pose and Warrior I, emphasizing attention to breath, posture, and alignment, contribute to this awareness (Kulkarni, 2023).Visualization techniques and sensory awareness exercises condition the mind and body to react swiftly.Neuroplastic changes induced by yoga lead to improved body awareness, heightened attention, and enhanced present-moment awareness (Castro, 2020).Additionally, the decrease in stress, anxiety, and depression observed after hatha yoga practice optimizes brain function, enhancing cognitive processing and reaction times (Shohani et al., 2018).Practitioners employ concentration techniques during yoga poses, enhancing attentiveness and decreasing reaction time, which holds significance in activities requiring quick responses, such as sports.Thus, the interplay of mindfulness, breath control, and physical postures in yoga creates a holistic foundation for improving reaction time and overall skill-related fitness.Utilizing yogic interventions like integrated yoga, meditation, and pranayama can enhance brain wave activity and cognition, offering an alternative medicine approach to boost performance in games and sports (De & Mondal, 2020;Islam, 2021). Yoga can have positive effects on cognitive functions, including attention, concentration, and overall mental well-being.Understanding the potential effects of yoga on auditory and visual response time may contribute valuable insight into the broader cognitive benefits associated with yoga practice.By comparing the reaction times of yogic and non-yogic practitioners, the study seeks to provide empirical evidence of how yoga may or may not affect sensory-motor responses.Therefore, the research aims to explore and examine thoroughly whether individuals engaged in yoga practice exhibit differences in auditory and visual reaction times compared to those who do not practice yoga. Selection of Subjects A cohort of forty (n = 40) male college-going students from the University of Delhi were selected at random to partake in this study.Among them, twenty individuals (n = 20) were yoga practitioners at the Indira Gandhi Institute of Physical Education and Sports Sciences (IGIPESS), actively participating in district and state-level yoga competitions.The remaining twenty participants (n = 20) were non-yogic practitioners from the School of Open Learning (SOL) and led a sedentary lifestyle.The subjects' ages ranged from 17 to 25, and the data was gathered at the yoga lab of IGIPESS, University of Delhi, India.All participants had normal vision, were free from acute or chronic illnesses and medication use, and abstained from smoking or alcohol consumption during the tests.These undergraduate students volunteered for the study, and it's noteworthy that all participants were right-handed. Instrument and Tools The study utilized the Medisystems audio-visual reaction (AVR) timer in India, which is ISO 9001:2015 (QMS) certified.This timer comes with dual functionality, catering to both the researcher and the subject.Each side has four switches and four lights.The switches on the researcher's side control the lights, while those on the subject's side are used to turn off the lights.Additionally, there is a mode featuring four different melodies or tones.The researcher initiates a sound, and the subject's task is to promptly turn it off in the shortest time possible.This setup allows the researcher to assess reaction times using both visual and auditory stimuli.The reaction time of the subject, measured in milliseconds, is considered indicative of their performance (Medi Systems India, n.d.). Test Administration The Audio-Visual Reaction (AVR) timer was placed on a table, with subjects comfortably seated in chairs.ART and VRT were quantified in a serene, acoustically controlled environment to optimize focus and eliminate distractions.The reaction time task was conducted between 9 am and noon, an hour after a light breakfast, to maintain consistent conditions.Participants maintained contact between their right index finger and the AVR timer switch while performing tasks.Visual reaction time (VRT) was assessed in response to illuminated lights, while auditory reaction time (ART) was gauged through reactions to low-and mediumpitched sounds.The test administrator, seated across from the subject, controlled the AVR machine, creating lights or sounds of the same amplitude.The AVR timer's LCD display showed reaction time in milliseconds.Each subject repeated the task three times, and the average score represented the experimental reaction time. Statistical Analysis After subjecting the data to Levene's test, the researchers confirmed a normal distribution.Statistical analysis involves calculating descriptive statistics, such as the mean and standard deviation (SD).An independent t-test was then performed to determine the significance level, using a preestablished threshold of p < 0.05. Results Table 2 descriptive analysis of reaction times among yogic and non-yogic groups revealed notable differences.In the yogic group, the mean and SD of VRT were 18.50±1.72,while the ART was 17.20±1.02.On the other hand, the nonyogic group exhibited a higher mean VRT of 19.65±1.93,with a mean ART of 17.90±1.07.In Table 3, the comparison between VRT and ART of yoga and non-yoga practitioners reveals that for yoga practitioners, t (38) = 2.91 and p = 0.006 (2-tailed).Similarly, for non-yogic practitioners, t (38) = 3.55 and p = 0.001 (2-tailed).The findings suggest that both yogic and nonyogic practitioners displayed significantly lower ART in comparison to VRT.According to VRT between yogic and non-yogic practitioners, t (38) = 1.99, and p = 0.054 (2-tailed), and ART between yogic and non-yogic practitioners, t (38 = 2.12, and p = 0.041 (2-tailed).The results show that the VRT between yogic and non-yogic practitioners' hand quickness is almost the same, and the ART of yogic practitioners was found to be faster than that of non-yogic practitioners. Discussion The findings presented in Table 3 highlight a significant difference in visual reaction time (VRT) compared to auditory reaction time (ART) among both yogic and nonyogic practitioners.This observation aligns with consistent research indicating that individuals engaging in yogic practices tend to have notably shorter ART in comparison to VRT.A noteworthy yoga training program demonstrated a significant decrease in both ART and VRT, with ART experiencing a more pronounced reduction (Ramanathan & Bhavanani, 2020).Further supporting this trend, Şenel and Eroğlu (2006) found significant distinctions between auditory and visual reaction times in male players, with auditory reaction time being superior.The study by Rahman and Islam (2021b) on male university team branch athletes and non-athletes revealed that ART exhibited greater statistical significance compared to VRT in both auditory and visual domains.The physiological aspect of these findings is reflected in the time taken for stimuli to reach the brain, with auditory stimuli taking approximately 8-10 ms and visual stimuli requiring 20-40 ms (Kemp, 1973;Marshall et al., 1943).Consistent with these temporal differences, research by Pain and Hibbs (2007), Shelton and Kumar (2010), and Ghuntla et al. (2014) supports the notion that auditory reaction time surpasses visual reaction time in terms of speed.Researchers Jain et al. (2015) further contributed to this body of knowledge by concluding, based on their study of college students, that auditory reaction time is generally quicker than visual reaction time. The results of this study indicate that the VRT in terms of hand quickness is nearly identical between yogic and nonyogic practitioners.However, the auditory reaction time (ART) of yogic practitioners was observed to be faster than that of their non-yogic counterparts.This aligns with existing research, such as a study suggesting that yoga practices can significantly improve reaction times, both visually and auditorily (Madanmohan et al., 1992).A meta-analysis further supports the positive impact of yoga on auditory and (Ghuntla & Dholakiya, 2023).Other studies have demonstrated that short-term yoga training can effectively reduce baseline values of auditory, visual, and cutaneous reaction times in healthy individuals (Begum et al., 2012).Additionally, a study implementing a deep relaxation technique reported a notable decrease in both auditory and visual reaction times (Naik, 2021).Contrastingly, studies on visual simple reaction times among different team sports show no significant variations (Reza et al., 2023).Interestingly, hand quickness in simple visual reaction time was found to be similar between university athletes and sedentary students in the population (Rahman et al., 2020).Notably, research has consistently indicated that individuals who do not engage in sports tend to exhibit poorer auditory response times compared to athletes participating in various sports (Atan & Akyol, 2014;Palashikar et al., 2014;Kaplan et al., 2019).The observed reduction in ART and VRT within the yoga group suggests enhanced sensorimotor skills and an improved processing capacity of the central nervous system among individuals practicing yoga (Shobana et al., 2021). The findings demonstrate that yoga has a positive impact on reaction times, enhanced sensorimotor skills, and improved central nervous system processing capacity due to yoga practice. Table 1 . The characteristics of the Participants (Mean ± SD) Table 2 . Descriptive statistics of reaction time for yogic and non-yogic practitioners Table 3 . Reaction time using an independent t-test of yogic and non-yogic practitioners	2024-05-04T15:32:17.630Z	2024-04-23T00:00:00.000	{ "year": 2024, "sha1": "7816dfa550dd47deb8ca72b534034a43a5ef18e3", "oa_license": "CCBY", "oa_url": "https://tmfv.com.ua/journal/article/download/2592/1703", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "58159591e2af5eefc9b2be4b08c12c811fb46c57", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
256116940	pes2o/s2orc	v3-fos-license	Bound state solutions of sublinear Schrödinger equations with lack of compactness We consider the following nonlinear Schrödinger equation -Δu+V(x)u=a(x)uq-1u+f(x),x∈RN,\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} -\Delta u+ V(x)u= a(x)\left\| u\right\| ^{q-1}u+ f(x), \quad x\in \mathbb {R}^{N}, \end{aligned}$$\end{document}where V is a non-symmetric bounded potential, a is an indefinite weight, 0<q<1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0<q<1$$\end{document} and f≠0\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f\ne 0$$\end{document} is a nonnegative perturbation such that f∈L2(RN)∩L2NN+2(RN)\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f\in L^{2}(\mathbb {R}^{N})\cap L^{\frac{2N}{N+2}}(\mathbb {R}^{N})$$\end{document}. Using variational methods, we prove the existence of two solutions with negative and positive energies, one of these solutions being nonnegative. Introduction The Schrödinger equation is central in quantum mechanics and it plays the role of Newton's laws and conservation of energy in classical mechanics, that is, it predicts the future behaviour Bucharest, Romania of a dynamical system. It is striking to point out that talking about his celebrating equation, Erwin Schrödinger said: "I don't like it, and I'm sorry I ever had anything to do with it". The linear Schrödinger equation is a central tool of quantum mechanics, which provides a thorough description of a particle in a non-relativistic setting. Schrödinger's linear equation is where ψ is the Schrödinger wave function, m is the mass of the particle,h denotes Planck's renormalized constant, E is the energy, and V stands for the potential energy. Schrödinger also established the classical derivation of his equation, based upon the analogy between mechanics and optics, and closer to de Broglie's ideas. He developed a perturbation method, inspired by the work of Lord Rayleigh in acoustics, proved the equivalence between his wave mechanics and Heisenberg's matrix, and introduced the time dependent Schrödinger's equation In physical problems, a cubic nonlinearity corresponding to p = 3 in equation (1.1) is common; in this case problem (1.1) is called the Gross-Pitaevskii equation. In the study of equation (1.1), Floer and Weinstein [26] and Oh [36] supposed that the potential V is bounded and possesses a non-degenerate critical point at x = 0. More precisely, it is assumed that V belongs to the class (V a ) (for some real number a) introduced in Kato [31]. Taking γ > 0 andh > 0 sufficiently small and using a Lyapunov-Schmidt type reduction, Oh [36] The change of variable y =h −1 x (and replacing y by x) yields where Let us also recall that in his 1928 pioneering paper, Gamow [27] proved the tunneling effect, which lead to the construction of the electronic microscope and the correct study of the alpha radioactivity. The notion of "solution" used by him was not explicitly mentioned in the paper but it is coherent with the notion of weak solution introduced several years later by other authors such as Leray, Sobolev and Schwartz. Most of the study developed by Gamow was concerned with the bound states ψ(x, t) defined in (1.2), where u solves the stationary equation for a given potential V (x). Gamow was particularly interested in the Coulomb potential but he also proposed to replace the resulting potential by a simple potential that keeps the main properties of the original one. In this way, if is a subdomain of R N , Gamow proposed to use the finite well potential It seems that the first reference dealing with the limit case, the so-called infinite well potential, was the book by the 1977 Nobel Prize Mott [35]. The more singular case in which V 0 is the Dirac mass δ 0 is related with the so-called Quantum Dots, see Joglekar [29]. In contrast with classical mechanics, in quantum mechanics the incertitude appears (the Heisenberg principle). For instance, for a free particle (i.e. with V (x) ≡ 0), in nonrelativistic quantum mechanics, if the wave function ψ(·, t) at time t = 0 vanishes outside some compact region then at an arbitrarily short time later the wave function is nonzero arbitrarily far away from the original region . Thus, the wave function instantaneously spreads to infinity and the probability of finding the particle arbitrarily far away from the initial region is nonzero for all t > 0. We refer to Díaz [25] for more details. The main results In this paper, we consider Schrödinger equations with sublinear nonlinearity and nonsymmetric potentials, which are affected by a nonnegative perturbation. We are interested in the multiplicity of solutions and we establish several sufficient conditions for the existence of two solutions. We point out that sublinear problems on the whole space do not have necessarily a solution. In fact, the existence of solutions is in relationship not only with the nonlinearity but also with the behaviour of a certain potential. Brezis and Kamin [18] pointed out a striking phenomenon, which asserts that a sublinear problem on the whole space has a solution if and only if a linear equation depending only on the potential has a solution. They considered the nonlinear problem , ρ ≥ 0. Brezis and Kamin [18] proved that the nonlinear problem (2.1) has a bounded positive solution if and only if the linear equation has a bounded solution. Their analysis showed that such a solution exists for potentials like while no solution exists if Consider the following class of sublinear Schrödinger equations 2) arises in the study of solitary waves in nonlinear equations of the Klein-Gordon or Schrödinger type. We look for the existence of two solutions for problem (2.2), where the potential V (x) and the weight a(x) are indefinite, that is, they are sign-changing functions in R N . Such problems, with indefinite linear and nonlinear terms, present challenging mathematical difficulties. In 2008, using the Nehari manifold method, Chabrowski and Costa [21] proved the existence of two solutions of problem ( 2), see [3,5,17,19,28,30,32,34] and the references therein. In a pioneering paper, Ahmad, Lazer and Paul [3] considered the resonant problem where λ k denotes the kth eigenvalue of the Laplace operator. They proved that a sufficient condition for the existence of a solution is where G(x, s) = s 0 g(x, t)dt and E 0 = Ker (− − λ k ). Brown [19] proved the existence of two solutions of problem (2.2) when V = f = 0 and a(x) changes sign. In the case of unbounded domains, many authors have studied the existence of solutions of problem (2.2) with superlinear subcritical nonlinearity (1 < q < 2 * = 2N N −2 ), see [1,2,4,33]. For instance, Li and Wu [33] treated problem (2.2) where f = μh, V = λ and a(x) ∈ (0, 1). The authors proved the existence of positive numbers > 0 and λ 0 , μ > 0 such that for any λ > λ 0 and μλ 2) admits multiple positive solutions. For the sublinear case and especially for the whole space R N , to our best knowledge, few results are known. We can for example quote the papers [5,[14][15][16]. For instance, Benrhouma [14] proved the existence of at least three solutions of problem (2.2), provided that a(x) < 0 and V changes sign. As far as we know, the only existence result for problem (2.2) where both a and V change sign in R N , is obtained by Tehrani [37]. He considered the equation under the following assumptions: Under these assumptions, by using bounded domain approximation techniques, Tehrani [37] proved the existence of at least one solution. In this paper, we prove the existence of two solutions for problem (2.2), provided that both a(x) and V (x) change sign in R N . We consider two classes of assumptions on the indefinite non-symmetric potentials a(x) and V (x). First class: We suppose that V satisfies (H 1 ) and the following hypotheses: has not a nontrivial solution. We suppose that a satisfies: and there exist α, R 1 > 0 such that Second class: We assume that a and V satisfy (H 1 ), (H 3 ), N ± and the following conditions: The main results in this paper are the following. then problem (2.2) has two solutions U 1 , U 2 ∈ E with I (U 1 ) < 0 and I (U 2 ) > 0. One of these solutions is nonnegative. (H 3 ) and N ± hold. Then there exists m 1 > 0 such that if One of these solutions is nonnegative. Remark 2.3 In fact, the assumptions are necessary only to guarantee that I (u) ≥ c > 0 on the sphere in Y (see Lemmas 3.3 and 3.4). Thus these hypotheses can be removed if there are other ways to get I (u) ≥ c > 0 on the sphere in Y . We divide our paper into four sections. In Sect. 2, we give some notations and preliminary results. In Sects. 3 and 4, we prove Theorems 2.1 and 2.2. The main difficulties that arise in treating this class of nonhomogeneous Schrödinger equations (2.2) are the following: (i) the lack of compactness due to the unboundedness of the domain; (ii) the sign-changing of potentials a(x) and V (x). To avoid the first difficulty, we employ the Del Pino and Felmer method [23]. To overcome the second difficulty, we control the positive mass in relation to the negative mass of the potentials a(x) and V (x). The key tool for obtaining the multiplicity of solutions is a suitable recurrent variational method. Notations and preliminaries We will use the following notations: If we equip E with the norm then E becomes a reflexive Banach space. On the Sobolev space H 1 (R N ) we consider the usual norm Define the following energy functional on Y (Y = E or H 1 (R N )): Under suitable assumptions on a, V and f (to be fixed later), the functional I is well defined, of class C 1 on Y and any critical point of I is a weak solution of problem (2.2). We recall that a Palais-Smale sequence for the functional I , for short we write (PS)sequence, is a sequence (u n ) n ∈ Y such that The functional I is said to satisfy the Palais-Smale condition if any (PS)-sequence has a convergent subsequence in Y . In the sequel we need the following auxiliary results. Proof If x = 0, the inequality (3.2) is trivial. Then Multiplying by \|x\| q+1 , we obtain the desired result. Lemma 3.2 Assume that A 1 holds. If u n u weakly in H 1 (R N ), then there exists a subsequence of (u n ) ∈ H 1 (R N ), also denoted (u n ), such that Proof Since a ∈ L 2 1−q (R N ), then for every > 0 there exists R 2 > 0 such that Observe that by Hölder's inequality we have Proof of Theorem 2.1 We split the proof into several steps. We first establish the existence of first solution with negative energy. Next, we show that problem (2.2) has a weak solution with positive energy. Existence of a nonnegative solution Consider the minimization problem where ρ 0 is defined in Lemma 3.3. Lemma 4.1 Assume that hypotheses Hence M 1 < 0. Thus we conclude the proof. Theorem 4.2 Assume that hypotheses (F) , (A 1 ) and (H 3 ) hold. Then there exists a weak nonnegative solution U Proof Let (u n ) n be a minimizing sequence of problem (4.1). Since (u n ) ∈ B(0, ρ 0 ), we can extract a subsequence, also denoted by (u n ), such that u n U 1 in E, u n → U 1 in L m loc (R N ) for all 1 ≤ m < 2 * and u n → U 1 a.e. in R N . Existence of a second solution with positive energy solution We start this subsection by showing that the functional I satisfies the Palais-Smale condition. For this purpose, we need the following auxiliary properties. Lemma 4.3 Assume that hypotheses (A 1 ), (H 3 ), (H 4 ) and (F) hold. Then any (PS)-sequence of I is bounded in E. Proof Let (u n ) ∈ E be a (PS)-sequence of I . We argue by contradiction, assuming that u n = t n → +∞. Re-normalizing, we set v n = u n t n . Thus up to a subsequence, v n v in E. We claim that v = 0. For this purpose, we take ϕ ∈ C ∞ c (R N ). Since (u n ) is a (P S)sequence of I , we have Dividing relation (4.7) by t n , we obtain (4.8) Using (A 1 ) and Hölder's inequality, we deduce that Thus since 0 < q < 1, we have As a consequence of hypothesis (F), we have From (4.9), (4.10) and passing to the limit in relation (4.8), we infer that Using now (H 4 ), we conclude that v = 0 a.e. in R N . This proves our claim. Substituting ϕ = v n in relation (4.8), we obtain It follows that Using now (A 1 ), (H 3 ) and (F), we obtain On the other hand, since (u n ) is a (PS)-sequence of I , we have Therefore (4.14) From (A 1 ), (F) and (4.14), we infer that lim n→+∞ v n q+1 = 0. (4.15) Combining (4.13) and (4.15), we conclude that v n → 0 in E as n → +∞, which contradicts v n = 1. The proof is completed. Proof Let (u n ) ∈ E be a (P S) sequence such that Using Lemma 4.3, (u n ) is bounded in E. Then up to a subsequence, u n U 2 in E, u n → U 2 in L m loc (R N ) for all 1 ≤ m < 2 * and u n → U 2 a.e. in R N . According to [23], it is sufficient to prove that for any > 0, there exist R 2 > 0 and n 0 ∈ N * such that \|x\|≥R 2 \|∇u n \| 2 + \|u n \| q+1 dx ≤ , for all R ≥ R 2 and n ≥ n 0 . This ends the proof. Proof of Theorem 2.2 We first observe that (H 2 ) and (H 3 ) imply that the Schrödinger operator L = − + V (x) is defined on H 2 (R N ) and 0 is an isolated eigenvalue of finite multiplicity. Let H − , H 0 and H + denote the negative, null and positive spaces of the quadratic form associated to the operator L. More precisely, We have the orthogonal decomposition For u ∈ E, we denote by u ∓ , u 0 the orthogonal projections of u on H ∓ , H 0 respectively. Then u = u − + u 0 + u + . Moreover, there is an equivalent norm · X on H 1 (R N ) such that For more details, we refer the reader to Costa and Tehrani [22]. Existence of a nonnegative solution of problem (2.2) Consider the problem where ρ 1 is defined in Lemma 3.4. Proof It is sufficient to replace Lemma 3.1 by Lemma 3.2 in the proof of Theorem 4.2. Lemma 5.3 Suppose that hypotheses A 1 , (H 2 ), (H 3 ), (F) and N ∓ hold. Then any (P-S)-sequence of I is bounded in H Proof Let (u n ) be a (PS)-sequence of I . Case 1. We suppose that a satisfies N + . Arguing by contradiction, suppose that u n X → +∞. Then there exist c > 0 and d > 0 such that for n large enough we have Indeed, from A 1 and for n large enough we obtain Similarly, from A 1 , we have for n sufficiently large. Therefore Therefore min (β, 1) R N \|∇u n \| 2 + u 2 n φ R dx ≤ R N V − + χ B(0,R 0 ) (x)φ R u 2 n dx + R N a(x)φ R \|u n \| q+1 dx + R N f (x)φ R (x)u n dx − R N u n ∇u n ∇φ R dx + 3 , The rest of the proof is similar to the second step of the proof of Lemma 4.4. Concluding remarks The content of this paper is in relationship with the recent contributions of Bégout and Díaz [9][10][11][12][13] for the understanding of the nonlinear stationary Schrödinger equation. We expect that the methods developed by Díaz [24,25] in the case of an unbounded potential V (x) of Hardy type can be applied to show that the nonnegative solutions of problem (2.2) established in Theorems 2.1 and 2.2 have a compact support (if f (x) is also with compact support). This case corresponds to the abstract setting of infinite well type potentials.	2023-01-24T15:32:30.658Z	2018-05-05T00:00:00.000	{ "year": 2018, "sha1": "2a983bb7939634347727ddc58ebad06580f7dde8", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s13398-018-0541-9.pdf", "oa_status": "HYBRID", "pdf_src": "SpringerNature", "pdf_hash": "2a983bb7939634347727ddc58ebad06580f7dde8", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [] }
254117650	pes2o/s2orc	v3-fos-license	Defect Analysis and Innovation Design of Synchronizer for Clutchless Automatic Mechanical Transmission The synchronizer is a key component of automatic mechanical transmission (AMT) equipped in electric vehicles, but the inertial lock-ring synchronizer (ILRS) commonly used there is not suitable especially for pure electric vehicles without a clutch because of big shift impact. To make the shifting process rapid and smooth, a new synchronizer named pressure-controllable friction ring synchronizer (PCFRS) was designed. Initially, the inevitable shortcoming of ILRS was verified by simulation and test. Furthermore, the mechanical characteristics and advantages of the new synchronizer over ILRS were analyzed. Then, the formulations describing the dynamic transmission based on the working mechanism of the PCFRS were established. Finally, the shifting simulation results with PCFRS and ILRS based on the same operating conditions were compared and analyzed. The research shows that the PCFRS can meet the main shifting evaluation index of an AMT without complex control methods, as well as it takes only 0.2406 s to finish the comfortable and zero-speed-difference shifting. The shifting quality of PCFRS is better than that of the ILRS. It lays a foundation for using the new synchronizer as a part of clutchless AMTs equipped in pure electric vehicles. Introduction Pure electric vehicles, as crucial parts of new energy vehicles, are the important researching objects in the automotive industry in response to the energy crisis and environmental pollution [1]. The automatic mechanical transmission (AMT) can enhance their power and economy, as well as improve the motor and controller unit's lifespan [2], so it is in vogue to put an AMT in the transmission systems of pure electric vehicles. For the motor of pure electric vehicles, the driving torque and speed can be controlled more easily, so the motor can reduce the need for clutches due to the active speed regulation, and the clutchless AMT is in popular [3,4]. The shifting quality of clutchless AMT directly affects the dynamic performance, economy and driving comfort of an electric vehicle [5]. Thus, a synchronizer with better shifting quality is required for the clutchless AMT because of the duty of achieving the speed synchronization between the sleeve and the gear ring [6,7]. To be specific, it can reduce the shifting impact and noise. In addition, it can make the shifting process steady and extend the service lifespan of AMTs [8]. As a core component, the performance has an important influence on the shifting quality, the research on synchronizers is significant. Different shifting control strategies based on the working mechanism of ILRS are proposed to improve the shifting quality. Shifting force is a key factor affecting shifting quality. Wang et al. [9] adopted a position and force switching control scheme to avoid the problem that the shifting force produces a large impact. For the lock position, as the shifting occurs, the friction coefficient Open Access Chinese Journal of Mechanical Engineering between the synchro ring and the friction cone ring will decrease and cause shifting failure [10]. Besides, the rapid adjustment of the motor speed and the rapid teeth feeding of the sleeve make the synchronizer fail to lock teeth. Qin et al. [11] adopted a gear shifting control strategy based on parallel coordinated control to realize reliable, smooth and fast gear shifting. In addition, the input shaft is directly connected with the drive motor, resulting in a greater inertia of the input shaft, therefore a greater impact when the gears are engaged. For the above questions, Lin et al. [12] designed an inverse system and an active disturbance rejection controller to reduce the impact and synchronization time. Zhang et al. [13] proposed a stage-by-phase multivariable combination controller based on the control of position, velocity and force of the actuators, which reduces the impact of mode switching and improves the dynamic performance of electric vehicles. Zhang et al. [14] proposed a synchronizer shifting trajectory tracking controller. Although the above researches have achieved effective results in gear shifting, the control algorithms may be too complex to complete system identification and parameters correction because of the very short shifting time. Besides, due to the randomness of shifting and the wear of mechanical parts, the reliability of the strategies need to be improved. The structural parameters of the synchronizer have various effects on the synchronization performance, locking performance, shifting quality and service lifespan [15]. Different parts of synchronization also have different degrees of influence [16,17]. Wang et al. [18] optimized the parameters of synchronizer to effectively shorten the synchronization time and increase the lifespan of the synchronizer. The researchers have effectively improved the shifting quality of the AMT using the inertial lock-ring synchronizers (ILRSs). However, in combination with the characteristics of the ILRS and the electric motor, the following problems still exist. First, the sleeve can not be moved before completing synchronization. Second, there is a break-through load when the sleeve breaks away from the slider [19], which is unpredictable and difficult to control. Next, the ILRS will cause two large impacts during the gear shifting. At last, combined with the shifting process of the ILRS, the synchro ring will not work after the synchronization phase, the speed difference will generate in the final turn-teeth phase, and then it will generate a huge impact [20]. With the application of motors in the new energy vehicles, the clutchless mechanical direct-connection motor-transmission systems are increasingly popular in the electric vehicles. The research on the direct-connection motor-transmission system mainly focuses on the application of various motor-controlling algorithms to achieve the speed synchronization of the sleeve and the gear ring, and the feasibility of using adapter sleeves to shifting gears. Bóka et al. [21] established the dynamic model of the jointing process of the synchroniserless system, and analyzed the influence of the relative speed and relative angle of the jointing sleeve and the jointing gear ring on the gear shifting. Yu et al. [22] studied the control of the synchronization speed during gear engagement and motion control of the gear-change actuator mechanism. The speed synchronization of the sleeve and the target gear is realized. The researchers have made great progress in the speed relationship between the sleeve and the target gear, which is a clue for the development of clutchless AMT. However, the direct shifting method using the sleeve requires high control accuracy and strict conditions, and the control algorithm requires wonderful real-time performance of the drive system, which makes it difficult to use the sleeve for high quality shifting. Regarding the electromechanical coupling of gear shifting in pure electric vehicles, some scholars have studied novel synchronization mechanisms to replace traditional synchronizers to achieve high quality shifting. A British innovation company-Zero shifting has developed a new system which is replace the synchronizer with the Zero meshing ring mechanism, and using two sliders to achieve shifting without power interruption [23]. However, two sets of shifting forks are needed to realize the shifting, which makes the shifting actuator more complicated and harder to control. Mo et al. [24,25] proposed a new type of synchronizer called harpoon-shifting, which is replaced the friction cone ring of the ILRS with a torsion spring to eliminate the speed difference. It overcomes the shortcomings of friction synchronous shifting. However, torsion springs greatly limits the range of the shifting speed difference. If the speed difference between the sleeve and the meshing gear ring is too large, the spring will be compressed to the maximum value and cause greater impact; if the speed difference is too small, there may be a speed difference in the gear advancement process. To sum up, the existing new synchronizers are still defective when applied to pure electric vehicles. In summary, the clutchless AMT of pure electric vehicles using traditional ILRSs is not suitable, the way of directly canceling the synchronizer and adopting the method of sleeve shifting is relatively difficult and requires high-precision sensors, all of them are terrible influencing factors. Therefore, the better shifting scheme is to adapt a suitable synchronizer with shifting control method for easily achieving shifting. In response to the above problems, a pressure-controllable friction ring synchronizer (PCFRS) is proposed in the paper, which realizes synchronization through compression springs and does not require lock teeth of gear during synchronization. There is no slider mechanism, that is, it is not necessary for the sleeve to apply the overtaking force that breaks off the constraint of the slider. In the gear-shifting stage, the generated speed difference can be eliminated synchronously to achieve no-speed-difference shifting. The structure of the paper is as follows. Section 2 provides the shifting simulation and experiment results about the ILRS. Section 3 analysis the working mechanism and shifting strategy of the PCFRS. Section 4 establishes the dynamics model of the meshing. Section 5 simulates gear shifting and analyzes the simulation results about the PCFRS and ILRS. Section 6 draws the conclusion, summarizes and looks forward to this paper. Modelling and Simulation Analysis of ILRS The whole structure of ILRS is represented in Figure 1. It is unable to eliminate the speed difference caused by the engagement of the shifting. Therefore, in order to achieve better shifting, the position control of the Proportional Integral Derivative (PID) is used to realize the shifting force control with different P values. Among them, the P value is a proportional reflection of the deviation signal of the control system. Once the deviation occurs, the controller immediately produces control effect to reduce the deviation. The P parameter quickly acts on the output. The larger the P value is, the stronger the effect will be, and the dynamic response will be faster. Different shifting modes are used for multiple sets of simulations, which are designed to simply and intuitively reflect the mechanical characteristics of the ILRS. The goal is to make it comparable to the corresponding mechanical characteristics of the PCFRS. The shifting process of the ILRS is shown in Figure 2, which shows the relationship between the input shaft speed and the output shaft speed and the displacement of the sleeve at all stages. Combined with the simulated speed curve and displacement curve, point A indicates that the ILRS completes the pre-synchronization phase and then enter the synchronization phase AB. The speed difference is eliminated at point B to complete synchronization. After the synchronization is completed, the synchro ring is turned in the BC section. At this time, the synchro ring and the target gear rotate at the same speed, and the turn-ring phase is completed at point C. The sleeve continues to move to contact the target gear at point D and turns the target gear teeth. Point E is the collision between the teeth of sleeve and the teeth of the target gear. F point completes the movement of the sleeve to realize gear shifting. It can be seen from the BEF stage that the speed difference produced by the ILRS during the turn-teeth phase will not disappear, and can only disappear through the collision between the teeth. Shifting Impact of ILRS Impact is an important indicator for evaluating shifting quality. It is expressed as Eq. (1). where v is the longitudinal speed of vehicle. a represents the longitudinal velocity of the vehicle. α is the wheel angular acceleration. T w is the wheel driving torque. I v is the moment of inertia of the wheel. R w is the wheel radius. The impact degree is derived from the change in the longitudinal speed of the vehicle [26]. The software realizes the shifting simulation with a certain simulation step length, so it is not exactly the same as the actual situation. The second derivation of the curve will make the deviation larger. The degree of impact can also be calculated by derivation of the wheel torque. At the moment when the sleeve teeth contacts the internal teeth of the friction cone ring, the impact torque is also generated by the teeth surface collision and the generation time is very short, which can reflect the impact characteristics, so it is also meaningful to measure the shifting impact. In addition, the impact torque only needs to derive the longitudinal velocity once, and the resulting error will be smaller. The torque is expressed as follows: Using the impact torque instead of impact to evaluate the shifting quality, the torque is expressed as follows: where α 2 is the angular acceleration of input shaft. i 0 is the gear ratio of the transmission. The shifting simulation with different shifting forces and different shifting modes is carried out to reflect the mechanical characteristics of the ILRS. The PID position control is used to directly realize the shifting, and different P values are used to realize different shifting forces, as shown in Figure 3, when the shifting force decreases, the shifting time is longer, and the shifting impact torque decreases. It can be seen from Figure 3 that in several sets of the shifting force (F), when the F value is 170 N, the synchronization time is 0.0813 s, which is close to the synchronization time of the PCFRS in Section 5, so the F value for the second shifting mode is 170 N. The second shifting strategy is that when the speed difference between the sleeve and the target gear is eliminated, the shifting force value achieved by the smaller gear shifting force. The simulation results are shown in Figure 4. The smaller the gear shifting force, the smaller the speed difference is produced by the shifting, the longer the shifting time will be. This shifting strategy will produce a smaller impact torque compared to the pure position control. In order to compare with the PCFRS simulation results in Section 5, a group that produces impact torque is found, and the time required to shifting is 0.2486 s when the impulsive torque is 69.8256 N·m. Sliding Power Work with ILRS Shifting The sliding friction work is mainly expressed in the form of heat, which directly affects the reliability of the synchronizer. It may even cause irreversible damage to the synchronizer and affect its lifespan. This section will verify whether the shifting mode meets the requirements. According to the definition of sliding work, sliding work L f can be expressed as follows: From Eq. (4), T f is: where F a is the axial contact force of the friction cone ring and the synchro ring. f is the friction coefficient of friction cone and synchro ring friction pair. θ is the relative rotation angle of the friction cone ring relative to the friction cone surface of the target gear during the synchronous friction process, dθ = \|ω g − ω r \|dt. β is the angle of friction cone. The analysis of the sliding work is shown as Eq. (6) [27]: where t T is the relative sliding time of friction cone ring and synchro ring. ω g , ω r are the rotation speed of the target gear and the synchro ring. In order to make the evaluation more objective, the specific sliding work and specific sliding power are introduced to evaluate the performance. The specific sliding work is expressed as follows: where A f is the contact area of the friction cone and the synchro ring. In the synchronization stage, the instantaneous specific sliding power is used. The calculation can be calculated by dividing the contact area by the product of the friction torque and the relative angular velocity difference, which is expressed as follows: The simulation of different strategies for the ILRS fully demonstrates its mechanical characteristics. As shown in Figures 5 and 6, they are the specific sliding work and specific sliding power generated during the shifting simulation process of the two shifting strategies. The specific sliding work and specific sliding power are far less than the allowable value 0.09 J/mm 2 and 0.45 W/mm 2 [27]. It shows that the shifting simulation results of the ILRS meet the requirements of the mechanism, and the simulation data is reasonable. PV Value Analysis with ILRS Shifting Regarding the working conditions of the synchronizer, the PV value is often used to check [28], that is, the product value of the surface pressure of the friction cone and the relative sliding speed. The PV value can intuitively reflect whether the shifting force and speed difference set during the shifting process are reasonable. The surface pressure P of the PV value is expressed as: The relative sliding speed V of the PV value is expressed as: Organizing Eqs. (9) and (10) get Eq. (11): According to ZF Friedrichshafen AG's recommendation, the allowable pressure in the contact area of the friction cone is 3 N/mm 2 , and the allowable relative sliding speed is 5000 mm/s. So, the PV value should not be greater than 15,000 N/(mm·s) [27]. The PV values corresponding to the two shifting strategies are shown in Figure 7, the simulated PV values are far less than the allowable value of 15,000 N/ (mm·s). Therefore, the shifting simulation results meet the working conditions of the synchronizer. Experiment Analysis of ILRS Shifting To verify the actual use effect of ILRS, a pure electric vehicle is adopted which equipped with a new type of Clutchless AMT and ILRS is selected as the shift actuator, they are shown in Figure 8. In order to reflect the working characteristics of the ILRS more intuitively, the experiment adopts a similar way to the simulation. For the sake of safety, the test shift speed is lower than the simulation shift speed. The test curves are shown in Figure 9. In the figure, the blue curve is the displacement of the sleeve. The speed of the input shaft and the target gear connected to the output shaft will fluctuate at the shifting time. At first, the sleeve is in the middle position, that is, the transmission is in neutral state. Then the input shaft speed decreases rapidly in the synchronization stage. In the stage of turn-teeth, the speed of input shaft decreases and the speed of output shaft increases. The experimental results are consistent with the simulation results. The speed difference produced by the shifting cannot be reduced or eliminated by the mechanism and will inevitably cause impact. Due to the gap between the teeth surfaces, the teeth surfaces will not contact quickly after completing the tooth entry. So in order to better complete the shifting, the speed of the motor connected to the input shaft is gradually increased to reduce the speed difference when the sleeve advance is completed. The analysis is based on the structural characteristics of ILRS. If the motor speed is not adjusted when the sleeve advance is completed, the speed difference generated by the tooth can only be eliminated by collision. In addition, as can be seen from the figure, although the speed of motor is adjusted, the speed of the output shaft will fluctuate. Therefore, according to the experiment, the simulation data is consistent with the actual phenomenon. The speed difference produced by turning teeth is not self-elimination will produce an impact. Motor speed regulation requires high precision and the speed of the input shaft also fluctuates to the speed of the output shaft when adjusting speed. In a word, ILRS has inevitable drawbacks. Working Mechanism Analysis of the PCFRS Based on the problem presented by ILRS, the PCFRS is designed. The structure of PCFRS is shown in Figure 10, which includes hub, sleeve, wave spring, synchro ring, friction cone ring. The synchro ring and the sleeve are used to eliminate the speed difference and engage the hub with the target gear. The hub is connected to the input shaft for combining the sleeve and the synchro ring. The shifting force is transmitted by the spring between the sleeve and the synchro ring. After the synchronization is completed, the sleeve directly compresses the wave ring close to the teeth to realize the turn-teeth and complete the shifting. If the rotational speed of the motor is directly adjusted enter the speed range of the gear advancement, the sleeve is directly controlled to complete the shifting. If the rotational speed does not reach the gear-advancing speed range, the sleeve will move a certain distance and wait until the gear-advancing speed range is synchronously reached before completing the gear shifting to ensure the reliability of gear shifting. The functions of PCFRS are as follows: The hub is fixed on the input shaft, which is connected to the drive motor, forces the synchro ring and the sleeve to rotate at the same speed. The sleeve is moved axially on the hub, connecting the target gear and the input shaft to achieve driving. The synchro ring is the friction unit. It rubs the friction cone ring fixed on the target gear to realize the synchronization of the input shaft and target gear speed. The two sides of the wave spring touch the sleeve and the synchro ring, respectively. During shifting, the sleeve is moved axially and pushing the wave spring touch the synchro ring. The synchro ring is touching with the friction cone ring under the action of spring compression. The wave spring is further compressed to complete the gear shifting. The friction cone ring is fixed on the target gear, the outer side rub the synchro ring to synchronize the speed, and the end surface is opened with internal teeth holes to guide the sleeve teeth to complete the gear shifting. Compared with the ILRS, the PCFRS has the following advantages: (1) There is no pre-synchronization and no need to squeeze the slider with excess force during the shifting process, which makes the shifting process control more stable and reliable. (2) There is no lock teeth and turn-ring phase, and there is no need to worry about the inability to synchronize the speed due to the failure of the lock teeth of the synchro ring, which saves shifting time, does not need to produce two shifting impacts and makes the synchronizer work more reliable. (3) The sleeve can move before the speed difference is completely eliminated in the synchronization phase, and it can be set to move when the speed difference is in the allowable range, which make shifting more efficient. (4) The working characteristics are related with the speed regulation characteristics of the motor, and the process is flexible and effective. According to different speed differences, it can adopt direct shifting or shifting after synchronization to the corresponding speed difference, which is more suitable for the shifting of pure electric vehicle transmissions. (5) Synchronous friction torque always exists during the shifting process, so that the speed difference generated after turn-teeth is quickly reduced, and impact-free shifting can be realized, which making shifting easier to control. (6) There are fewer components than ILRS and no sliding block mechanism and sudden shifting force. The limitation of the design is that the friction is still needed to synchronize firstly and then to shifting. The friction will bring decrease the lifespan of friction elements. However, according to relevant literature studies, pure electric vehicles are suitable for two-speed transmissions [29][30][31]. The low-gear transmission of pure electric vehicles does not shifting frequently, thus reducing the drawbacks of friction loss in shifting. In order to highlight the mechanical characteristics of the PCFRS, this paper will compare the shifting characteristics of the PCFRS and the ILRS. In order to verify that the PCFRS adopts wave springs to meet the working requirements during the entire shifting process, this paper will verify the working conditions of each shifting. In addition, the shifting actuator will always bear the pressure brought by the wave spring. The problem can be solved by adopting a worm gear shifting actuator. According to the self-locking characteristics of the worm gear, the synchronizer can help the vehicle shifting easily. The simple shifting control strategy of the pressure controllable friction ring synchronizer is as follows: (1) The sleeve moves a certain distance, and then the wave spring is compressed to make the synchro ring rub the friction cone ring for synchronization; (2) When the speed difference is enter the required range, the sleeve will continue to move at a certain speed; (3) After the sleeve touches the internal teeth of the friction cone ring, the contact force of the teeth surface causes a certain speed difference, and then the latter two are separated; (4) The sleeve continues to move at a certain speed, and the friction element eliminates the speed difference at a faster speed while the sleeve moves, and finally realizes a zero-speed-difference shifting. Phased Dynamics Modeling of the Synchronization Process The model of the transmission system with a PCFRS is shown in Figure 11, where s represents the sleeve, r represents the synchro ring, and g represents the target gear. The PCFRS is mounted on the input shaft. This section will combine the transmission system to analyze the dynamics characteristics of the PCFRS. In order to illustrate the analysis of the shifting process of the PCFRS, the sleeve, the wave spring, the synchro ring and the friction cone ring are used to articulate the working mechanism. The whole process of the shifting of the sleeve is divided into multiple stages. In order to express the dynamics relationship of the gear meshing process more clearly, the gear contact model is adopted [32], as shown in Figure 12. Where R a and R b are the basic radius of the gear, θ a and θ b are the torsional freedom of the driving gear and the driven gear, c y1 , c y2 , k y1 and k y2 are the supporting damping coefficient and supporting stiffness of the driving gear and the driven gear, respectively. c m and k m are the damping coefficient and stiffness of the driving gear and driven gear meshing. I a , I b are the moment of inertia of the meshing gear. e is the static transmission error of the gear pair. The relative displacement of the driving gear and the driven gear due to torsion in the normal coordinate system is In fact, the process of synchronously eliminating the speed difference and the timing of shifting are both random. This section selects the most representative stages that can cover all situations. Combined with the dynamics model establishment method proposed [33], it is expressed as follows. Stage 1: The sleeve moves to make the synchro ring contact the friction cone ring. (12) The state of PCFRS in the first stage is shown in Figure 13. At this stage, the sleeve is in the middle position of the synchronizer, and the wave spring is in the free length state. The sleeve moves axially toward the target gear under the guiding action of the hub, pushing the wave spring to contact the synchro ring. The spring force pushes the synchro ring to move axially toward the target gear under the guidance of the hub and the synchro ring quickly contact the friction cone surface on the outside of the friction cone ring. When the sleeve continues to move and reaches the set position, the wave spring gets a certain degree of compression and the shifting process is in stage 2 or stage 3. where I s is the moment of inertia of the hub and the sleeve. I r is the moment of inertia of the synchro ring. θ i is the rotation angle of the corresponding component, θ i is the angular velocity of the corresponding component, θ i is the angular acceleration of the corresponding component, where i = s, g, x, f, v, 1. R j is the basic radius of the corresponding gear, y j are the translational displacement of the corresponding gear. ẏ j are the translational velocity of the corresponding gear, where j = g, x, e, f. y 01 and y 02 are the relative displacement of meshing gear. ẏ 01 and ẏ 02 are the relative velocity of meshing gear. e y1, e y2 are the static transmission error of the gear pair. ė y1, ė y2 are the static transmission velocity error of the gear pair. F shift is the force of shifting. Stage 2: Synchro ring and friction cone ring contact for synchronization. The state of PCFRS at this stage is shown in Figure 14. The sleeve stops moving in the axial direction, and the spring force generated by the spring compression makes the friction cone surface of the synchro ring and the friction cone ring keep in touch. The friction surface will be (13) produced friction torque, because there is a speed difference between the synchro ring and the friction cone ring. The synchro ring is constrained by the hub, so that the rotational speed of the PCFRS and the input shaft gradually become consistent. At this stage, the shifting motor stops driving, the stop of the sleeve is realized by the self-locking characteristic of the worm gear. When the speed difference is reached the allowable teeth-entering range by the friction torque, this stage ends and the shifting process will proceed to stage 3. where F f is the friction force between the synchro ring and the friction cone ring. R m is the friction equivalent radius. Stage 3: Before the speed difference is eliminated, the sleeve enters the teeth and the friction cone ring teeth contact. The state of PCFRS at this stage is shown in Figure 15. According to the shifting characteristics of the PCFRS, (19) the sleeve can move again without completely eliminating the speed difference. When the speed difference reaches the allowable range, the sleeve moves to contact the internal teeth of the friction cone ring to turn teeth, and the contact between the sleeve and the friction cone ring generates surface contact force. At this time, there is a friction torque between the friction cone ring and the synchro ring. There is also a turn-teeth torque between the friction cone ring and the sleeve. Both friction torque and turn-teeth torque promote the elimination of the speed difference. Under the action of the helical teeth, the sleeve moves to the internal teeth hole of the friction cone ring. At the end of this phase, the shifting process proceeds to stage 4 or stage 5. where F t is the tangential component of the turn-teeth force. F N is the positive pressure on the bevel. R t is the turn-teeth radius. γ is the teeth surface angle. Stage 4: After the speed is eliminated, the sleeve teeth moves in contact with the friction cone ring teeth. The state of PCFRS at this stage is shown in Figure 16. This situation exists when the speed difference between the sleeve and the friction cone ring is eliminated, but (26) the sleeve has not been fully meshed. Therefore, stage 4 is required. The sleeve continues to turn the friction cone ring teeth and advance the teeth. The gear shifting method at this stage is that the sleeve teeth are slowly advanced so that the teeth surface is in continuous contact, so that no excessive great speed difference is generated, and finally a successful meshing without a speed difference is achieved. Stage 5: After the speed is eliminated, the contact between the sleeve and the teeth surface of the friction cone produces a speed difference. The generated speed difference is used to complete the teeth advancement. The state of PCFRS at this stage is shown in Figure 17. It exists in the situation of stage 4, but it is not the slow gear advancement. The teeth advancement according to the teeth advancement state of the stage 3, resulting in (32) the speed difference, and then the slow teeth advancement is used. The difference in speed reduces the relative angle between the sleeve and the teeth hole of the friction cone ring to realize shifting. At this stage, there may be impact during meshing, but the difference in speed is small, the impact produced is within an acceptable range. In addition, the speed difference can also be eliminated when the teeth is advanced to realize the speed difference-free shifting. (37) −R e c m ẏ 02 +ẏ e −ẏ f −ė y2 +k m y 02 + y e − y f − e y2 , The relative angle of the friction cone ring teeth hole is also random, and there may be a small or zero relative angle to the friction cone ring teeth hole during the teeth engagement process, so the shifting is completed directly after the gear is dialed or the gear is directly engaged. Simulation Analysis of the PCFRS and Comparison with ILRS Simulation Results In this paper, ADAMS is used to establish to perform the multi-body dynamics modeling and debugging. The model is shown in Figure 18. In order to simplify the simulation process, the target gear replaces the gear meshing system in the transmission as the output shaft, converts the wheel inertia to the target gear, and sets the specified initial speed and moment of inertia for the target gear, the (42) input shaft and the synchronizer. After that, the ADAMS and MATLAB/Simulink co-simulation was used to realize the shifting simulation. Combining the structural characteristics of the PCFRS, firstly, the PID position controller is used to move the sleeve to the specified position, and after synchronization is completed, another PID speed controller is used to control the shifting motor to realize shifting. PCFRS simulation adopts the same parameters and conditions as ILRS simulation. Table 1 shows the main parameters of the simulation of the PCFRS and the ILRS. Simulation Analysis of the PCFRS Shifting The shifting process of PCFRS is shown in Figure 19, which shows the relationship between the rotational Combining the rotational speed curves of the input shaft and the displacement curve of the sleeve, point A in the figure is that the gap between the components of the shifting process is eliminated and the idle stroke of the sleeve moving in the direction of the target gear is completed. As the movement of the sleeve enters the synchronization phase, point B indicates that the sleeve to reach the set position, and then the speed difference is determined. When the speed reaches the range of sleeve advancement, the sleeve advancement continues. Point C indicates that the sleeve is in contact with the friction cone ring teeth. Then it enters the turnteeth phase and the teeth surface contact in the gear shifting stage produces a force and the friction cone ring to accelerate the synchronization process. The synchronization is completed at point D, at the same time the sleeve is moved at a certain speed. At point E, the sleeve teeth are separated from the friction cone ring teeth, and the friction torque continues to be synchronized while the engagement sleeve is moving. At point F, the sleeve teeth contacts the friction cone ring gear again, and after the contact continues to turn teeth, which produces a speed difference and the teeth separate. The sleeve teeth still advances at a certain speed, and the friction torque quickly eliminates the speed difference. At point G, the advancement of the sleeve is completed, and there is still a small speed difference at this time. Due to the small gap between the sleeve teeth and the teeth groove of the friction cone ring, synchronization is still performed after the advancement of sleeve is completed. At point H, the speed difference is completely eliminated, achieving no speed difference shifting. Figure 21 The specific sliding work and specific sliding power of the PCFRS under different shifting speeds Shifting Impact Analysis As shown in Figure 20, different shifting effects with different moving speeds of the sleeve are adopted, and it takes a long time to achieve a shifting without a speed difference of 0.2406 s, but the impact torque of teeth collision is not generated. When the shifting produces the impact torque of 66.5673 N·m in the shifting mode, the shifting time is only 0.2031 s. The result will be compared with the simulation results of the ILRS in Section 2. Sliding Power Analysis This section will verify whether the shifting process meets the standard through the sliding work to further determine the rationality of the PCFRS shifting simulation, which makes the comparison between the PCFRS and the ILRS more convincing. Figure 21 shows the specific sliding work and specific sliding power between the friction elements during the shifting process. The allowable values of specific sliding work and specific sliding power are 0.09 J/mm 2 and 0.45 W/mm 2 [27]. It can be seen from the figures that the specific sliding work and specific sliding power of the PCFRS in different shifting modes meet the allowable value. PV Value Analysis The simulation curves are shown in Figure 22. When the sleeve moves at different speeds, the working conditions of the synchronizer all meet the allowable value and meet the requirements of use. The simulation of the above shifting modes is reasonable. Comparison between PCFRS and ILRS Shifting This paper aims to highlight the advantages of the PCFRS from the aspect of mechanical characteristics by comparing the PCFRS and the ILRS. If there is a speed difference to realize the shifting, the impact torque will be generated. In Table 2, the shifting data of the ILRS and the PCFRS with similar impact torque are selected, and the data of the PCFRS without speed difference shifting data is selected for comparison and explanation. The ILRS cannot eliminate the speed difference generated during turn-teeth phase, and it will eventually generate impact torque. In order to produce a smaller impact torque, a smaller shifting force must be adopted at the expense of shifting time. It can be seen from the table that although the ILRS takes 0.0813 s to complete the synchronization, when it produces an impact torque similar to that of the PCFRS, the shifting time is longer and the impact torque is still greater. The shifting quality of PCFRS is much better than that of ILRS. Conclusions As a universal synchronizer in AMT, ILRS is not suitable for pure electric vehicles without a clutch. The simulation and test researches in this paper prove the conclusion. ILRS has very high requirements on the speed regulation and response time of the motor. To ensure the quality of shifting, the motor must be able to achieve accurate and rapid speed adjustment. In addition, even if the performance of the motor can be guaranteed, the tooth profile of the synchronizer itself will cause a secondary impact on the tooth, which is unavoidable. The above factors will reduce the shifting quality of ILRS. PCFRS is suitable for clutchless AMTs of pure electric vehicle, which is verified by mechanical characteristics and work rationality simulation analysis. The research results show that the impact torque can eliminate the speed difference in the turn-teeth phase to achieve no speed difference shifting. Through the verification of evaluation indexes such as specific sliding work and PV value model, it is concluded that the shifting process of PCFRS meets the allowable value. The shifting simulation results of the PCFRS and the ILRS are compared. The wave spring can act on the PCFRS during the shifting process to make the synchronous friction torque always work. Moreover, the PCFRS does not have the turn-ring phase and the lockteeth phase, which makes the time for shifting without speed difference to be as short as 0.2406 s. When the PCFRS adopts the mode of shifting with a smaller speed difference, the required shifting time is shorter to 0.2031 s. Compared with the ILRS that shifting time is 0.2486 s, the impact torque is smaller as well as the time required is shorter. In addition, the mechanism studied in this paper still need to be proved by bench or actual vehicle loading test. Affected by the epidemic, the production of PCFRS has not been completed, the next step will be to test and verify. Besides, the structure of the PCFRS has been determined, but the corresponding size is only preliminary determined in combination with the structural characteristics. In the next step, the specific size will be further optimized.	2022-12-01T14:16:09.476Z	2022-03-05T00:00:00.000	{ "year": 2022, "sha1": "a26f6ae8daaf6b0282082c95dbdbeaa233acca28", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1186/s10033-022-00690-8", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "a26f6ae8daaf6b0282082c95dbdbeaa233acca28", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [] }
130085411	pes2o/s2orc	v3-fos-license	From turf to table : Grass seed to edible grains in the Willamette Valley Western Oregon’s Willamette Valley has a rich history of agricultural production and, like an increasing number of regions globally, a growing local food movement. Recent declines in grass seed markets and an increased consumer interest in local grains have raised the possibility of a transition from grass seed land to edible grain production for local markets. We used geographic information systems (GIS) to determine if the Willamette Valley population’s dietary grain needs could be met if current grass seed land were converted to production of soft white winter wheat. In order to explore transitional obstacles and opportunities, we conducted interviews with local farmers, a wholesaler, an agriculture extension worker, and seed developers. The GIS analysis indicated that such a transition could exceed the recommended grain needs of the region’s 2008 population. The interviews revealed technical and cultural aspects of transitioning from grass seed production to wheat and other edible crops, identifying insufficient infrastructure (storage, processing, distribution, and market outlets) as the primary barrier to producing for local markets. This combination of GIS analysis (predictive of the food-producing capacity of a region) with in-depth contextual information and practical insights from farmers’ voices provides a robust model for planners seeking to analyze and address local food system challenges and possibilities. Our research, while focusing on the Willamette Valley’s transition toward a more locally based food system, explores the potential steps for any region looking to transition from nonedible to edible crop production for local consumption. a Department of Environmental and Earth Sciences, Willamette University, 900 State Street, Salem, OR 97301 USA; kgiombolini@gmail.com b Corresponding author: Kimberlee J. Chambers, Department of Environmental and Earth Sciences, Willamette University, 900 State Street, Salem, OR 97301 USA; +1-503-370-6816; kchamber@willamette.edu c Department of Environmental and Earth Sciences, Willamette University, 900 State Street, Salem, OR 97301 USA; jbowerso@willamette.edu d Department of Environmental and Earth Sciences, Willamette University, 900 State Street, Salem, OR 97301 USA; phenry@willamette.edu Journal of Agriculture, Food Systems, and Community Development ISSN: 2152-0801 online www.AgDevJournal.com 142 Volume 2, Issue 1 / Fall 2011 Local Food Eating locally has been endorsed in popular literature by authors such as Michael Pollan (2006) and Barbara Kingsolver (Kingsolver, Hopp, & Kingsolver, 2007), as well as in a growing body of research promoting local food and assessing community food production and consumption capacities (see Allen, FitzSimmons, Goodman, & Warner, 2003;Colasanti & Hamm, 2010;Delind, 2006;Feagan, 2007;Feenstra, 1997;Giombolini, Chambers, Schlegel, & Dunne, 2010;Herrin & Gussow, 1989;Hinrichs, 2000;Hinrichs, 2003;Ilbery, Watts, & Simpson, 2006;Marsden, 1995;Selfa & Qazi, 2005).However, recent studies have shown that current local food production may be insufficient to meet local food needs (Desjardins, MacRae, & Schumilas, 2009;Giombolini et al., 2010;Peters, Bills, Lembo, Wilkins, & Fick, 2008).One avenue to increasing local food production may come from transitioning cultivation from nonedible to edible crops, thus strengthening local food systems1 for consumers and producers.Understanding the obstacles to and opportunities for such a transition requires analyzing yield potentials and examining the challenges that may be faced by those involved.Our research addresses these goals by exploring a transition from nonedible grass seed to edible grain production for local consumption in Oregon's Willamette Valley.While we acknowledge the importance of growing a diversity of crops and a variety of edible grains, we have chosen wheat for our case study because of its importance as a dietary staple, the history of wheat production in the region, the absence of wheat in common local food venues, the relative similarities in grass seed and edible grain production, and the availability of research on wheat yields. According to the United States Department of Agriculture (USDA) and United States Department of Health and Human Services (USHHS) 2005 Dietary Guidelines for Americans, a balanced diet should consist of a combination of grains, meat and beans, vegetables, fruits, dairy, and oils, with grains making up the majority of a healthy diet.In the context of local food venues such as farmers' markets and community supported agriculture (CSA), grains in large quantities are frequently absent, compared to seasonal fruits and vegetables, dairy, and meats.While grains may be available through food cooperatives, retail stores, and bakeries, the relative absence of local grains from these venues as well speaks to a gap in our local food systems.This lack largely stems from grains, such as wheat, generally being produced as largescale commodity crops for export from regions known for high yields, such as the Great Plains in the central United States (USDA, 2009a).When looking at how to transition to increased local food production, it is important to consider the issue of scale of production and the argument for competitive advantage in grain production on larger fields with more mechanization.This, however, does not diminish how the relative lack of local grains creates challenges for communities and individuals working to build local food systems.This research focuses on crop transitioning to wheat and other edible grains within Western Oregon's Willamette River Basin due to the region's history of rich agricultural production and its vibrant local food movement.Much of the region's agricultural land is currently in nursery crop, hayseed, and grass seed production (ODA, 2008a) (see table 1 for 2009 Willamette Valley crop data in acreage and value). Wheat is an important cash crop in Oregon and is predominantly grown in the eastern part of the state.The Willamette Valley also produces wheat for national and international markets, although the amount harvested fluctuates greatly from year to year (ODA, 2009a) in response to national and international markets.Based on the recommended dietary requirements of the USDA and USHHS 2005 Dietary Guidelines for Americans, edible grain production in the Willamette Valley growing region would not have met the 2008 population's requirements for any of the last five years of production.In 2004 crop yields equaled 73% of the 2008 population's dietary requirements; in 2005 it met 34%; in 2006 and 2007 it met 29%; and in 2008 it met 67% (Giombolini et al., 2010).Within these fluctuations, even the relatively high numbers can be deceiving.In 2006, 92% of the wheat produced in Oregon was exported, principally to Asian markets where it was used to make such items as steamed buns and noodles (ODA, 2007).This last fact is not to recommend that international trade should cease, but to illustrate that while Willamette Valley grain yields have the potential to meet a significant percentage of the local population's recommended dietary requirements, local consumers are not benefitting from it. The demand and marketing of local food is expanding beyond farmers' markets and community supported agriculture to community organizations, large and small grocers, cooperatives, and supermarkets (Blake, Mellor, & Crane, 2010;Borst, 2008;Dunne, Chambers, Giombolini, & Schlegel, 2010;Guptill & Wilkins, 2002;Morris & Buller, 2003).As Feagan (2007) has noted, community is an important component of local food systems because food is intertwined with community.There are several emerging community organizations in the Willamette Valley that support the expansion of a local food system and play an important role in expanding production and markets for local edible grains. In the southern Willamette Valley two community groups, The Ten Rivers Food Web2 (TRFW) and Willamette Valley Farm and Food Coalition3 (WVFFC), have partnered to support the Southern Willamette Valley Bean and Grain Coalition (SWVBGC). 4These groups publish blogs to document their meetings at which they discuss successes and challenges in production as well as provide information on growing and purchasing edible grains (for example, see Armstrong, 2008;MacCormack, Kise, & Augerot, 2008).Both the TRFW and WVFFC focus on community initiatives, grower networking, and food education.The SWVBGC consists of farmers, distributors, activists, and community members interested in developing economically sound organic bean and grain production methods, as well as local markets for their sale.According to the SWVBGC's blog, the group formed and has grown in response to a number of perceived issues, including the increased cost of petroleum products, fluctuating world grain prices, and concern over nonexistent local bean and grain distribution infrastructure (Armstrong, 2008).It is through community organizations such as these that much research, education, and policy initiatives about community food systems are conducted. From Grass Seed to Grains Grass seed -cool season forage and turf grasshas been an important commodity for Oregon's economy as well as its landscape.Oregon growers produce essentially all of the U.S. production of annual ryegrass, perennial ryegrass, bentgrass, and fine fescue.Smaller amounts of Kentucky bluegrass, orchardgrass, and tall fescue are also grown in Oregon (OSU, 2009).It is the third highest value commodity crop grown in Oregon, grossing over US$500 million in 2008 (ODA, 2008a).The temperate climate of Oregon's Willamette Valley, with wet winters and arid summers, makes it one of the world's most productive regions for grass seed farming (Young, 2003).According to 2008 crop production data from Oregon State University Extension Service's (OSUES) Oregon Agriculture Information Network database (OAIN), over 450,000 acres (180,000 hectares) of agricultural land in the Willamette Valley is in grass seed production; in 2003 this represented more than one third of the growing region's cropland (Young, 2003).In 2009, the numbers dropped slightly to just over 410,000 acres (170,000 hectares) of grass seed cultivated in the Willamette Valley growing region (OSUES, 2008). Grass seed production in the region faces challenges as new laws influencing agricultural practices for producing crops as well as declining market values cause farmers to consider possible alterna-tive crops.The near-total ban on field burning that passed Oregon's legislature in the summer of 2009 (SB-528) may speed a change in the percentage of land producing grass seed (Oregon Legislative Assembly, 2009).Field burning has been a popular grass seed farming technique since its implementation in 1948.It is used to control weeds, remove straw residue, and eliminate crop diseases (Chilcote, 1969).Although limited burn restrictions have been in place since the late 1980s, the recent legislation is a far stricter ban, which creates more obstacles to grass seed production (ODA, 2008b).The greatest effects of the ban will be on land currently in annual ryegrass, the most commonly grown but lowest value grass seed variety (Young, 2003).According to an OSU extension service field crops agent, because annual ryegrass is the most successfully grown but has the lowest returns, the increased costs of inputs and maintenance as a result of being unable to burn the fields will make growing annual ryegrass economically unfeasible. Recent global economic conditions have also influenced the grass seed market. A 2009 article in The Oregonian highlighted decreased demand due to reduced planting of lawns and golf courses as one of the challenges grass seed farmers face (Read, 2009).Market prices for annual ryegrass seed in August 2009 hovered around US$0.18 per pound, while grass seed costs approximately US$0.26 per pound to produce (Dietz, 2009).Different varieties of grass seed command different prices.In spring 2010, annual ryegrass sold for US$0.15 per pound while perennial ryegrass sold for US$0.40 to US$0.50 cents a pound (T.Silberstein, Oregon State University Extension Service field crops agent, personal communication, February 4, 2010).Due to adverse market conditions, economic factors such as the decline in housing starts, and legal restrictions on field management practices, the future of the grass seed industry is unclear (Repko, 2009).This has spurred many regional grass seed farmers to begin to seek out alternative crops (Lies, 2009). Given the widespread use of wheat in the United States, the growing market demand for local foods, and the similarities in cropping techniques to non-edible grains, wheat has potential as a grass seed replacement crop.In the past, it was grown widely throughout the Willamette Valley (Bunting, 1995).According to Brumfield (1968), the region was one of the primary wheat-growing areas in the Pacific Northwest during early European settlement.Wheat milling and processing facilities were built throughout the area beginning in the 1830s.Wheat production was phased out over time due to competing grass seed markets.Malone (2010) provides a detailed history of the rise of grass seed production in the lower Willamette Valley, describing it as resulting from economic and social changes (e.g., World War II and increased demand for turf and forage seed).Figure 1 illustrates the change in wheat yields in the Willamette Valley over the past century. For our research, we used the Willamette Valley as a case study for transitioning grass seed acreage to wheat production.Given the potential for this growing region to produce its own grain, as well as its population's interest in purchasing local foods, it is uniquely suited to testing strategies for creating local markets for grains, a staple not commonly sourced locally.Using geographic information systems (GIS) analysis, we projected estimated soft white winter wheat yields for land currently in grass seed production to determine whether wheat production on transitioned lands could meet the regional population's dietary grain requirements.Interviews were conducted in order to more holistically illustrate the necessary steps and attendant challenges in transitioning from grass seed to edible grain production.Local food system planning must address all aspects of grain production -cultivation, processing, transportation, distribution, and policy -if it is to support these agricultural and societal transitions.This research illustrates a method of investigating transitions to more local food production and the importance of including many voices in the research, planning, and policy processes.An important finding of our research for building more resilient local food Study Area The Methods To visually represent current grass seed crop production land in the Willamette Valley and provide numerical projections for soft white winter wheat yields from land in grass seed, we used the GIS software ArcMap (ESRI, 2008) to analyze crop production data.We used the yield projections, along with recommended dietary requirements for the 2008 population in the region (based on the USDA and USHHS's 2005 Dietary Guidelines for Americans), to determine if yields from areas converted from grass seed to wheat production could meet the dietary grain needs of the local population. In order to better understand the process of transitioning from grass seed to wheat, we conducted semistructured interviews with farmers either transitioning their land or currently growing wheat, edible grain, and/or beans for both local and commercial markets, as well as individuals connected to increasing local food production in the Willamette Valley.Interviewees represented the most central characters in the transitioning process in the growing region at the time of the research (2009)(2010). GIS and Crop Production Analysis Datasets.We used three publically accessible datasets to assess the potential for soft white winter wheat production in 2007 of fields planted in grass seed in the Willamette Valley. 5We began with a National Agricultural Statistics Service (NASS) raster-based file for 2007 Oregon cropland that was clipped to the Willamette River Basin.Secondly, we used a personal geodatabase file based on Soil Survey Geographic (SSURGO) surveys that give the predicted weighted average soft white winter wheat yields for each soil type in bushels per acre 6 (NRCS, 2009).Soil productivity (measured in bushels per acre) was obtained from soil survey data conducted by the NRCS, which used a variety of methods, including interviews with agricultural producers, review of crop yield data collected by USDA Farm Service Agency county offices, interviews with Oregon State County extension agents who are familiar with wheat yields on soils in their counties of responsibility, and rod row sampling, to determine soil productivity.Another geodatabase file was used to intersect the Willamette Valley SSURGO wheat yields feature class with the polygon grass seed shapefile converted from a raster.This feature class contained the SSURGO soil survey polygons and weighted average soft white winter wheat yields for all areas identified as grass seed land in the NASS 2007 crop cover raster dataset.This final dataset was used to calculate the potential soft white winter wheat yields for areas currently in grass seed production. Crop production potential calculation.Each of the classified soils had specific weighted average soft white winter wheat yields (in bushels per acre) that were used to calculate total projected yields.We used only land yielding 100 bushels per acre or greater to calculate total potential soft white winter wheat crop production because economically viable land in western Oregon must produce an average of at least 100 bushels of wheat per acre (T.Silberstein, Oregon State University Extension Service field crops agent, personal communication, February 11, 2010).The benchmark of 100 bushels per acre used in this study is not, however, presented as a prescription to farmers; there is a complexity of factors that influence a farmer's decision to grow particular crops.The total number of bushels was then converted to pounds of wheat flour based on the most recent version of the USDA and NASS Agricultural Statistics (2007), a publication of commodity conversion factors for various agricultural crops and livestock.Using the conversion factor for bushels of wheat to pounds of wheat flour (2.3 bushels yields to 100 pounds of flour), we determined how many pounds of flour would be produced.Finally, in order to determine if the yielded number would match the 2008 Willamette Valley population's recommended dietary requirements for grain we converted the pounds to grams, because serving sizes are designated in grams (see the following equation): Total # of bushels X 45359.24÷ 30 = Total servings 2.3 produced It is important to note that this conversion factor is based on hard red wheat bread flour (i.e., white unbleached flour).There is a difference in weight between white and whole-wheat flour.The process of making white wheat flour retains only approximately 75% of the original grain weight after key nutritional components such as the bran and germ are removed from the grain kernel (Kansas State University Extension Service, 1997).The actual weight depends on the processing technique (stone ground, steel bur ground, removal of germ and bran, etc.).With this in mind, the final produced weight of whole-wheat flour may be higher. Population and dietary grain requirements. We acquired detailed population data from the 2008 Oregon Population Report, an annual publication of Portland State University's Population Research Center (Proehl, 2009).This population data was used in conjunction with USDA and USHSS (2005) Dietary Guidelines for Americans recommended daily requirements to calculate grain servings for the region's population.These calculations were based on data used in previous research conducted by Giombolini et al. (2010). Soil ratings and cartography.In order to visually represent the potential growing regions for soft white winter wheat in the Willamette Valley, we created a weighted map highlighting the areas of greatest wheat yields.Some soils exhibited much higher wheat yields than others.3). Interviews We conducted semistructured interviews to create a broad overview of the grass seed industry, regional agriculture, and the process of transitioning to edible grain production in the Willamette Valley.The semistructured format allowed for comparability and consistency.As the goals of this component of our research were qualitative in nature rather than quantitative, participants were chosen using purposeful sampling (Bickman & Rog, 1998;Patton, 1990).We focused primarily on a group of farmers, distributors, and community members in the southern Willamette Valley dedicated to local food security and transitioning to a more localized food system. Most of the farmers interviewed were key informants who represented the core of the transitioning movement at the time of our research (2009-2010) and were associated with the Southern Willamette Valley Bean and Grain Coalition (SWVBGC).Three of the participants interviewed were large-scale grass seed farmers (their acreage ranged from 800 acres to 9,300 acres, or 300 hectares to 3,800 hectares) transitioning to edible grain production for local markets.One interviewee was a small-scale organic farmer engaged in growing test plots of different wheat varieties to determine their suitability to the region before recommending their use to large-scale grass seed farmers.We also interviewed a wholesaler spearheading the transition's marketing aspect.Additional interviews were conducted with four others not directly connected with the SWVBGC: one agriculture extension representative, two grass seed growers not transitioning, and one small-scale farmer/seed researcher. The number sampled is representative of the key actors and reflects the majority of attitudes of those involved with this small movement and initiative. Interview questions included a variety of survey, specific, attribute, and structural questions (Bickman & Rog, 1998) focused on grass seed and wheat production, farming ideology, and marketing. Farmers were asked different questions from those asked of local distributors and other community members working to facilitate the transition from nonedible export crops to edibles grains for local markets.Interviews were held at participants' offices, farms, or public locations of their choosing. Each interview lasted about 60 minutes.They were recorded and transcribed with the consent of the participant.Triangulation was used when possible in order to verify the validity of the interviews by comparing the information provided to other sources such as statistics or alternative references (Bickman & Rog, 1998). Results After summing the total areas of each soil type (for Legend those yielding 100 bushels per acre or greater) and multiplying this number by the associated soft white winter wheat yields (bushels/acre), we combined the totals to give projected bushels of soft white winter wheat from land in grass seed production.Of the total area, 264,581 acres (107,072 hectares) yielded less than 99 bushels, and 250,537 acres (101,389 hectares) yielded 100 bushels or greater.Based on calculations determining the total yields of winter wheat in bushels from land in grass seed production in 2007, the recommended dietary grain needs of the Willamette Valley's 2008 population would be met two times over.The total projected number, 25,324,934 bushels of soft white winter wheat, converts to 16,648,112,453 servings.The recommended dietary grain needs (based on gender and age) for the 2008 population of the Willamette Valley is 6,836,647,100 servings. Discussion: The Transitioning Process The projected numbers from our GIS model have shown that it is possible to meet the recommended dietary grain needs for the Willamette Valley's 2008 population by transitioning from grass seed to wheat production.The GIS model, however, is based on predicted outcomes without taking into account the various and complex factors that influence crop production.With this in mind, what are the perceived obstacles to this transition?The following discussion uses information gathered through interviews to contextualize the calculated numbers for potential wheat production. Farmers interviewed described transitioning as a holistic process with a need to focus not only on transitioning to different crops but also to different farming techniques and marketing strategies.They saw that transitioning is not limited to changing from grass seed to wheat, but from grass seed to other edible grains, beans, and seeds as well, bringing crop rotation particularly into focus due to the potentially higher yields to which it can lead.Farmers also discussed transitioning from conventional agriculture to more organic-based production.Interviewees stressed their reasons for feeling that a transition to organic production was important to make, how this influenced their farming practices, and the attendant risks and barriers.Members of the SWVBGC have coalesced around organic production due to the environmental and health benefits of organic food, in addition to their sense that many consumers interested in local food prefer that their food be organic (Armstrong, 2008).Production by members of the SWVBGC has grown from humble beginnings of less than 50 acres (20 hectares) of transitional or organic beans and grains to more than 600 acres (250 hectares) transitioning to organic, and over 100 acres (40 hectares) certified organic (Armstrong, 2010a;MacCormack et al., 2008). Our interview results reveal that transitioning from grass seed to edible grains in the Willamette Valley would involve building local food systems, technical farm changes, and a cultural shift.We believe that these practical insights from local voices on the requirements for transitioning from nonedible crops to edible grains are not unique to the Willamette Valley.The following insights represent individuals' perspectives and provide contextual information and a robust model for planners in other communities seeking to analyze and address local food system challenges and opportunities. Building Local Food Systems Our interview findings reveal the need for the transition from grass seed crops to edible grains and beans to extend beyond the farmers and their fields to building local food systems with increased infrastructure, along with market creation that includes expanded community involvement. Rebuilding infrastructure.According to the interviewees, one of the greatest barriers to the transitioning process is the lack of infrastructure that is needed to adequately promote local food production, processing, distribution, and consumption.Without these infrastructure elements, creating a reliable market where local crops can be sold is difficult.Most farmers do not have the time or skills to create infrastructure or develop markets.While several farmers currently provide the storage facilities, process, and distribute their crops out of necessity, many made a point of emphasizing that they were farmers -not processors.While multiple roles may be evolving for farmers, each part of the food system requires different skill sets.Farmers represent only one part of that system.Grain production is highly regionally specific and generally requires primary or secondary processing before marketing to consumers (USDA, 2009a).This presents what some perceive as a barrier to creating local food systems for grains, as appropriate infrastructure must be created to process harvests from raw and often inedible states (MacCormack et al., 2008;Merlo, 2005).On one hand this can be considered a barrier, but growing a local food system also creates an opportunity for new businesses and entrepreneurs.The Willamette Valley currently has little infrastructure to support the storage, processing, and distribution of local grains, beans, and edible seeds.That which existed historically disappeared with the increase in grass seed production. Storage is a particularly significant issue.One farmer observed: Storage, to a big degree, is going to rely on the farmer.For us, we are taking bins at our seed warehouse that would normally be for grass seed and we are going to be storing different grains. Farmers themselves, especially those who clean seed and have extra storage space, will initially house the seed before it enters the market.One farmer who owns a 17,000-acre (7,000-hectare) grass seed farm commented that he is increasing his facilities for wheat storage as a method of avoiding the saturated wheat harvest market and commanding a higher price during other times.Malone (2010) identifies grain storage and processing facilities located in Oregon and notes that only one elevator in the Willamette Valley is licensed to store and transport organic wheat.Lack of storage space is a critical factor in making it difficult for farmers with limited storage space to grow for the local market.Farmers are taking on multiple roles since current conditions are leaving them without many options, but as operations grow increased infrastructure will be needed.The SWVBGC blog notes that members express concerns that additional storage infrastructure will need to be developed in order to accommodate larger future harvests of grain and beans (Armstrong, 2010a). In addition to lacking sufficient storage, the Willamette Valley has few processing plants and mills.Its dominant flour mill is Cereal Food Processors, Inc., a privately held corporation and America's largest independent flour milling company.This mill processes 760,000 pounds (340,000 kg) of flour per day.The majority is produced from hard red wheat grown not in Oregon, but in Montana (Cereal Food Processors representative, personal communication, April 21, 2010).While there are smaller processors such as Bob's Red Mill in Milwaukie and Grain Millers in Eugene, they typically do not process the small quantities of grain that many producers are looking to sell locally.In 2009, approximately 500,000 pounds (over 200,000 kg) of wheat produced in the Willamette Valley was available to be milled for the local market.A small mill7 with a grinding capacity of 750 pounds (340 kg) of wheat a day would only need to operate 12 to 14 hours a week to meet the processing needs of the local population (J.Henderson, sales coordinator for wholesaler, personal communication, April 21, 2010).Farmers note that the lack of small processing plants makes it hard to market local wheat, but wonder at what point is enough grain produced to justify investing in this infrastructure. Without mills, wheat is sold as a whole grain, a form which is inconvenient as well as unfamiliar to the many who prefer flour for cooking and baking.One option may be to sell whole grain to consumers and develop the infrastructure for personal grinding.Located in Corvallis, Oregon, the First Alternative Co-op has installed two flour grinders, one for whole wheat bread flour and one for whole wheat pastry flour, giving customers the ability to grind whole grain wheat in quantities suitable for home use.Approximately five pounds (2.3 kg) of bread flour and two pounds (0.9 kg) of pastry flour are processed daily.This is an example of a positive, if very small step, as a wide range of milling options including both small and larger scale grinding facilities may be needed in order to seriously promote wheat locally. In their blog posts, the SWVBGC farmers detail how in 2010 they took steps toward infrastructure development with the addition of four organic seed cleaning facilities and two small organic grain mills (Armstrong, 2010a;Rea, 2010).They note, however, that despite these steps, securing adequate facilities for storing harvested grains and processed flour remains essential if grain products are to be kept free of vermin and mold (Armstrong, 2009).While SWVBGC members have thus far been able to overcome structural barriers to the production, processing, and sale of organic grains and dry beans, increased production and markets (with the ultimate goal of profitability for farmers) will require expansion of critical local food system infrastructure components. Market Creation.In the Willamette Valley, the development of local food markets for grains has begun through the work of community organizations, a wholesaler, and individuals who share risks with the farmers.This step is critical, as farmers will not produce if market demand is not there.Some local organizations are working to develop markets, such as the SWVBGC, which has the stated intention of helping farmers transition and sell locally and is considering producer cooperatives as a potential option for increasing farmers' capacity to do so (Armstrong, 2008;Armstrong, 2010a;MacCormack et al., 2008). Many questions surround the long-term strategies needed to develop a local market for grains.For example, although producers report that demand continually outstrips supply, the local market's ability to absorb these crops may be tested in the next couple years as more than 500 acres (200 hectares) of grass seed are transitioned to beans and grains (Armstrong, 2010a).The question of how to manage production so that the supply of grains and beans does not flood the market has been raised for the future of the SWVBGC (Armstrong, 2010a;Armstrong, 2010b).To avoid overproduction it is important to develop a diversity of markets and different avenues to market the increasing supply of beans and grains. A wholesale company based in Eugene has been working diligently alongside local farmers to provide markets for their crops.The CEO of the company notes: If we had enough market there would be a lot more farmers interested in growing these [crops].If we could provide contracts for the farmers then they would definitely grow. The wholesaler is interested in establishing contracts with farmers in order to have stable agreements between both parties with an agreed upon price and quantity.Economic incentives to providing local crops exist because they tend to command higher prices, but reliable markets are needed in order to sell the grain.Farmers rely on the support from such companies to create avenues for distribution, in order to successfully transition from grass seeds to edible crops for local markets. Personal interaction plays an important role in gaining customer trust and support for establishing alternative food systems (Watts, Ilbery, & Maye, 2005).The wholesaler in question has invested time, energy, and resources into understanding its customer base and creating new markets for the local crops being grown.It has sent a questionnaire to customers asking if they would be willing to buy transitional, not organic, products locally during the three years the farmer transitions to organic.Overall, it found customers supportive of buying transitional crops. Given the risks inherent in the transitioning process, financial support from wholesalers is crucial to transitioning farmers.The CEO states, We go in with the farmer where we give the farmer money down, plus we also provide the seed; we really want to take the risk with the farmer if we know that we can sell the crop. Such companies share risks with farmers to make the system work by providing financial support through a difficult growing season or providing some of the inputs.In general, a farmer is riskaverse and does not want to take transitional risks alone; partnerships with companies such as wholesalers are crucial.Many farmers commented that the transition from grass seed to edible grains and beans would not be possible without support from the wholesaler: it was the catalyst for the transitioning process.Future support from community members and other entities such as buying clubs may be another option to help mitigate some of each farmer's risk. All farmers necessarily take some risk as a poor crop year could lead to financial hardship, but for farmers transitioning to edible grains for local markets the risks are unique in that they are doing something different from the norm.One farmer interviewed stated, We need guaranteed income or we can't make it; it is a really scary feeling like we could lose everything if we have a bad year. For many farmers grass seed has provided a relatively risk-free crop for decades, if not generations.Yet farmers often operate on the margin and the current situation with grass seed sales is reducing some farmers' opportunities to diversify.A grass seed farmer who is not transitioning to edible grain crops for the local market commented on those who are transitioning away from grass seed: It's how much risk you can take.When times are good, you can set aside some acres to experiment with.Right now we are kind of hunkering down and scraping through until times get good. Farmers frequently find it difficult to take the initial steps toward moving outside of conventional practices because growing something different and failing may be worse than waiting it out and continuing production of crops that in the past have been dependable. Many SWVBGC farmers have relied upon a single wholesaler to purchase and sell their transitional organic beans and grains (Armstrong, 2010b).Sole reliance on this one distributor for their product may result in overlooking the possibility of large contracts with other significant consumers, such as bakeries and restaurants.While the incremental steps taken by the SWVBGC have thus far been effective at growing and distributing organic beans and grains, more long-term market strategies will be needed (see Armstrong, 2010c).Continued growth and networking between organizations will be instrumental in supporting the transitioning process from grass seed to edible grains and beans. On-farm Technical Transitions Farmers interviewed described the relative ease of transitioning from farming grass seed to raising wheat and other crops, but also outlined the obstacles to such a transition.Farming contains many technical elements, and transitioning farmers must consider not only the change in crop types, but also technical transitions involving farm equipment, marketing and transport tools, seed stock, and organic production methods. Equipment.Grass seed equipment requires few significant changes in order to process wheat and other such crops.According to farmers we interviewed, the main change involves investing in combine headers designed to harvest wheat.In general, given suitable header selection, wheat and grass seed (as well as most other edible grains, beans, and seeds) can be seeded, harvested, and cleaned using the same large equipment.One farmer interviewed commented: That's the beauty.Beans, grains, and edible seeds we can harvest using grass seed equipment.We don't have to change anything. Economically, the initial mechanical transition does not require great financial inputs.Malone (2010) presented an alternative view based on her research that found grain is more costly to produce than grass seed.Grain requires more processing (e.g., crushing and grinding the grain), but farmers interviewed said that cleaning and harvesting wheat should require few mechanical alterations.Growing dry farmed beans presents further difficulties due to the Willamette Valley's relatively short summers and inconsistent weather patterns.Having reliable harvests has been and continues to be a challenge for farmers trying to bring beans into the crop rotation and to the local market.(These comments are expressed in detail in the SWVBGC blog; see Armstrong, 2010c.)The scale of edible crop acreage can be a determining factor in equipment selection.The farmers we interviewed were transitioning anywhere from 2 to 400 acres (0.8 to 160 hectares) of land.One couple employed a 1965 combine to harvest their hard winter wheat because: It's probably 25% the size of our conventional combines.The older combine works perfect because we're only doing 30 or 20 acres [12 or 8 hectares]. In considering equipment changes, farmers face relatively few barriers; the real challenges concern the lack of available infrastructure for distributing, marketing, and transporting other crops. Marketing and transport. Marketing and transporting grass seed is different than marketing and transporting wheat.Grass seed, although a commodity crop, is not sold on the commodity market and tends to be produced under contracts, which serve as a type of risk-management plan.Grass seed companies create contracts with farmers each year to determine the type and amount of grass seed to be planted.The farmer then grows the seed and holds it until the seed contractor picks up the seed for distribution.In this way, the farmers do not own their seed, but grow it.Two of the grass seed farmers we interviewed said that with the decline of the grass seed market, contractors are not completely fulfilling their contracts, leaving many grass seed farmers to store grass seed from the past year that the contractor could not sell. As wheat is a commodity, farmers are responsible for selling, transporting, and distributing the wheat they produce.Wheat value depends on volatile market prices.The break-even price for wheat grown on land yielding 100 bushels per acre is approximately US$5.50 to US$6.00 per bushel (T.Silberstein, Oregon State University Extension Service field crops agent, personal communication, February 11, 2010).Wheat prices in 2007 peaked at a high of US$10.30per bushel, which inspired many Willamette Valley grass seed farmers to grow more wheat (USDA, 2010).The spike in wheat prices proved temporary, however, and by 2009 wheat prices hovered around US$4.50 to US$5.00 per bushel (USDA, 2010).The volatility of market prices is an important consideration when providing recommendations from the 100 bushel yield benchmark used in our GIS model.The current marketing structures in place for wheat will need to be altered to establish a more stable market price, perhaps to a contract-based system similar to grass seed, in order to serve the local market. Seed stock.The question of seed stock and seed varieties suited to the Willamette Valley is also critical to transitioning farmers, particularly which varieties of wheat to grow and what the availability of organic seed supplies might be. The projected bushel yields from the GIS data are for soft white winter wheat varieties as opposed to hard wheat varieties.The main difference between the two varieties has to do with their respective protein levels (although gluten and ash levels are also components).Hard wheat is typically used for breads and is primarily grown in the Midwestern states, whereas soft wheat is commonly used for pastries and flatbreads and is frequently grown in the Pacific Northwest (USDA, 2009a).There is a potential market for both soft and hard wheat to meet local demand.Soft wheat can address local pastry needs, while hard wheat can address local bread baking needs.Cultivating the knowledge and ability to use both appropriately in the long term will support increased production and consumption of local grains. Protein levels and uniformity dictate the types of wheat grown, and grade and quality standards have limited the number of commercially produced varieties of wheat due to farmers' inability to receive government funding and loans for "undesirable" seed (Malone, 2010).Farmers interested in growing grains for the local market are diverging from these past influences on seed selection and are not necessarily relying on government subsidy programs, as they are growing seed varieties that are more rigorous for the Willamette Valley climate.During the historic boom in wheat production, both hard and soft wheat varieties were grown in the Willamette Valley.Brumfield (1968), in describing popular wheat types grown in the late 1800s and early 1900s, mentions names such as Turkey Red, Bluestem, and Marquis, all of which are hard red varieties (Carleton, 1916).These heirloom hard wheat types are now being re-introduced into the Willamette Valley as viable options. Several farmers interviewed commented on the commonly held belief that hard wheat cannot be grown in the Willamette Valley because the climate does not allow it to develop protein levels sufficient for bread making (12% to 14% protein).Trials with hard spring wheat done by some of the farmers interviewed refuted this idea.One farmer commented: People say you can't raise high enough protein wheat here in the valley to make good protein.We have been successful in doing that one field, one year. "One field, one year" speaks both to the farmer's optimism and realism, as the possibility exists but there is still uncertainty about consistency.Given that these experiments are in their infancy, it bears mentioning that the consistency of such local hard wheat's protein levels from year to year is unknown.Many factors may affect these levels, particularly weather and soil; further research and variety development is needed. Oregon State University has continually developed different cultivars of wheat suited to successful growth in the region (Ross, 2007).While the majority are types of soft white wheat, the variety commonly grown by farmers in the Willamette Valley, within the past decade researchers have also developed cultivars of hard wheat (Peterson, 2008).Although hard spring wheat trial yields are much lower than for soft white wheat, they have demonstrated that it is possible to produce adequate protein levels (Peterson, 2008).Over time and through various factors such as farm management, selection, and soil development, these yields could increase.Some farmers also participate collaboratively in seed development.One farmer interviewed stated that he is testing out many new varieties developed by Washington State University and other breeders, both nationally and internationally.Others have dedicated small plots of land to growing out several varieties in order to gauge their success in the local climate.One farmer interviewed is growing three new hard red wheat varieties from three distinct regions -Argentina, Washington state, and North Dakota -to look at protein levels and milling qualities.In determining the type of crops to grow in the Willamette Valley on large-scale farms, farmers will have the added task of assessing a wider variety of crops with some level of trial and error.Partnerships with universities and small and large-scale farmers, as well as with community members, will be important in developing successful seed varieties. In order to find truly suitable wheat varieties, experimentation with growing a greater diversity of crops is important.Also important is the need to recognize that achieving standard protein levels, which Malone (2010) cites as a significant barrier to local processing, does not necessarily need to be viewed as the goal.Part of the advantage of organizing on a local level is that it allows for transparency and open communication between the producer and consumer.Farmers can account for fluctuating protein levels while consumers still find the product usable.It is innovative ideas and experimentations that will create successful new crop varieties for the Willamette Valley growing region.What is needed now is more research into growing out hard wheat varieties developed for this climate. Organic production.The shift from conventional to organic agriculture requires changing farming techniques.Hanson, Dismukes, Chambers, Greene, and Kremen (2004) describe the steep learning curve farmers face in the conventional-to-organic transition as they learn biological pest controls, manage nutrient cycles without synthetic fertilizers, plant different crops, and supply new markets.Two major changes noted by farmers transitioning to organic edible grains were reduced use of chemical pesticides and the substitution of crop rotation in place of synthetic fertilizer.One farming couple transitioning part of their land stated: Conventional farmers can learn a lot more from organic farmers than we can teach them.Chemicals are not an option with them.They look at strictly keeping that plant healthy.It's just easier to spray it with a chemical pesticide and say we've done everything we can; well, we haven't.It's not the easiest thing to do. When discussing motivations for transitioning to organic, farmers also mentioned the impact of the recent ban on field burning: We just can't afford the pesticide anymore.We were burning some of these fields and field burning was taken away, so we had to replace field burning with more pesticides or more crop rotation. Burning increased yields by killing pests that conventional farmers are now treating through the use of more chemical pesticides.Farmers we spoke with also emphasized the importance of diverse organic production, with one noting: Diversification became important; cutting down on fertilizer and chemical use brought crop rotation into focus. The use of crop rotation and changing farming techniques to a greater focus on soil health is a key part of organic production.Transitioning to organic production of edible grains and beans may benefit Willamette Valley grass seed farmers by decreasing costly chemical and synthetic inputs. Establishing the best organic practices for a specific farm or field such as the correct crop rotations takes time and experimentation.What works at one farm may not work at another, as they may have different soil types supportive of different crops.One farmer noted that much of the farm's soil lacks the proper drainage to grow highprotein spring wheat, and conditions in such marginal, poorly drained land is better suited to grass seed and other crops.While wheat was the focus of the present GIS analysis, this more marginal land potentially could be used to grow other edible crops, such as barley and rye.In addition to time and experimentations, the use of GIS and soil survey data will be helpful in identifying successful rotations for specific soil types.Farmers, however, must make the choice for their individual property based on a variety of complex factors. Cultural Transition Interviewees drew attention to additional challenges beyond technical transitions to farming.In addition to the actual technical transitioning away from grass seed production, farmers may experience a kind of cultural shift with regard to their agricultural experiences.This shift can relate to changes in which types of crops are grown, what is perceived as being an ideal field, the scale of farming, and novel market interactions.A number of benefits may accompany this cultural change, including the expansion and diversification of markets, increased food security, enhanced support for local communities, and greater opportunities to connect directly with consumers. Quality grass seed production has a long and dignified history in the Willamette Valley, with some farming families focused on this form of agriculture for generations.Many farmers take great pride in their weed-free green fields and large store-houses of grass seed.One farming couple we interviewed said of their acres of grass seed: It was kind of nice in a way if you like the green lawn/golf course look.It was like "wow, we have the world's biggest lawn."Then you think about what's really gone into it.Now the occasional weed popping up doesn't bother us at all.This couple's statement exemplifies the changes in thought and values which many such transitioning farmers confront.They realize that the chemical fertilizers and pesticides needed to maintain the aesthetic of a weed-free field is not worth the cost and possible environmental and health consequences.However, new forms of agriculture, which may carry some risk of failure, are difficult to undertake.Learning to build soil health, plant on a smaller scale, rotate crops, and decrease pesticide use challenge many in the transitional process. Transitions in scale require a different mindset and demand greater attention to detail.Most farmers we interviewed were accustomed to large fields, often hundreds of acres in size.Although the goal in the Willamette Valley is transitioning large amounts of acreage from grass seed to edible grain production, the process will begin with smaller acreage -20, 50, 100 acres or 8, 20, 40 hectares, rather than thousands.In transitioning, particularly to organic production, that kind of reduction to smaller plot sizes is a dramatic change for farmers used to planting thousands of acres of grass seed.One farmer interviewed observed that farmers "are not comfortable with 10 or 20 acres at a time."Smaller scale dictates a different interaction with their crops, as each 10 or 20 acre (4 or 8 hectare) plot requires greater attention and manual input than far larger grass seed plots. Additionally, farmers may need to change how they view the established export-focused market system.One farmer said the following about the need to reconsider marketing: [Marketing] locally, regionally, then internationally as opposed to now where you sell it to these big outfits that sell it to Asia and whatever you've got left you dump off locally.If you flip that around you get paid more and food security will be increased. Growing for the local market, a farmer is diversifying his or her operation by selling through a variety of outlets, while prioritizing local markets.Transcending economics, transitioning is also about supporting the local community.Our research demonstrates that farmers could produce more grain than the Willamette Valley's population requires.While dismissing the idea of selling surplus product on the global market would be short-sighted, providing for the needs of the local community is an important consideration. Farmers are proud of their products and of their ability to produce for a local market; to see the face of their customer represents an important ideal to many of the farmers interviewed.In the current grass seed and commodity market system, farmers have lost the connection to the final consumer of their product.One farmer reflected: As a grass seed producer, I miss having my customer right here.We're really quite proud of what we produce; it would be nice to see how our customer appreciates it or not.So we could adjust or whatnot.We hardly ever see the end customer, and so you don't get that satisfaction. In grass seed farming, there simply is no real connection with the consumer.The end product belongs by contract to seed companies who ship it in bulk to clients around the globe.Similarly the soft white winter wheat currently grown in the Willamette Valley is generally exported from the Port of Portland to Asian nations for milling and processing.In a local market system the farmer can have a connection to a local bakery and its customers.The ability to associate and form human connections with the end consumer is an important motivator in the transitioning process.The son of one farmer reflected on this personal motivation for selling directly to customers: "Knowing that you had something to do with what everyone is having for dinner is kind of cool."Connecting local farmers with consumers is viewed as one of the broader benefits of local food systems, promoting positive community engagement, connecting people to each other through shared connection to place, and thereby creating an inclusive sense of community (Feagan, 2007;Hendrickson & Heffernan, 2002;Martinez et al., 2010). Conclusion: Transformation of Agriculture in the Willamette Valley In order to predict the food-producing capacity of the Willamette Valley, we used GIS analysis in conjunction with interviews to highlight practical issues and provide deeper contextual information.The combination of these methods provides a robust model for planners to analyze and address local food system challenges and opportunities.Viewed alone, GIS data is disconnected from the practical issues of implementation and the culture of agriculture, and is limited by scale and complexity.Thus interviews give greater depth to the model and open a more complex dialogue about transitioning land from nonedible to edible crops for local markets.With this type of GIS research, it is imperative to include the voices and insights of individuals because they not only provide possibilities for personal investment in the research or planning, but also give a more holistic perspective on the barriers and opportunities involved. The Willamette Valley acts as an exciting model for how communities are organizing to support the transition to a more local food system as farmers, consumers, distributors, planners, marketers, and entrepreneurs come together to promote the wellbeing and resilience of their community.All actors in the food system need to be involved -from farmers transitioning to growing edible grains for local consumption rather than global grass seed markets, to organizations like the Southern Willamette Valley Bean and Grain Coalition connecting farmers, to wholesalers helping develop new markets, to community members buying local grains in order to support local production.This research digs deeper into the process of building local food systems, focusing on growing staple foods for local populations and the importance of incorporating a new demographic of farmer outside the traditional direct-market, small organic producer.Thinking broadly, this research directs our focus on transitioning land by promoting largescale farmers currently growing inedible crops to growing edible crop production for the local market, as well as looking at the role that organizations and all actors in the food system must play to make this transition possible.This research lays out several next crucial research areas as scholars, planners, and nongovernmental and community organizations continue to create and experiment with new frameworks to build local food systems.Specifically, further research needs to be done on how to increase infrastructure, develop markets for producers, and expand community involvement.What are the most pressing infrastructure needs and what are strategic ways to meet those needs?What will be the characteristics of a local market?How can ownership and prioritization be ensured in a local market?How can we foster greater community involvement and awareness about local food systems and food security?These questions are critical in furthering local food system research.These questions and others will best be answered through interdisciplinary research that combines quantitative and qualitative methods and includes the voices of the local participants.Through models such as the one that we have presented with this case study, researchers, policy advocates, and policy-makers can partner with communities to build resilient, strong local food systems for the future. Figure 3 . Figure 3. Map of the Willamette Valley's (WV) Projected Soft White Winter Wheat Yields (ww) in Bushels per Acre on Grass Seed Land	2019-04-25T13:11:35.209Z	2011-10-30T00:00:00.000	{ "year": 2011, "sha1": "321babb6e83ab959269a19ce04974330e09e5671", "oa_license": "CCBY", "oa_url": "https://foodsystemsjournal.org/index.php/fsj/article/download/87/pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "321babb6e83ab959269a19ce04974330e09e5671", "s2fieldsofstudy": [ "Environmental Science", "Geography", "Agricultural and Food Sciences", "Economics" ], "extfieldsofstudy": [ "Geography" ] }
26445190	pes2o/s2orc	v3-fos-license	Application of the Regional Water Mass Variations from GRACE Satellite Gravimetry to Large-Scale Water Management in Africa Time series of regional 2° × 2° Gravity Recovery and Climate Experiment (GRACE) solutions of surface water mass change have been computed over Africa from 2003 to 2012 with a 10-day resolution by using a new regional approach. These regional maps are used to describe and quantify water mass change. The contribution of African hydrology to actual sea level rise is negative and small in magnitude (i.e., −0.1 mm/y of equivalent sea level (ESL)) mainly explained by the water retained in the Zambezi River basin. Analysis of the regional water mass maps is used to distinguish different zones of important water mass variations, with the exception of the dominant seasonal cycle of the African monsoon in the Sahel and Central Africa. The analysis of the regional solutions reveals the accumulation in the Okavango swamp and South Niger. It confirms the continuous depletion of water in the North Sahara aquifer at the rate of −2.3 km/y, with a decrease in early 2008. Synergistic use of altimetry-based lake water volume with total water storage (TWS) from GRACE permits a continuous monitoring of sub-surface water storage for large lake drainage areas. These different applications demonstrate the potential of the GRACE mission for the management of water resources at the regional scale. OPEN ACCESS Remote Sens. 2014, 6 7380 Introduction Satellite gravimetry remains the only technique that provides information on the total water storage change at continental scales and gives access to groundwater variations when a priori information on surface and sub-surface reservoirs is available [1][2][3].Data of the Gravity Recovery and Climate Experiment (GRACE) mission were widely used to estimate changes in land water storage and fluxes over Africa at basin to regional scales.By using the first two years (April 2002 to May 2003) of GRACE data, the study of [4] show that seasonal total water storage (TWS) variations vary between ±50 mm of TWS in the Congo and Niger basins.Time series of GRACE data allow us to estimate inter-annual variations and trends in TWS, as well as the contributions of TWS to sea level change for the largest drainage basins and lakes of Africa [5][6][7][8][9][10][11][12].They were also used for comparisons, validation and calibration of hydrological model outputs at the basin scale and over large bio-climatic regions, such as the Sahel or West Africa [13][14][15][16].Combined with external datasets in situ, GRACE data offer the unique opportunity to estimate groundwater storage variations for lake drainage areas [17,18] and large river basins [19] or at a regional scale [20], river discharges [21], evapotranspiration at the basin scale [22][23][24] and the water budget [25].They were also used to determine the specific yield [26,27] and loading effects in the Sahel [28]. In the following section, the principle of classical satellite gravimetry and the particularities of the regional method for recovering water mass variations from GRACE satellite data are presented.Time series of 10-day 2° by 2° maps of water mass variations over Africa (30°W-60°E; 40°S-40°N) are computed from accurate K-band range rate (KBRR) residuals along daily GRACE orbits for the whole period of GRACE (2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012).Then, global solutions used for comparing our regional solutions computed over Africa are listed.The spatial averages of our African solutions over hydrological units (see the drainage basins and desert aquifer area in Figure 1) versus time are computed to establish water mass balances, and, thus, sea level contributions, for the recent period covered by the GRACE mission.For isolating the African regions that produce the largest contributions to the sea level mass balance (and the ones in water deficit), the first space and time modes of the variability of GRACE data are extracted using a principal component analysis (PCA).Then, the PCA modes are compared to pure seasonal, semi-seasonal and multi-year linear trend variations (e.g., African monsoon), so that multi-year water mass gains (or losses) can be located in Africa and quantified.Finally, combining water volumes derived from regional TWS solutions and radar altimetry measurements of the level of the lakes enables us to estimate the soil and groundwater variations over the East African Great Lakes.6) Volta (~408,000 km 2 ); (7) Senegal (~270,000 km 2 ); as well as the areas contributing to the Atlantic Ocean (blue dots), India Ocean (red dots), Mediterranean Sea (green dots) and the endorheic ensemble (purple dots).The driest part of the Sahara Desert area in South Algeria (8) (~1.1 million km 2 ) and ( 9) the North Western Sahara Aquifer System (NWSAS) (~1.6 million km 2 ) are also displayed. Recovery of Surface Water Mass Changes by Space Gravimetry Since its launch in 2002, the Gravity Recovery and Climate Experiment (GRACE) space mission has measured, for the first time, changes of total water storage (TWS), including surface water, soil, moisture and groundwater, with unprecedented centimeter accuracy in terms of geoid height.GRACE data have already demonstrated a strong potential for estimating hydrological system information, such as river discharges [29], evapotranspiration rate [22,30], groundwater variations [31][32][33] and the detection of extreme climate events, such as floods and droughts [34][35][36][37][38][39][40].Several analysis centers, such as the Center for Space Research, University of Texas (CSR) in Austin (TX), the Jet Propulsion Laboratory (JPL) in Pasadena (CA), the GeoForschungsZentrum (GFZ) in Potsdam (Germany) and the Groupe de Recherche en Géodésie Spatiale (GRGS) in Toulouse (France), use Level 1-B GRACE observations to produce lists of monthly and 10-day global Stokes coefficients (i.e., spherical harmonics of the geopotential) up to harmonic degree 60 for CSR Release 5, 80 for GRGS RL03 and 90 for JPL and GFZ RL05; in other words, at the maximum surface resolution of 250-300 km.During the estimation process of the dimensionless Stokes coefficients from orbit data, the static gravity field and its time variations (i.e., atmospheric and ocean mass changes, including the effects of the periodic tides) are removed through a priori models describing these known gravitational accelerations.Therefore, the residuals correspond to the unmodeled contributions of mass to the observed gravity field and, mainly, the continental hydrology component. Unfortunately, the correction models remain imperfect due to their lack of completeness in the description of water mass movements by omission and/or the lack of resolution, which represent important sources of error in the recovery of continental hydrology variations.As GRACE-based residual Stokes coefficients are averages over constant time intervals of 10 days or a month, errors in the correction models with periods from hours to days contaminate these GRACE solutions by aliasing and, thus, degrade their accuracy [41].These effects of signal distortion deteriorate the quality of true water mass signals into other time frequencies and make these signals indistinguishable by sampling. In the case of the GRACE orbit, hydrology-related signals are measured mainly along satellite tracks in the nearly latitudinal direction, but they are projected onto global spherical harmonics (SH) functions, which ensure the best spatial frequency representation.Because of this polar plane geometry of the GRACE orbit, this particular distribution of measurements creates north-south "stripes" in the 10-day and monthly GRACE solutions.Moreover, the determination of the SH coefficients leads to underdetermined systems of normal equations to be solved by creating correlations between SH coefficients of high degrees (i.e., >10-15) [42] and amplification of this orbit error and data noise [43]. Another problem while using SH is the "leakage" of energetic signals propagating over the entire sphere, as these global undulations come to pollute the water mass estimated in the region of interest.This is particularly the case of small regions that are not fully represented by the degree 60 truncated SH spectra of the GRACE solution (i.e., error by omission).Besides, different low-pass filtering techniques have been proposed, but they can partly cancel some of these effects [42,44,45].The simplest way to increase the signal-to-noise ratio in estimation remains to average the signals over large surfaces of more than one million square kilometers, such as tropical drainage basins, to cancel the effects of the short wavelength SH undulations. An alternative approach for estimating surface water mass densities in a region from GRACE data has been recently proposed by [46,47].This new strategy is based on the optimal localization in space, instead of the best localization in spatial frequency, and leads theoretically to better spatial localization and resolution [48].The authors of [47] have shown that this regional method offers a reduction of both north-south striping due to the distribution of GRACE satellite tracks and the temporal aliasing of correction models over South America [36] and Australia [49].According to these two latter studies, regional maps present more realistic spatial and temporal patterns than the global solutions when compared to independent datasets of rainfall.The main modes of variability in South America coincide with the geographical limits of known hydrological units, such as individual groundwater layers [36].In the present article, 10-day regional solutions over Africa are analyzed and compared to other datasets. Methodology of the Regional Approach The two main steps of this regional method are: (1) using the principle of mechanical energy conservation to deduce the variations of difference potential anomalies (DPA) between the twin GRACE satellites, representing mainly the continental hydrology contribution, from the accurate along-track KBRR measurements; and (2) adjusting the equivalent water heights (EWH) of a network of juxtaposed 2° by 2° surface tiles by the linear inversion of the DPA passing over the considered region every 10 days [46,47].This regional approach differs from the NASA "mascons" [50][51][52] as, instead of classical band-limited SH, the regional method imposes the geometry and the best spatial localization of surface hydrology structures by construction.Schematic view of the processing for estimating global and regional solutions from a given 10-day or monthly period of Gravity Recovery and Climate Experiment (GRACE) observations.GINS, Géodésie par Intégrations Numériques Simultanées; GRGS, Groupe de Recherche en Géodésie Spatiale; SVD, singular value decomposition; KBRR, K-band range rate. In the first step, KBRR observations are reduced by removing the contributions of known gravitational accelerations related to large-scale mass variations (i.e., atmosphere and ocean mass variations, polar movements, solid and oceanic tides, as well as the static gravity field of the Earth that represents 99% of the observed signals).This operation is made by iterative least squares adjustment of daily dynamical orbits using the Géodésie par Intégrations Numériques Simultanées (GINS) software [53,54].Thanks to the measurements of on-board GRACE accelerometers, the effects of non-conservative forces are also removed from the KBRR observations in the orbit adjustment.KBRR residuals represent the cumulated contributions of unmodeled phenomena and, mainly, water storage change over continents.These residuals are related to the different accelerations of the two GRACE vehicles resulting from the gravity signals of continental hydrology, and they are easily converted into variations of kinetic energy differences.According to the principle of energy conservation, these kinetic energy difference variations directly correspond to potential energy differences, or in other words, DPA.To reduce unrealistic orbit errors at fractions of the satellite revolution periods and, thus, to avoid numerical instabilities in the following linear inversion, DPA arcs passing over Africa are linearly de-trended.It locally absorbs orbit error and keeps a subset of DPA short and medium wavelengths that are less than the latitudinal dimension of the considered region.The missing long-wavelength information of water mass change is from the first degrees of the GRGS solutions, and these large undulations are added to the DPA-derived regional solutions to complete the water mass signals after the inversion of residual DPA [36,47,49]. In the second step of the method, the Newtonian matrix A is defined from the positions of the two GRACE satellites and of the surface tiles, in a geocentric reference frame, according to Newton's first law of attraction.This matrix relates each unknown EWH to the DPA observations inside the region during 10 successive days.As gravimetry inversion does not usually provide a unique solution, regularization strategies should be applied to find numerically-stable solutions, either based on the truncation of singular values [46] or by introducing an averaging radius [47]. This latter type of regularization consists of adding a spatial constraint matrix block C to the Newtonian matrix A. The coefficients of this extra matrix C are obtained by imposing each equivalent water height to be a linear combination of its neighbors weighted by the inverse of their angular distances and inside a maximum geographical radius r.In the case of spatial "averaging", the coefficients for a given radius r should equal 1/P, as P is the number of surface tiles located at a distance lesser than r, and 0 elsewhere.Introduction of these linear constraints enables the ill-conditioned matrix A to be inverted.A good compromise for keeping enough hydrological details with no smoothing and limiting the increase of numerical noise was earlier found by considering a radius of r = 600 km over continental areas [47].A simplified flowchart summarizing the estimation process of regional solutions is presented in the following Figure 2. 10-Day Regional GRACE Solutions for Africa Daily arcs of five-second sampled K-band range (KBR) measurements of the inter-satellite velocity have been used in the GINS software [53,54] to adjust dynamical reference orbits.GRACE data are corrected from the known gravitational accelerations related to atmosphere and ocean mass redistributions, including tides and polar movements, using a priori global models.KBR rate residuals that represent mainly the continental hydrology have been converted into residual differences of potential (RDP), according to the conservation of the mechanical energy of the two GRACE vehicles versus time.Satellite tracks flying over Africa are selected, and each one is corrected from a least squares-adjusted linear trend.Following the two-step regional method explained in the previous section, time series of successive 10-day and 2° by 2° solutions of water mass change have been inverted over the whole African continent (30°W-60°E; 40°S-40°N) from RDP and, then, completed with the long wavelengths (>6700 km) of the GRGS GRACE solutions, or equivalently, the SH of degrees less than six, for the period 2003-2012.One complete year of regional solutions is displayed in Figure 3. Global GRACE Solutions from Official Centers CSR, GFZ and JPL Three processing centers, including the Center for Space Research (CSR), Austin, TX, USA, the GeoForschungsZentrum (GFZ), Potsdam, Germany, the Jet Propulsion Laboratory (JPL), Pasadena, CA, USA, and the Science Data Center (SDC) are in charge of the processing of the GRACE data and the production of Level-1 and Level-2 products.These products are distributed by the GFZ's Integrated System Data Center (ISDC) [55] and the JPL's Physical Oceanography Distributive Active Data Center (PODAAC) [56].Preprocessing of Level-1 GRACE data (i.e., positions and velocities measured by GPS, accelerometer data and KBR inter-satellite measurements) is routinely made by the SDC, as well as monthly global GRACE gravity solutions (Level 2).These latter solutions consist of time series of monthly averages of Stokes coefficients (i.e., dimensionless spherical harmonics coefficients of geopotential) developed up to a degree between 50 and 120 that are adjusted from along-track GRACE measurements.A dynamical approach, based on the Newtonian formulation of the satellite's equation of motion in an inertial reference frame, centered at the Earth's center, combined with dedicated modeling of the gravitational and non-conservative forces acting on the spacecraft, is used to compute the monthly GRACE solutions [57].During the estimation process, atmospheric and ocean barometric redistribution of mass variations are removed from the GRACE coefficients using European Centre for Medium-range Weather Forecasts (ECMWF) and National Centers for Environmental Prediction (NCEP) reanalysis for atmospheric mass variations and ocean tides, as well as global ocean circulation models.The GRACE coefficients are hence residuals that should represent mainly continental water storage, but also errors from the correction models and noise.The monthly GRACE solutions differ from one official provider from another due to the differences in the data processing, the choice of the correction models and the data selection for computing the monthly averages. Global GRACE Solutions Provided by GRGS These Level-2 European Improved Gravity model of Earth by New techniques solely from GRACE Satellite data (EIGEN) Release 04 10-day gravity models are derived from Level-1 GRACE measurements, including KBRR, from LAser GEOdynamics Satellites (LAGEOS) 1 and 2 Satellite Laser Ranging (SLR) data for the enhancement of lower harmonic degrees [47] and using an empirical stabilization approach without any post-processing smoothing or filtering.The 10-day Stokes coefficients are converted into terms of water mass coefficients from degree 2 up to degree 50-60 (i.e., spatial resolution of 400 km), expressed in EWH.Regular one-degree 10-day maps of surface water mass for the period 2002-2012 are derived from these latter SH water mass coefficients and made available for the last release (RL03) [58]. Independent Component Analysis of CSR, JPL and GFZ GRACE Solutions Since the global GRACE solutions are unfortunately dominated by striping, our idea is to combine monthly solutions from different centers of analysis to extract the continental hydrology component from noisy sources by redundancy.A post-processing method based on independent component analysis (ICA) was applied to the Level-2 GRACE solutions from official providers (i.e., University of Texas-Center for Space Research (UTCSR), JPL and GFZ).Pre-filtered with 400-km radius Gaussian filters before applying an ICA, the Level-2 GRACE solutions need to be somehow low-passed filtered to not have a Gaussian distribution.When they are not filtered enough, they are still dominated by striping, and their distribution keeps being Gaussian.If they are too low-pass filtered, they correspond to the long wavelengths of the continental hydrology, and they also exhibit Gaussian properties.A compromise of ~400 km to ensure no Gaussianity, and, thus, an efficient separation, has been proposed by [59] after several tests to extract most of the parts of the continental hydrology.Time series of ICA-based global maps of continental water mass changes computed over the period of March 2003-December 2010, are used for the comparison in this study [45].For a given month, the ICA 400-km filtered solutions only differ by a scaling factor, so that only the GFZ-derived ICA 400-km filtered ones are presented. Time Series of Altimetry-Derived Water Level of Lakes in East Africa Satellite altimetry was originally designed to provide accurate measurements of the dynamic topography of the ocean [60].Radar altimeters demonstrated strong capabilities to accurately estimate water levels over land and are now used for systematic monitoring of lakes [61,62], large rivers [63,64], wetlands and floodplains [65]. In this study, we used the time series of water levels derived from satellite altimetry measurements made available by the Hydroweb database at Laboratoire d'Etudes en Géophysique et Océanographie Spatiales (LEGOS)-Observatoire Midi-Pyrénées (OMP) [66] for four large lakes of East Africa (i.e., lakes Turkana, Victoria, Tanganyika and Malawi).All details about the processing of altimetry data and the computation of time series of water levels can be found in [61].As these lakes do not have significant changes in area, the time series of water levels were simply converted into time series of water volumes using the mean surface of the lakes, as in [18].The mean surfaces of the lakes are 8860, 68,800, 32,600 and 22,490 km 2 for Turkana, Victoria, Tanganyika and Malawi lakes, respectively [67]. TRMM 3B43 Monthly Rainfall In this study, we used the Tropical Rainfall Measuring Mission (TRMM) 3B43 product which is a combination of monthly rainfall at a spatial resolution of 0.25° from January 1998 to December 2012, and other data sources.This dataset is obtained by combining satellite information from the passive microwave imager (TMI) and precipitation radar (PR) onboard the Tropical Rainfall Measuring Mission (TRMM), a Japan-U.S. satellite launched in November 1997, the Visible and Infrared Scanner (VIRS) onboard the Special Sensor Microwave Imager (SSM/I) and rain gauge observations.The dataset results from the merging of the TRMM 3B42-adjusted merged infrared precipitation with the monthly accumulated Climate Assessment Monitoring System or Global Precipitation Climatology Center Rain Gauge analyses [68,69]. Residual Errors Estimated in the Arid Sahara Region Figure 4 presents a time series of TWS in a desert area in the south of Algeria.As no hydrological variations are expected in such a very dry region, the residual water mass signals can be interpreted as a good indicator of the error in estimating water mass variations from GRACE orbit data.These recovery errors do not exceed 15-19 mm EWH RMS when GRACE solutions are averaged over a surface of ~1 million square kilometers, consistently found by [70] from one year of global solutions.They also exhibit a seasonal cycle, in particular for the global and regional solutions (see Figure 4), which may result from leakage effects from surrounding areas polluting stronger water mass signals from the neighboring drainage basins, as well as errors from the a priori models used for correcting GRACE data and isolating the continental hydrology variations.For the global solutions, this latter effect is due to the spectral SH truncation of the global solutions at degree n = 60-90 (and of the underlying correcting models developed in SH for our regional solutions); in other words, the lack of spatial resolution to describe small objects, which creates unrealistic undulations propagating over the entire terrestrial sphere (i.e., leakage), as explained in Section 2. 1) considering the regional GRACE solutions (top) and global solutions from different pre-processing centers (bottom).Amplitudes over this region are typically less than 20 mm of equivalent water height (EWH) RMS.RL, release. Pre-Analysis of the 10-Day Maps of Water Mass over Africa Annual, semi-annual amplitudes and linear trends for each surface element have been least squares adjusted from the complete series of 10-day regional solutions over the period 2003-2012.Dominant seasonal amplitudes of ±200 mm EWH are well located in the Sahel latitudinal band (i.e., 5°N-15°N), as expected, and in the Congo Basin (Figure 5).The seasonal signature of the West African monsoon can also be seen [14].It is slightly greater for the global GRGS and 400-km ICA solutions.Besides, negative linear trends are found in the Tigris-Euphrates region of −25 mm EWH per year, consistent with the recent depletion of water mass shown by previous studies [71]; there are important gains of water mass over the Okavango swamps, reaching more than +30 mm EWH per year (Figure 6).In the Niger basin, the gain remains close to +15 mm EWH per year.There are also slight depletions in the south of Lake Chad and in the south of Mozambique.Unrealistic north-south striping in the trend estimates is more important for the global GRGS solutions than for 400-km ICA solutions, as the combination of different global solutions by ICA reinforces the hydrological signals versus the noise.Striping does not appear in the linear trend map related to the regional solutions at all (e.g., see the residuals over the oceans). Principal Component Analysis of the GRACE Datasets Principal component analysis (PCA), or discrete Karhunen-Loève transform, consists of decomposing the time series of water mass maps (i.e., maps of regional water mass over Africa) into main space and time "modes" that are projections onto orthogonal directions (or the principal axis) of variability [72] (e.g., see [73] for the computational aspects).Before PCA decomposition, the time series of the regional solutions has been corrected from the dominant seasonal oscillation related to each surface element (see Figure 5) using 13-month window averaging. First Mode of Variability: The Long-Term Behavior This mode corresponds to multi-year variations of water mass, since its temporal mode is characterized by a regular increase from 2006 to 2010 (Figure 7).All of the spatial modes range in ±100 mm EWH.This represents 50%-70% of the explained variance of the regional and smooth ICA solutions and 71% of the GRGS solutions.Thus, the corresponding spatial modes and the linear trend map presented in Figure 6 show similar patterns.The spatial mode associated with the monthly GRGS solutions contains unrealistic, short-scale undulations on the oceanic areas, as they are now derived from an empirical singular value decomposition (SVD) process of stabilization, and not a low-pass filtering, as for the GFZ, CSR and JPL solutions, which eliminates short wavelength more efficiently.The spatial components of the global solutions are affected by striping, especially the GRGS solutions, whereas no striping is visible in the regional water mass maps.There has been a dominant increase of mass in the Okavango swamp in the Zambezi River basin since 2006.The Okavango water system is an endorheic basin with no outlet to the sea, and it empties into the Kalahari Desert (~18,000 square kilometers), known as the Okavango Delta.Water storage also increases regularly over Volta Lake in Ghana, which is the largest artificial lake in the world.An important mass loss is observed in the Congo basin, centered on the extensive floodplains known as the "Cuvette Centrale" (see [74] for their spatial extent), especially in the regional solutions.The signature of water mass depletion in the NWSAS region is also clearly visible in this first PCA mode associated with the regional solutions.The temporal component of the first mode of the TRMM rainfall also exhibits multi-annual variations (Figure 7).The corresponding spatial mode of the precipitation shows patterns that coincide with the modes of the PCA of regional/global GRACE solutions, in particular the long-term deficit of water for the Niger Delta and further over the land of Cameroon, as well as the increase along the coast of Guinea, Sierra Leone, Liberia, Ivory Coast and Ghana. Second and Third Modes of Variability Second and third modes represent only 10%-15% and 6%-10% of the explained variances of the water mass signals, respectively (Figures 8 and 9).As the amplitudes of the third modes are smaller, it is more complicated to interpret its spatial and temporal patterns.The modes of global solutions still suffer from unrealistic striping that is visible over oceanic areas.In particular, the modes related to the GRGS solutions are affected by short wavelength noise.Except the JPL solutions, the temporal characteristics of the second PCA modes are two-year oscillations, as well as the maximum amplitudes of the spatial patterns on Central and Western Africa (i.e., northern part of the Congo River basin), including the Sahel, and along the Ivory Coast.As seasonal and semi-annual components have been removed before applying PCA, these patterns correspond to the bi-annual or quadrennial water mass variations related to the West African monsoon.Residual six-month and even multi-year oscillations appear for temporal modes of JPL.As for the first mode of variability, similarities between PCA modes of GRACE and TRMM rainfall exist.The second PCA mode of the TRMM data is characterized by important amplitudes of precipitation in the southern tropics, in particular over the Congo Basin.This strong signal also appears in the second modes of GRACE datasets, but it is less visible in the case of GRGS solutions.The temporal mode of the rainfall data is shifted; it occurs ~6 months sooner than the GRACE solutions (Figure 8).The third mode of rainfall is related to the African monsoon, as its main signature is located in the Sub-Saharan band, as for the spatial PCA modes of GRACE solutions (Figure 9). Time Series of TWS Averaged over African River Drainage Basins The masks used in this study come from the five-minute, 1/2° and 1° datasets of the continental watersheds and river networks for use in regional and global hydrologic and climate system modeling studies [75].The 1° dataset is used for the drainage regions and the river watersheds, while the 0.5° dataset is used to define the drainage regions around the lakes.The coordinates of the drainage basin limits for each lake are obtained using the lake boundaries and the drainage network at 1/2°.Time series of water mass for the main drainage basins of Africa over 2004-2012 (see Figure 1) have been computed as masked averages versus time and then converted into mm of equivalent sea level (ESL) profiles.In this latter operation, the water volume variations (i.e., equivalent water heights times the basin surface) are divided by the surface of the oceans (~360 million square kilometers) and multiplied by −1.The per-basin time series of ESL are presented in Figure 10.Positive multi-year contributions to the sea level concern the Congo, Nile and Orange river basins, and they represent +0.044 mm/y in total, whereas the total negative contribution from the other basins is more important in magnitude (i.e., −0.123 mm/y), where the multi-year sea level contribution of the Zambezi Basin is the largest in magnitude (i.e., −0.1 mm/y).This region centered on the Okavango swamps and floodplains has been storing more and more water during the last decade. As displayed in Figure 11, the sea level contributions averaged over large drainage areas to the Atlantic and Indian oceans and the Mediterranean Sea exhibit clear seasonal oscillations of amplitudes ±2, ±1 and ±0.5 mm of ESL, respectively.The seasonal contribution of land waters to the Indian Ocean (i.e., runoff along the southeastern coast of Africa) is in opposite phase with the two others.The sum of the contribution of these three large areas is negative (i.e., −0.088 mm per year); thus, the water mass balance of Africa for the period 2004-2012 indicates a gain of mass on the continent.In particular, the strongest multi-year trend magnitude is the one related to the Indian Ocean (i.e., −0.094 mm/y), as it shows a clear acceleration of the gain of water mass on land during recent years, passing from −0.034 mm/y before 2008 to −0.123 mm/y after 2008.A comparison with Figure 5 indicates that the latter contribution is driven by the long-term variations of the hydrology in the Zambezi River Basin.For the Atlantic and Mediterranean contributions, the positive ESL trends have been decreasing since 2008 from +0.138 mm/y and +0.030 mm/y down to +0.071 mm/y and +0.027 mm/y, respectively.Once the global isostatic adjustment (GIA) of the solid Earth from mainly post-glacial rebound and representing −0.3 mm/y is corrected [76,77], the net contribution of African rivers to sea level for 2004-2012 remains small, since it represents only ~3% of the global increase of the sea level of 3.3 ± 0.4 mm/y measured by altimetry.This latter comparison suggests that the multi-year contribution of Africa hydrology remains in the total sea level error bar. Detection of Recent Groundwater Withdraw of NWSAS Aquifer The NWSAS presented in Figure 1 (Area 9) is characterized by a transboundary "fossil" aquifer (i.e., with no meteoric water recharge) shared by three countries (i.e., 60% Algerian, 30% Libyan and 10% Tunisian surfaces) and in a critical situation of depletion by intense water pumping.While the groundwater imbalance is −2.2 km 3 per year as estimated from historical records of piezometry before the year 2000 (see the report of [78]), our regional GRACE solutions averaged over the NWSAS indicate that the depletion of groundwater for the most recent decade is twice that, suggesting a worrying acceleration of the withdrawing of drinking water (Figure 12).In particular, a sudden loss of −25 km 3 lasting a few months appears in the year 2007 between the periods January 2004-December 2007, and January 2008-January 2012.A comparison with the latter trends estimated considering the total GRACE period suggests that the rapid drop of groundwater in 2007-2008 remains exceptional (according to the dashed line).These are consistent with the recent estimates of groundwater loss from −2.2 km 3 /y in 2000 [78] to −2.75 km 3 /y in 2010 [79].Time variations of TWS from GRACE appear reliable to directly and efficiently estimate the region-wide groundwater changes in a large aquifer system in arid areas and to provide useful information on groundwater recharge. Subsurface Water Storage Changes by Combining Regional Solutions and Radar Altimeter Data Time series of the subsurface waters (i.e., soil plus ground waters) were estimated by removing altimetry-based water levels of the main African lake, or reservoir waters, from the TWS measured by GRACE.As altimetry-based water levels from [78] have a temporal resolution of one month, the ten-day regional solutions were consequently averaged on monthly time periods before computing the mere difference.Anomalies of these residual subsurface waters, including groundwater, were estimated at a monthly time-scale as the difference between TWS and surface storage over the common period of availability of two datasets (i.e., mid-2003-2012).These anomalies are presented in Figure 13 for the largest East African lakes: Turkana, Victoria, Tanganyika and Malawi.They temporally and/or spatially extend the previous studies from [17,18,80], but using regional solutions instead of classical global ones.Figure 13.Time-series of the anomaly of the water storage of TWS (black), of the surface reservoir (blue) and of the subsurface reservoir, including groundwater (red), in the left column, and their inter-annual variations, in the right column, for Lake Turkana (a), Lake Victoria (b), Lake Tanganyika (c) and Lake Malawi (d). Time variations of TWS and subsurface waters are well correlated for the three largest lakes, as the correlation coefficient R equals 0.75, 0.76 and 0.76 for Lake Victoria, Lake Tanganyika and Lake Malawi, but not for the smaller Lake Turkana, with R equal to 0.28.If the surface water storage represents only a small part of TWS variations, as for Lake Turkana over 2003-2013, its inter-annual variations are larger than the inter-annual TWS signals, similarly to what was found by [18].The inter-annual signals of subsurface water presents three successive phases: from mid-2005 to mid-2008: they decreased at an average rate of −4 km 3 /y from mid-2008 to mid-2009 and remained stable; and since then, they present a bi-annual cycle of ±5 km 3 of amplitude (Figure 13a).For Lake Victoria, the variations of the anomaly of water storage are greater for the surface reservoir than for the TWS.A larger decrease is observed for the anomaly of surface storage (−60 km 3 /y from 2004 to the beginning of 2006) than for TWS (−70 km 3 /y from 2004 to mid-2006) and the opposite up to the end of 2007, with increases of 40 and 20 km 3 /y for the surface water storage and TWS, respectively.Consequently, subsurface water is varying as the difference over the same time periods (Figure 13b).The results obtained with the regional solutions are very similar to those obtained by [18,81].Nevertheless, [18] found larger variations of TWS than these from surface water, most likely because these authors included in the drainage areas of Lake Victoria the Kivu, Edouard, Albert and Kyoga lakes and their drainage areas, without adding their volume variations to the surface storage. The TWS variations are dominated by the subsurface water component for Lake Tanganyika.A large variation of TWS, strongly impacting the subsurface water reservoir, is observed from 2005 to 2008, with a minimum reaching −40 km 3 in the beginning of 2006 and a maximum of 30 km 3 in the beginning of 2007.Smoother variations are present in the surface reservoir (Figure 13c). As for Lake Victoria, TWS variations of Lake Malawi are dominated by the surface component, but the amplitudes of the surface reservoir are slightly greater than these from TWS at an inter-annual time scale.The time variations of the surface reservoir exhibit a steep decrease from 2004 to the beginning of 2006, followed by a significant increase up to mid-2009, whereas they are lower for TWS (Figure 13d). These results demonstrate the strong capacities of multi-satellite observations to monitor quantitatively the changes in the storage of the surface and sub-surface reservoirs associated with climate variability and human activities at a regional scale.These remotely-sensed datasets are likely to have huge importance in regions where in situ data are sparse, while they have huge importance in terms of water supply for dense human populations.In the case of Lake Victoria, it directly supports 30 million people in terms of a freshwater supply [8] and indirectly another 340 million people along the Nile Basin [82], being the source of the White Nile. Conclusions In this paper, we present 10-day regional solutions of water mass change over Africa for the period 2003-2012, revealing the dominant seasonal and African monsoon signals (±250 mm EWH).Principal component analysis (PCA) of the GRACE datasets provided the main modes of variability of the surface water mass.Temporal and spatial patterns are consistent for the regional and global solutions.However, the regional solutions offer a better geographical localization of hydrological structures, while global solutions remain affected by aliasing errors (i.e., north-south striping).This is probably due to the benefit of details brought by GRACE short tracks into the regional solution, instead of considering band-limited spherical harmonics defined on the entire Earth. Monitoring water supply by using these regional solutions enables us to confirm the long-term drought of the NWSAS aquifer and even to reveal the sudden water loss occurring in early 2008.In terms of large-scale mass balance, the small contribution of the African hydrology changes to global sea level rise for the period 2004-2012 remains negative, especially due to the gain of water mass in the swamp regions of the Zambezi River basin that represents −0.123 mm/y of ESL as a continuous deficit of the level for the Indian Ocean.Principal component analysis of the complete time series of regional GRACE solutions has been made to identify the separate contributions of the different African regions.In particular, the first mode of PCA, representing 50%-70% of the explained variance, reveals the long-term drought in East Africa and in the region of Lake Chad and, alternatively, the increase of water mass in the Okavango and Niger regions.The second PCA mode of 10% corresponds to an oscillation of two years over the Sahel and the central African regions.This latter mode is clearly related to the residuals of the dominant African monsoon.GRACE-derived TWS variations demonstrated strong capabilities for land water management in terms of monitoring the sub-surface changes alone over arid and semi-arid areas, such the NWAS basins, or in combination with altimetry-based water levels for lake drainage areas. Figure 2 . Figure 2. Schematic view of the processing for estimating global and regional solutions from a given 10-day or monthly period of Gravity Recovery and Climate Experiment (GRACE) observations.GINS, Géodésie par Intégrations Numériques Simultanées; GRGS, Groupe de Recherche en Géodésie Spatiale; SVD, singular value decomposition; KBRR, K-band range rate. Figure 3 . Figure 3. Example of one-year series of regional maps of water mass changes presented at 10-day intervals from June 2007 to June 2008 (see also the next section), for the two semesters. Figure 4 . Figure 4. Total water storage (TWS) time series for the dry region of South Algeria (8 in Figure1) considering the regional GRACE solutions (top) and global solutions from different pre-processing centers (bottom).Amplitudes over this region are typically less than 20 mm of equivalent water height (EWH) RMS.RL, release. Figure 5 . Figure 5. Maps of the seasonal amplitudes of the water mass changes adjusted by least squares adjustment of a pure annual sinusoid at each grid cell of the 10-day regional solutions over Africa (2003-2012): (a) Regional solutions; (b) GRGS RL03 global solutions; (c) ICA 400-km filtered solutions.Units are mm of EWH.Note the strong signals due to the African monsoon in the Sahel latitudinal band. Figure 6 . Figure 6.Maps of the linear trends of the water mass changes by fitting a linear trend for each grid cell from all of the 10-day regional solutions over Africa for 2003-2012: (a) Regional solutions; (b) GRGS RL03 global solutions; (c) ICA 400-km filtered solutions; (d) TRMM Precipitation 2003-2012.Units are mm of EWH per year. Figure 7 . Figure 7.The first spatial and temporal modes of principal component analysis (PCA) of the regional, global GRGS and 400-km ICA GRACE solutions (top).The corresponding linear trends of the precipitation from TRMM for comparison (bottom). Figure 8 . Figure 8. Second spatial (top) and temporal (bottom) modes of the PCA of the regional, global GRGS and 400-km ICA GRACE solutions.Units are mm EWH. Figure 9 . Figure 9. Third spatial (top) and temporal (bottom) modes of the PCA of the regional, global GRGS and 400-km ICA GRACE solutions.Units are mm EWH. Figure 10 . Figure 10.Time series of equivalent sea level (ESL) for the chosen main African river basins (solid line) and the corresponding 13-month low pass filtered profiles (dashed line). Figure 11 . Figure 11.Time series of the main African river basins contributions to the Atlantic and Indian oceans, as well as to the Mediterranean Sea, expressed in mm of ESL.	2016-03-22T00:56:01.885Z	2014-08-07T00:00:00.000	{ "year": 2014, "sha1": "0f5c7b8b66e88812bdbdf81d48966bf55366a083", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2072-4292/6/8/7379/pdf?version=1407399997", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "0f5c7b8b66e88812bdbdf81d48966bf55366a083", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Geology", "Computer Science" ] }
226242585	pes2o/s2orc	v3-fos-license	TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes. Introduction Target of rapamycin (TOR) is an atypical serine/threonine protein kinase that belongs to the family of phosphatidylinositol-3 kinase related kinases. TOR kinases are highly conserved throughout evolution and control several aspects of metabolism, cellular growth and survival. Consequently, deregulation of TOR activity is associated with pathological outcomes, including cancer, diabetes and neurological defects [1][2][3]. TOR kinases are found in two highly conserved complexes, known as TORC1 and TORC2, which were first identified in the budding yeast Saccharomyces cerevisiae [4,5]. The mammalian TORC1, mTORC1, which contains the mTOR kinase together with the Raptor protein, is well-known for its positive role in promoting anabolic processes, while also inhibiting catabolic and starvation responses [3,6]. These TORC1 cellular functions are conserved in model organisms, including the budding yeast, Saccharomyces cerevisiae and the fission yeast, S. pombe [5,7]. The mammalian TORC2, mTORC2, is characterized by TOR together with the Rictor and mSin1 proteins and acts downstream of PI3K/insulin signaling to regulate cellular growth and metabolism [1,8]. The cellular roles of the TORC2 complexes and the extent of their evolution conservation are less well-understood compared with those of TORC1 [7]. The subcellular localization of TORC2 is also a subject for ongoing research. Like in higher eukaryotes, S. pombe TORC2 has been localized to the plasma membrane, but it is also found in the nucleus (reviewed in [9]). TOR complexes mediate many of their functions via phosphorylation and activation of downstream AGC kinases, a group that was named after three representative families, the cAMP-dependent protein kinase A (PKA), the cGMP-dependent protein kinase (PKG) and the protein kinase C (PKC) families [10]. mTORC1 directly phosphorylates and activates the p70 ribosomal S6 kinase-1 (S6K1), which subsequently regulates growth, metabolism and translation [11][12][13]. The S. cerevisae orthologue of S6K1 is Sch9 [14], while three AGC kinases, Psk1, Sck1 and Sck2, act downstream of the S. pombe TORC1 [15]. mTORC2 mediates its effects via phosphorylation and activation of three different members of the AGC kinase family: the three isoforms of AKT (also known as PKB) [16][17][18], SGK1 [19] and PKCα [20,21]. In S. cerevisiae, there are three AGC kinases that act downstream of TORC2, Ypk1, Ypk2 and Pkc1, which are involved in a wide variety aspects of membrane homeostasis, regulation of the actin cytoskeleton, cell cycle progression and genome integrity ( [22][23][24][25][26][27][28][29][30] and reviewed in [31,32]). In S. pombe, TORC2 is responsible for the phosphorylation and activation of the AGC kinase Gad8 [33], thereby regulating several aspects of cell cycle progression, promoting survival under a large variety of stress conditions and enabling cells to enter the sexual development pathway in response to starvation [33][34][35][36][37][38]. Disruption of gad8 + results in a phenotype very similar to disruption of TORC2, including delayed entrance into mitosis, defects in growth under conditions of high temperature, osmotic, oxidative stress [33] or genotoxic stresses [39]. The activity of S. pombe TORC2 is acutely inhibited by glucose depletion or exposure to osmotic stress [40], but this inhibition is transient [41,42]. TORC2-Gad8 activity is also regulated under nitrogen starvation and in response to quiescence [43,44]. The majority of members of the AGC kinase that lie downstream of TOR complexes share a common mechanism of activation that involves phosphorylation at three major conserved sites (reviewed in [10,45,46]). One phosphorylation site is located at the activation loop (also known as the A-loop), which is also present in the kinase domain of other conventional protein kinases. Two additional sites are characteristic for AGC kinases and are located C-terminally to the kinase domain at the so called turn motif (TM) and hydrophobic motif (HM). Phosphorylation events at the C-terminal extension are mediated by TOR complexes, while the activation loop site is phosphorylated by PDK1 in higher eukaryotes, or by PDK1-like kinases: Pkh1 and Pkh2 in S. cerevisiae [47] or Ksg1 in S. pombe [33,48]. Phosphorylation at the HM in several AGC kinases, including SGK1 and S6K1, but not AKT, leads to docking into a regulatory hydrophobic pocket in PDK1, called the PIF (PDK-interacting fragment) motif. This intermolecular interaction between PDK1 and its downstream target kinases facilitates substrate recruitment and induces the kinase activity of PDK1 towards the activation loop of downstream kinases [49][50][51]. Several AGC kinases, including S6K1, AKT and SGK1 employ the PIF pocket for intramolecular interaction with the phosphorylated hydrophobic motif, thus promoting their kinase activation [52,53]. The role of the PIF pocket in allosteric regulation of the ATP-binding and kinase activity of PDK1 could provide valuable sites for selective inhibitors [54,55]. Similar to other AGC-kinases, full activation of Gad8 requires phosphorylation at all three sites. The catalytic subunit of S. pombe TORC2, Tor1, is responsible for phosphorylation of S546 in the HM and S527 in the TM [33]. Previous studies suggested that TORC2-dependent phosphorylation at S546 facilitates the access of Ksg1 (S. pombe PDK1) to phosphorylate Thr387 in the activation loop [33]. In this study we demonstrate that under stress conditions, point mutations in the PIF pocket of Gad8 render its kinase activity independent of TORC2, possibly by modifying the protein conformation of the activation loop. Our studies suggest structural roles for specific amino acids in keeping Gad8 activity in check, while also identifying a separation of function of Gad8 activity under distinct sets of stress conditions. Isolation of Tor1-independent mutant alleles of gad8 + Tor1, the catalytic subunit of S. pombe TORC2, is responsible for the phosphorylation (and therefore activation) of the Gad8 kinase [33]. Consistent with previous studies that showed that strong overexpression of gad8 + suppressed phenotypes associated with Δtor1 [56], strong overexpression of gad8 + under the regulation of the nmt1 + promoter from the multicopy plasmid pREP1 [57] rescued the high temperature (37˚C) or osmotic stress (KCl) sensitivities of Δtor1 cells (pREP1-Gad8, Fig 1A). Thus, Gad8 that lacks Tor1-dependent phosphorylation retains a certain level of activity when strongly overexpressed. In contrast, low overexpression of gad8 + from the pREP81 plasmid, which contains a weak version of the nmt1 + promoter [57] is unable to suppress the sensitivity of Δtor1 cells at high temperature or under osmotic stress conditions (pREP81-Gad8, Fig 1A). Therefore, we reasoned that the pREP81-gad8 + construct, Isolation of tor1-independent alleles of gad8. A, Serial dilutions of wild-type (WT) and Δtor1 cells transformed with the indicated plasmids. pREP1, in grey, allows strong overexpression, while pREP81, in black, allows weak overexpression of the cloned gene. Cells were spotted onto minimal medium with, or without 0.8M KCl and were incubated for 4 days at 30˚C (no stress) or at high temperature (37˚C). B, Schematic representation of the screen carried out to isolate tor1-independent gad8 alleles. The gad8 + open reading frame was amplified under PCR mutagenesis conditions using primers that contained sequences homologous to pREP81 sequences (in red) and sequences homologous to gad8 + (in blue). The mutagenized gad8 fragments were co-transformed together with linearized pREP81 plasmids into the tor1-L2045D strain and transformants were selected on minimal plates at 37˚C. C, Serial dilutions of wild-type (WT) and Δtor1 cells as described in A. D, Schematic representation of Gad8. Indicated in red are the tor1-independent gad8 mutations, T260C and K263C, and in black the Ksg1-dependent phosphorylation site, T387, and the Tor1-dependent phosphorylation sites, S527 and S546. E, Alignment of the amino acid sequences surrounding the T260C and K263C point mutations in several members of the AGC kinases. The tor1-independent gad8 mutations are in red. The K263, but not T260, residue is conserved in all the examined AGC kinases. in combination with random mutagenesis, could be useful for the isolation of TORC2 (Tor1)independent gad8 mutant alleles. We obtained a library of mutant alleles of gad8 by random mutagenesis using error-prone PCR with pREP81-gad8 + as template. We then co-transformated the resulting PCR fragments together with linearized pREP81 plasmids into yeast cells (see schematic presentation in Fig 1B). Since Δtor1 cells are poorly transformed, we used mutant cells harboring a point mutation in tor1 + , tor1-L2045D, which renders the mutant cells sensitive to high temperature or to osmotic stress [58]. We screened 23,000 colonies and isolated 3 plasmids that suppressed the temperature-sensitive phenotype of tor1-L2045D at 37˚C. We re-transformed these three plasmids, pREP81-gad8-m1/m2/m3, into naive tor1-L2045D or Δtor1 cells and found that they fully suppressed their high temperature and KCl sensitive phenotypes (S1A Fig). DNA sequencing indicated 5-9 mutations within the open reading frame (ORF) of gad8 + in each of the three isolated plasmids (S1B Fig). By a combination of sub-cloning and functional analysis, we identified two mutations, lysine 263 to cysteine (K263C) and threonine 260 to cysteine (T260C), which alone suppressed the temperature or KCl sensitive phenotypes of Δtor1 cells ( Fig 1C). The K263C mutation was found in two independently isolated plasmids (pRE-P81-gad8-m1 and pREP81-gad8-m2), while the T260C mutation occurred only once (pRE-P81-gad8-m3) (S1B Fig). The K263C and T260C mutations are located at the N-terminal edge of the kinase domain of Gad8 ( Fig 1D). The K263 residue is highly conserved among members of the family of AGC kinases, while T260 is not ( Fig 1E). The gad8-K263C or gad8-T260C mutations suppress a range of defects associated with the loss of TORC2 We next examined the ability of gad8-T260C or gad8-K263C to suppress additional defects associated with loss of TORC2. Cells disrupted for tor1 + are unable to grow under low-glucose conditions [41] and are severely defective in undergoing sexual development in response to starvation cues [34,35], a process in which two opposite mating-type cells undergo conjugation to form the diploid zygote that subsequently undergoes meiosis and sporulation. We found that the gad8-T260C or gad8-K263C mutant alleles suppressed low-glucose stress sensitivity in Δtor1 cells (Fig 2A) and partially suppressed the inability of Δtor1 cells to undergo sexual development pathway (Fig 2B). Expression of gad8-T260C or gad8-K263C had no detectable deleterious effects in wild-type cells (Fig 2A). Disruption of any of the essential subunits of TORC2, ste20 + (Rictor in higher eukaryotes) or sin1 + (mSin1), also results in pleiotropic defects under stress conditions (S2A and S2B Fig). Similar to the suppression observed in Δtor1 cells, the gad8-T260C or gad8-K263C mutation suppressed the sensitivity of Δsin1 or Δste20 cells to high temperature, osmotic stress or low-glucose (S2A and S2B Fig) and partially suppressed the sterility of Δste20 cells (S2C Fig). Thus, gad8-T260C or gad8-K263C suppresses phenotypes associated with loss of TORC2 and is not specific for loss of Tor1. We chose to further focus on the K263C mutation, since this mutation occurs in a highly conserved residue in the family of AGC kinases. We integrated the gad8-K263C allele into its natural chromosomal locus, under the native promoter of gad8 + . The chromosomally integrated mutation suppressed the high temperature, osmotic stress or low-glucose sensitivities in Δtor1 cells (Fig 2C and 2D) and partially suppressed the mating deficiency in Δtor1 cells ( Fig 2E). The kinase activity of Gad8-K263C is independent of TORC2 Tor1 phosphorylates Gad8 at S546 in the HM and at S527 in the TM, whereas Ksg1 phosphorylates Gad8 at T387 in the activation loop [33]. Previous studies have shown that the in vitro So far the only kinase that is known to phosphorylate Gad8 at S546 is Tor1. To our surprise, Gad8-K263C was phosphorylated at S546 in wild type cells, as well as in Δtor1 cells (S546-P, Fig 3A). This finding suggests that the Gad8-K263C mutant is phosphorylated by a kinase that normally does not recognize Gad8 as a substrate. Gad8-K263C was also phosphorylated at S546 under conditions that compromise Tor1 activity, such as osmotic or low glucose stress (S3A Fig), further supporting Tor1-independent phosphorylation of Gad8-K263C by an as yet unknown kinase. The only conditions under which we did not detect phosphorylation of Gad8-K263C were in Δtor1 cells in the presence of hydroxyurea or camptothecin (S3B Fig), a finding that may suggest that the activity of the kinase responsible for Gad8-K263C phosphorylation is inhibited under genotoxic stress conditions. In order to examine whether Gad8-K263C requires the phosphorylation at TORC2-dependent sites for its activity in vivo, we combined TORC2-dependent non-phosphorylatable mutations in gad8, S546A and S527A, together with the K263C mutation. As previously described [33], mutating both Tor1-dependent phosphorylation sites, S546 and S527, to alanine, abolished the ability of cells to grow at high temperature or in the presence of osmotic stress ( Fig 3B). In contrast, cells carrying the non-phosphorylatable mutations in gad8 together with the K263C mutation, gad8-K263C/S527A/S546A, were able to grow at high temperature or in the presence of osmotic stress ( Fig 3B). Thus, the K263C mutation reversed the temperature and osmotic stress sensitivities of gad8-S527A/S546A. We conclude that the phosphorylation at the TORC2-dependent sites is not required for the activity of the Gad8-K263C kinase. Previous findings suggest a model in which Tor1-dependent phosphorylation of Gad8 on S546 facilitates the interaction with Ksg1 and enhances subsequent phosphorylation by Ksg1 at T387 [33]. This suggestion is primarily based on the finding that mutating S546 into aspartic acid mimicked the effect of phosphorylating Gad8 in vivo and induced a stronger activity of Ksg1 towards phosphorylation of Gad8 in vitro [33]. Using specific antibodies against the phosphorylated form of Gad8-T387, we found that Gad8-K263C is phosphorylated at T387 in Δtor1 cells under normal or low-glucose growth conditions ( Fig 3C). The wild type Gad8 is not phosphorylated at T387 in the absence of Tor1 and is only weakly phosphorylated under low-glucose conditions ( Fig 3C). This finding suggests that the K263C mutation allows Ksg1 to interact with and to phosphorylate Gad8 in the absence of Tor1. In order to check whether phosphorylation of T387 by Ksg1 is required for Gad8-K263C activity in vivo, we introduced the T387A mutation into the chromosomal gad8 locus, either alone or in combination with the K263C mutation. As previously described, gad8-T387A cells failed to grow under stress conditions [33]. Strains carrying both the T387A and K263C mutations, gad8-T387A/K263C, . Cells were grown to mid-log phase and left untreated in YES medium or shifted for 1 h to EMM containing no carbon source (EMM-Glu) or to YES containing 1M KCl or 1 M NaCl. Gad8-HA and Gad8-K263C-HA were immunoprecipitated and assayed in vitro for their kinase activity using the Fkh2-GST peptide as a substrate. Phosphorylation of Fkh2-GST was detected with anti-PAS antibodies recognizing the phosphorylated form of the consensus sequence of AGC kinases (Fkh2-P). Phosphorylation of Gad8 at S456 was detected with anti-Gad8-S546-P phsophospecific antibodies (S546-P). B, Serial dilutions of the indicated strains spotted onto YES (no stress) or YES medium containing KCl and incubated at 30 0 C or 37 0 for 3 days. All mutations are chromosomal. C, Western blot analysis of protein extracts isolated from wild-type (tor1 + ) or Δtor1 cells grown in YES medium to mid-log phase or shifted for 1 hour into EMM containing no carbon source (-Glu). The status of phosphorylation of Gad8 at T387, the target site for Ksg1 (PDK1) was detected with anti-Gad8-T387-P phosphospecific antibodies. D, Serial dilutions of the indicated strains as described in B. https://doi.org/10.1371/journal.pgen.1009196.g003 PLOS GENETICS TORC2-independent Gad8 alleles also failed to grow under stress conditions (Fig 3D), indicating that Gad8-K263C still requires activation by Ksg1. The gad8-K263C mutation is located at the PIF pocket and induces higher flexibility at Gad8-T387, the PDK1-dependent phosphorylation site In the absence of a three-dimensional X-ray structure of S. pombe Gad8, a ligand-supported homology model of Gad8 in its un-phosphorylated state was generated using its homologue SGK1 kinase (PDB code: 2R5T, 47% sequence identity). The αC-helix and the C-terminal extension regions were modeled based on AKT1 and PKCα kinases (PDB codes: 6NPZ and 4RA4, respectively). The final model is presented in Fig 4A. The model was shown to have high quality stereochemical profile (94% of the amino acids in the core regions of the Ramachandran plot). The general 3D organization of Gad8 comprises an N-lobe domain of beta sheets, and a C-lobe domain mainly consisting of alpha-helices and loops. The two lobes are connected to each other by a hinge region that defines the activation loop. At the interface of the two domains there is a deep hydrophobic cleft that binds ATP and a Mg 2+ ion. The point mutation K263C is located at the small N-lobe between the αB-helix and the β4 beta-sheet, that together with the αC helix delimit the PIF-pocket. As described above, Gad8-K263C bypasses the need for phosphorylation by Tor1 at the TM and HM motifs, S527 and S546, respectively. However, there is still a need for a phosphorylation by Ksg1 at the activation loop (Thr387) to get a full activation of Gad8. Therefore, we speculated that the mutation K263C may alter the protein structure in a manner similar to that affected by Tor1 phosphorylation. In order to get insights into the effect of the mutation on the protein structure, three constructs, namely un-phosphorylated Gad8, Gad8-K263C, and Gad8 phosphorylated at both the HM and TM (Gad8-pS527/pS546) were subjected to molecular dynamics (MD) simulations. Each construct was simulated three times for 50 ns. Each replica was started from a different random seed. The stability of the resulted trajectories was tested based on the root mean square deviation (RMSD) of the backbone atoms with respect to the equilibrated structures. Next, the root mean square fluctuation (RMSF) was calculated in order to determine the degree of flexibility of each residue in each construct. RMSF plots of protein backbone atoms of un-phosphorylated Gad8, Gad8-K263C and Gad8-pS527/pS546 during the simulations are shown in Fig 4B. It is evident from this analysis that the activation loop in Gad8-K263C and Gad8-pS527/pS546 is more flexible than in un-phosphorylated Gad8. Furthermore, the PIFpocket region shows higher flexibility in Gad8-K263C and Gad8-pS527/pS546 compared to un-phosphorylated Gad8. We have identified an H-bond interaction between K263 and Q298 in WT-Gad8, which occurs in~62% of the simulation time. Interestingly, this H-bond interaction appears in Gad8-pS527/pS546 only in~22% of the simulation time, and does not appear at all in Gad8-K263C as a result of the point mutation. This may explain the higher flexibility of the PIF-pocket region in Gad8-K263C and Gad8-pS527/pS546. The Q298 residue is located at the β4 sheet in the PIF pocket and is also highly conserved in evolution (S4 Fig). Mutating the Q298 residue into leucine, gad8-Q298L, partially suppressed the mating deficiency of Δtor1 cells when expressed from the pREP81 plasmid (Fig 5A), suggesting that the MD analysis is useful in predicting additional gad8 alleles that bypass the requirement for TORC2. The gad8-Q298L did not suppress osmotic, high temperature or low glucose sensitivities in Δtor1 cells ( Fig 5B). Moreover, over-expression of gad8-Q298L in wild type cells showed sensitivity to low-glucose conditions (Fig 5B). It is possible that mutation at Q298 interfere with substrate (s) recognition motif or docking interaction with substrate(s) that are specifically required for viability under low glucose conditions. Possible reasons for the differential suppression activities in gad8 mutant alleles are further discussed below. The protein is shown as a ribbon diagram (grey), the ATP is shown as sticks, and the Mg ion is shown as a green sphere (ATP is colored according to atom types). The activation loop (A-loop) is shown in red. The highly conserved structures in AGC kinases, the DFG-motif and the Gly-rich loop are colored in blue and magenta, respectively. The Cterminal extension in cyan. The residues K263, T387, S527, and S546 are shown in balls and sticks. B, RMSF plot of the backbone atoms for non-phosphorylated Gad8 (blue), Gad8-pS527/pS546 (yellow, p denotes phosphorylation), and non-phosphorylated K263C-Gad8 (orange). gad8-K263C or gad8-T260C, but not gad8-Q298L cells, are sensitive to DNA damage or DNA replication stress We have previously shown that cells lacking TORC2 or gad8 + are highly sensitive to DNA replication stress or DNA-damaging conditions [39,56]. We employed the kinase-dead gad8 allele, gad8-K259R [33] to examine whether the kinase activity of Gad8 is required under these conditions. Cells carrying gad8-K259R are sensitive to genotoxic stresses, indicating that the kinase activity of Gad8 is required under these conditions (S5A Fig). Additionally, the phosphorylation sites of Gad8 are required for genotoxic stress, since gad8-S527A/S546A mutant alleles are also sensitive to DNA damage and DNA replication stress (S5B Fig). We therefore examined the ability of pREP81-gad8-K263C or pREP81-gad8-T260C to suppress the sensitivity of TORC2 mutant cells to genotoxins. We first examined the ability of gad8-K263C or gad8-T260C to suppress the sensitivity of Δtor1, Δste20 or Δsin1 to hydroxyurea (HU) or camptothecin (CPT). HU generates replication stress by depleting the dNTP pools, while CPT stabilizes covalent DNA topoisomerase-I complexes, leading to collision of DNA replication forks with the drug-enzyme-DNA complex and inducing double strand breaks [61]. gad8-K263C or gad8-T260C expressed from the pREP81 plasmid failed to suppress the sensitivity of Δtor1 (Fig 6A) or the sensitivity of Δste20 or Δsin1 to HU or CPT (S6A Fig). Moreover, gad8-K263C or gad8-T260C expressed from the pREP81 plasmid in wild-type cells conferred sensitivity to HU or CPT (Fig 6A). The chromosomally integrated gad8-K263C mutation, in an otherwise wild-type background, also conferred a strong sensitivity to HU or to CPT ( Fig 6B). Compared with Δtor1 or Δtor1 gad8-K263C cells, the chromosomally integrated gad8-K263C mutation exhibited a slightly less severe genotoxic stress sensitivity (Fig 6B), which may suggest that gad8-K263C is not fully impaired under genotoxic stress conditions or that Tor1 also acts independently of Gad8 in promoting genotoxic resistance. Notably, heterozygous gad8 + /gad8-K263C diploid cells are as sensitive to HU or CPT as gad8-K263C/ gad8-K263C cells, suggesting that the K263C mutation acts in a dominant-negative manner with respect to genotoxic stress sensitivity (Fig 6C). Mutating the K263 into alanine or arginine (negativelly charged residues, similar to lysine), K263A or K263R, resulted in suppression of the high temperature or high osmolarity sensitivity of Δtor1 cells, but conferred genotoxic stress sensitivity in wild-type cells (S6B Fig). Thus, K263 itself appears to be crucial for genotoxic stress responses. Unlike the K263C or T260C mutations, the Q298L mutation did not impair the ability of Gad8 to survive genotoxic stress conditions and showed a partial ability to suppress the sensitivity of Δtor1 cells to HU or CPT (Fig 6D). We also found that the gad8-S527D/S546D allele, containing aspartic acid residues at the phosphorylation sites of TORC2 [33] suppressed the sensitivity to high temperature and osmotic stress, as well as the sensitivity to CPT or HU (S6C Fig). Thus, constitutive activation of Gad8 in itself does not confer sensitivity to either of the stress conditions examined. We conclude that mutations in the PIF pocket of Gad8 bypasss the requirement for TORC2 under specific sets of stress conditions, but depending on the specific alteration, these muations may also impair the protein function under other stress conditoins. Under genotoxic stress conditions, the gad8-K263C mutation confers similar phenotypes to loss of function of gad8 + Dominant mutations often exhibit a phenotype that is different from that of loss of function of a given gene. Therefore, the finding that gad8-K263C cells shows similar defects to Δtor1 or Δgad8 cells with respect to genotoxic sensitivity is somewhat surprising. We further explored possible underlying mechanisms for the sensitivity of gad8-K263C mutant cells to genotoxins. Under DNA replication stress, S. pombe cells induce a group of~20 genes that are under the transcriptional regulation of the MluI cell cycle box-binding factor (MBF) complex [62]. We have previously shown that in mutant cells lacking TORC2 or Gad8, the induction of the MBF target genes in response to replication stress is significantly reduced compared with wild-type cells [63]. Examination of the level of induction of three representative MBF target genes, cdc22 + , cdc18 + , cdt2 + , revealed that these are also poorly induced in response to replication stress (HU) in gad8-K263C, or Δtor1 gad8-K263C mutant strains (Fig 7A). For the cdc18 + gene, the level of induction in gad8-K263C cells is not as impaired as in cells carrying Δtor1, yet, they are significantly reduced compared with wild-type cells (see p values and compare grey with green bars in Fig 7A). Exposure of cells to DNA damage leads to the activation of the main DNA damage checkpoint kinase, Rad3 (ATR), resulting in the activation of the downstream effector kinases Chk1 or Cds1 [61]. In response to CPT, Chk1 becomes activated by phosphorylation, which is detectable as a slow migrating band in Western blot analyses, and is essential for propagating the checkpoint activation signal [64]. As previously reported, Δtor1 mutant cells show a delay in the phosphorylation of Chk1 in response to CPT compared with wild-type cells ( [39] and see Fig 7B). Here we show that gad8-K263C cells showed an intermediate defective phenotype with respect to phosphorylation of Chk1 in response to CPT. Thus, while wild-type and Δtor1 Fig 6. gad8-K263C or gad8-T260 confer sensitivity to DNA damaging conditions. A, Plasmids containing gad8-K263C or gad8-T260 confer genotoxic stress in wild-type cells and are unable to suppress genotoxic sensitivity in Δtor1 mutant cells. WT or Δtor1 cells transformed with empty vector (pREP81), or with vector carrying tor1 + , gad8 + , gad8-T260C or gad8-K263C were serially diluted and spotted onto minimal medium with no stress or containing 5mM HU or 5μM CPT. Plates were incubated at 30 0 C for 3 days. B, The chromosomal K263C mutation confers sensitivity to genotoxic stress. WT, Δtor1, Δgad8, gad8-K263C or Δtor1gad8-K263C cells were serially diluted and spotted onto minimal medium with no stress or containing 5mM HU or 5μM CPT and incubated at 30 0 C for 3 days. Cells spotted on 0.8M KCl are used as control. C, gad8-K263C confers dominant-negative effect under genotoxic stress conditions. Serial dilutions of diploid cells were spotted onto the indicated plates. Plates were incubated at 30 0 for 3 days. D, The gad8-Q298L partially suppresses genotoxic stress sensitivity in Δtor1 cells. Serial dilutions of WT or Δtor1 cells harboring the indicated plasmids were spotted onto minimal medium or minimal medium containing 5mM HU or 5μM CPT and incubated at the indicated temperatures for 4 days. Fig 7. gad8-K263C cells show similar defects to Δtor1 cells with respect to DNA damage responses A, Expression levels of cdt2 + , cdc18 + and cdc22 + in WT, Δtor1, gad8-K263C and Δtor1gad8-K263C cells were determined by qRT-PCR. Total RNA was prepared from untreated cells or cells treated with 12mM HU for 3 h. The level of act1 + mRNA was used as a reference. Error bars (SD) were calculated from biological triplets and significant differences were determined by the Student's t-test ( �� P < 0.001). B, Chk1 phosphorylation is delayed in gad8-K263C in response to CPT. Wild-type, Δtor1 or gad8-K263C cells containing an HA-tagged Chk1 were grown to mid-log phase and treated with 30 μM CPT. Protein extracts from each of the indicated time points were analyzed by Western blotting. Membranes were probed with anti-HA to visualize Chk1 and with anti-Actin as a loading control. The upper band observed with anti-HA antibodies represent the phosphorylated and activated band of Chk1. C, De-phosphorylation of Chk1 is delayed in gad8-K263C mutant. Cells were arrested for 3 h in 30μM CPT before being released into fresh medium. Protein samples from the indicated time points were analyzed by Western blot as describe above. https://doi.org/10.1371/journal.pgen.1009196.g007 PLOS GENETICS TORC2-independent Gad8 alleles cells show phosphorylation of Chk1 15 and 120 minutes following exposure to CPT, respectively; gad8-K263C cells start showing phosphorylation of Chk1 60 minutes following exposure to CPT (Fig 7B). Despite the delayed phosphorylation of Chk1, Δtor1 or Δgad8 arrest cell cycle progression in response to CPT treatment and survive short exposures to the DNA damaging conditions [39]. In contrast, continuous exposure to CPT and other DNA damages conditions is detrimental to TORC2 mutant cells [39]. This differential sensitivity to short versus continuous exposure to DNA damage suggests that TORC2 mutant cells are defective in later responses to DNA damage, such as DNA damage repair and/or re-entrance into the cells cycle. Consistently, there is a marked delay in de-phosphorylation of Chk1 in Δtor1 cells following removal of the DNA damage [39]. Again, gad8-K263C cells showed an intermediate level of defect in de-phosphorylation of Chk1 following damage removal: Chk1 becomes de-phosphorylated 120 minutes following removal of CPT in gad8-K263C cells, compared to 60 minutes in wildtype cells and 240 minutes in Δtor1 cells (Fig 7C). In conclusion, under genotoxic stress conditions, despite its dominant nature, the gad8-K263C mutation shows a similar, albeit less severe phenotype to loss of function of gad8 + . Discussion TOR (Target of Rapamycin), which can be found in two structurally and functionally distinct complexes, is a major regulator of growth, proliferation and survival [1][2][3]. While initially TORC1 has been the focus of the majority of studies of TOR signaling, the recent decade has seen a growing interest in TORC2. This has been fueled by the findings that dysregulation of mTORC2 plays an important role in several human diseases [1]. TORC2 in S. pombe mediates the phosphorylation and activation of the downstream AGC-family protein kinase Gad8, which is orthologous to AKT1 and SGK1 [33]. Here, we report the isolation of gad8 mutant alleles that bypass the requirement for TORC2-mediated phosphorylation under distinct sets of physiological conditions. These mutations map into the PIF pocket of Gad8, a conserved hydrophobic pocket in AGC kinases, which regulates both inter-and intra-molecular interactions that affect ATP-binding and kinase activity and has been shown to specifically affect the docking of PDK1 to its downstream AGC kinases [45,46]. We isolated the gad8-T260C and gad8-K263C mutations by a genetic screen designed to identify Gad8 mutant proteins that do not require TORC2 for growth under high temperature conditions. The gad8-T260C and gad8-K263C mutant alleles, as well as gad8-K263A or gad8-K263R, suppress the inability of TORC2 mutant cells to grow under high temperature, osmotic stress or low-glucose conditions and partially suppress the requirement for TORC2 for sexual development. gad8-T260C, gad8-K263C, gad8-K263A or gad8-K263R did not suppress the sensitivity of TORC2 mutant cells to DNA replication stress or DNA damaging conditions. Indeed, these mutations confer sensitivity to genotoxic stresses when expressed in wild-type cells, demonstrating a dominantnegative effect in heterozygous diploid cells. Cells that chromosomally express the gad8-K263 mutation show a phenotype similar to the one conferred by loss of function of TORC2 with respect to genotoxic stress sensitivity, including low levels of MBF-gene induction and defects in both activation and inactivation of the DNA damage checkpoint. In future experiments it will be interesting to explore the possibility that mutations at K263 interfere with substrate(s) recognition motif or docking interaction with substrate(s) that are specifically required for the genotoxic stress response. The observed dominant negative phenotype could result from higher affinity of a defective Gad8 mutant protein to its substrate. Further studies identifying the substrates of Gad8 are required to support such speculations. Based on our MD analyses, we introduced another mutation in the PIF pocket of Gad8, Q298L. The Q298L mutation could partially suppress the mating deficiency and genotoxic stress sensitivities of cells lacking TORC2, but did not suppress the high temperature or osmotic sensitivities. The Q298L appears to confer a dominant negative effect under condition of low glucose. These findings suggest that while mutations in the PIF pocket can render Gad8 independent of TORC2, they may also impair the protein function under specific stress conditions, possibly by interfering with interactions with specific substrates. Similar to other AGC kinases, Gad8 requires at least three types of phosphorylation events; by TORC2 at its hydrophobic (HM, S546) and turn-motif (TM, S527) and by the PDK1-like protein, Ksg1, at its activation loop (T387) [33]. We demonstrate that the in vitro kinase activity of Gad8-K263C is maintained in the absence of Tor1 and that combining the K263C mutation together with mutations in the phosphorylation sites of TORC2, S527A and S546A results in a mutant allele, gad8-K263C/S527A/S546A, that is able to grow at high temperature or in the presence of osmotic stress. In contrast, combining the K263C mutation with mutations in the phosphorylation sites of Ksg1 results in a mutant allele that is sensitive to these stresses. Thus, our genetic and biochemical analyses suggest that the K263C mutation bypass the requirement for TORC2-dependent phosphorylation at the HM and TM, but not the phosphorylation at the activation loop by Ksg1. How, then, does the mutation K263C mutation in the PIF pocket of Gad8 bypass the requirement for TORC2? Our MD studies suggest that the K263C mutation causes a higher flexibility of the activation loop and the PIF region. The higher flexibility of the activation loop may allow it to explore wider conformational space, thus facilitating the phosphorylation of Thr387 by Ksg1. MD analysis of Gad8 phosphorylated at both the HM and TM similarly resulted in higher flexibility at the PIF domain and the activation loop, further suggesting an intramolecular activation path that involves the C-terminal phosphorylation sites, the PIF domain and the activation loop. Additionally, our MD analysis indicated the presence of an H-bond between K263 and Q298 in the predicted non-phosphorylated structure of Gad8. This H-bond may contribute to the rigidity of the PIF pocket. Mutating the Q298 residue into leucine resulted in partial suppression of the sexual development defect or genotoxic stress sensitivity of Δtor1 cells, suggesting that Q298 is important to render Gad8 activity dependent on TORC2. Our ability to predict mutations that bypass the requirement of TORC2 under stress conditions supports the validity of our MD analyses and calls for future studies of the role of the PIF pocket in rendering Gad8 dependent on activation by TORC2. Since the PIF pocket and the mode of activation of AGC kinases by TOR complexes are both highly conserved in evolution, similar mutations in AGC kinases may also result in TORC2-independet activities. The PIF pocket residues K263 and Q298 are highly conserved in the family of AGC kinases. The SGK1-K131 residue, which is equivalent to K263, was suggested to play a role in localization of SGK1 as part of a nuclear localization sequence (NLS) [65]. However, the NLS in SGK1 conforms to the consensus bipartite NLS (KKAILKKKEEK), while the equivalent Gad8 sequences does not (KKAHIVSRSEV). Analysis of the equivalent residues in PDK1 and AKT suggested that when the HM is phosphorylated, the equivalents of K263 and Q298 in PDK1 and AKT bind the first two phenylalanine residues in the HM [49,52]. Mutations analyses of PDK1-K115 indicated that K115 is important for PDK1 binding to artificial PIF peptides [49] and is critical for binding and phosphorylation of the PDK1 target kinases, S6K, RSK SGK, but not other substrates [51]. Thus, PDK1-K115 is important for docking to substrate AGC kinases but does not significantly alter the conformation of the active site of PDK1 [49,51]. PDK1 is different compared with other AGC kinases as it interacts with and phosphorylates, downstream AGC kinases. The role of the PIF pocket in other AGC kinases has been less-extensively studied. Our studies expand our understanding of the PIF pocket and suggest that it plays a role in keeping Gad8, and possibly other AGC kinases, dependent on TORC2 activation. Materials and methods Yeast strains, media and growth assays S. pombe strains used in this paper are listed in S1 Table. Yeast cells were cultured in rich YES medium supplemented with adenine and uracil or in Edinburgh minimal medium (EMM, 5 g/ liter NH 4 Cl), as described in [66,67]. EMM was supplemented with amino acids according to the auxotrophic requirements of the strains used. EMM-G is EMM containing no glucose. For cell growth assays, logarithmic growing cells were either streaked or serially diluted and spotted on solid YES or EMM plates. Stress sensitivity of S. pombe cells was assessed by streaking or spotting on YES or EMM plates supplemented with KCl, hydroxyurea (HU) or camptothecin (CPT), or on plates with reduced levels of glucose, or plates that are incubated at a relatively high temperature (37˚C). Strains that contained plasmids were assessed on EMM plates in order to select for the plasmid, while strains that contained chromosomal integrations were assessed on YES plates. Unless otherwise specified, plates were incubated at 30˚for 4 days before being photographed. Genetic screens for the isolation of Tor1-independent gad8 mutant alleles We obtained gad8 mutant alleles by using random PCR-based mutagenesis. 5 ng of pREP81-gad8 + plasmid was taken for PCR amplification of the gad8 + ORF with primers: #1324 (5'TCAATTGAATAAGTTGAATTAATTATTTCAATCTCATTCTCACTTTCTGACTTAT AGTCGCTTTGTTAAATCATATGTCCTGGAAACTTACAAAGAGTATGTATTCCATA) and #1282 (5'ATAGTTTGAAAGAAAAACCCTAGCAGTACTGGCAAGGGAGACATTC CTTTTACCCGGGGATCCGCTAGCCCATGGGTCGATCACCTAATGACACTTCCAG GTGCT). The underlined sequences are homologous to pREP81 sequences and the rest of the primer is homologous to gad8 + sequences. Four separate PCR reactions were performed, and in each reaction the concentration of a different dNTP was decreased from the standard 2mM concentration to 2μM. Using Phire Hot Start II DNA Polymerase (ThermoFisher #F-122L), four cycles of PCR were performed with the following temperature profile: 94˚C 1 min, 54˚C 1 min, 72˚C 2 min. The PCR products of the four separate PCR reactions were mixed together and 34 cycles of PCR were performed with the following temperature profile: 94˚C 1 min, 48˚C 2 min, 72˚C 2 min. The resulting PCR fragments were co-transformed together with the pREP81 plasmid into tor1-L2045D cells (TA3110) using the lithium acetate procedure. Transformant cells were incubated at 37˚C for 6 days. Approximately 23,000 colonies were screened. Plasmid DNA was isolated from cells that grew at 37˚C and then used to transform bacterial cells for plasmid amplification. Plasmid DNA extracted from bacteria was retransformed into tor1-L2045D (TA3110) cells and into Δtor1 (TA1291) cells. Plasmids that suppressed the temperature sensitive phenotype of Δtor1 and tor1-L2045D cells were subjected to DNA sequence analysis. Chromosomal integration of the K263C mutation into gad8 + We integrated the K263C mutation into the chromosomal locus of gad8 + using standard techniques based on homologous recombination. We first obtained a fragment containing the K263 residue using wild-type genomics DNA as template for two separate PCR reactions. One reaction used primer #1388 (5'TATCTATGCTTTAAAAACTATGAAATGCGCCCACATT GTATCTCGCAGTGAA) together with primer #252 (5'TCC CCCGGG TCACCTAATGA-CACTTCCAGG) and a second reaction using primer. #251 (5' GGAATTC CAT ATGTCCTGGAAACTTACAAAGAG) together with #1389 (5' CTTCACTGCGAGATACAATGTGGGCGCATTTCATAGTTTTTAAAGCAT AGATA). The underlined sequences indicate the site encoding for K263C. For the PCR reaction, 34 cycles of PCR were performed with the following temperature profile: 94˚C 1 min, 54˚C 1 min, 72˚C 2 min. The resulting two PCR fragments were stitched together using 1μl of PCR product from each PCR reaction as template and primers #251 and #252. PCR amplification was achieved by 34 cycles of PCR with the following temperature profile: 94˚C 1 min, 48˚C 1 min, 72˚C 2 min. The PCR product was transformed into the gad8::ura + (TA1029) strain and plated onto 5-FOA plates for selection against ura4 + cells. Genomic DNA was isolated from cells that grew in the presence of 5-FOA and were further subjected to DNA sequence analysis to ensure the presence of the K263C mutation at the correct locus. Construction of gad8 mutant alleles The gad8-K263A, gad8-K263R and gad8-Q298L mutant alleles were constructed by sitedirected mutagenesis using pREP81-gad8 + as the template and a pair of phosphorylated synthetic oligonucleotide primers (see S S2 Table) in a single-step plasmid amplification method, employing ALLin Mega HiFi Red Mastermix, 2X (HighQu # HLM0301). The amplified product was circularized by a ligation step and transformed into an Escherichia coli host. The resulting construct was confirmed by nucleotide sequencing. Assays for mating efficiency Cells were grown at 30˚C in EMM with adequate amino acid supplements to a density of approximately 5x10 6 cells/ml. Cells were then spotted on EMM-N plates and incubated for three days at 25˚C. An aliquot was taken for inspection under a light microscope and the numbers of cells, zygotes, and spores were counted. The percentage of mating was calculated by dividing the number of zygotes, asci, and free spores by the number of total cells. One zygote or one ascus was counted as two cells, and one spore was counted as a half cell. In each experiment 500 to 1,000 cells were counted. Protein extraction and immunoprecipitation (IP) assays Cells were grown to mid-logarithmic phase, washed once with water, and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM DTT, 125 mM potassium acetate, 12.5% glycerol, 0.1% Triton X-100, protease inhibitor mixture, and 1 mM phenylmethylsulfonyl fluoride). Cells were broken for 45 min with glass beads, centrifuged for 10 min at 10,000 × g and the supernatant was collected. 20 μg of total protein extract was resolved on SDS-PAGE using 10% acrylamide gels. For immunoprecipitation, 1,000 μg of proteins were prepared and pre-cleared with 20 μl of protein A-Sepharose and protein G-Sepharose beads mixture (GE Healthcare). 2 μl of hemagglutinin (HA) antibodies were added to the cleared extract and incubated overnight at 4 0 C. The beads were washed once with lysis buffer, once with lysis buffer containing 0.5 M NaCl, and twice with buffer A (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1% βmercaptoethanol). The resulting immunoprecipitates were used for in vitro kinase assays. In vitro kinase assays We applied a nonradioactive in vitro kinase assay for Gad8 [59], based on the use of GST-Fkh2 as a substrate [37]. For the Gad8 kinase assay, a DNA fragment encoding amino acid residues 291 (Gln) to 411 (Pro) of Fkh2 was expressed in Escherichia coli BL21 strain as GST fusion, using the pGEX-4T1 expression vector and purified. Cells expressing Gad8-HA extracts were immunoprecipitated, and the resultant immunocomplexes were resuspended in 30 μl of kinase buffer (10 mM MgAc, 100 mM ATP, and phosphatase inhibitor mixture, Sigma) containing 0.1 μg of GST-Fkh2. After incubation for 10 min at 30˚C, the reaction was terminated by addition of 7 μl of 5 × SDS-PAGE sample buffers and incubated for 5 min at 80˚C. The reaction was detected by Western blot analysis using anti-phospho-AKT substrate antibody (Cell Signaling Technology). Western blotting Proteins were resolved by SDS-PAGE 10-15% acrylamide gels and transferred to nitrocellulose membranes, blocked with 5% milk in TBST and immunoblotted with the indicated antibodies. Detection was carried out using the ECL SuperSignal detection system (Thermo Scientific). Gad8 Thr387 phosphorylation events were detected using total protein extracts by phosphospecific antibodies. Antibodies against Gad8 Thr387-P were raised against the Gad8 phosphopeptide CRFANWpSYQRPT [59]. To follow Chk1 phosphorylation, proteins were extracted with trichloroacetic acid (TCA) as described previously and resolved by SDS-PAGE using 8% acrylamide gels. Real Time Quantitative PCR (qRT-PCR) RNA extractions and qRT-PCR analysis were performed as described in [59]. 50 ml of each strain were grown to an A600 of �1 in minimal medium. RNA was prepared using the hot phenol method and treated with RNase free RQ1 DNase I (Promega) to remove DNA prior to reverse transcription. 1μg of RNA was reverse transcribed with ImProm-II Reverse Transcriptase (Promega), followed by Real-time PCR with Precision Fast qPCR Master mix kit (Primer Design). Reactions were performed in triplicates and run on the Step One Plus Real-Time PCR (Applied Biosystems). Threshold cycle (Ct) values for the cDNA of interest were normalized to the Ct values of act1 + and relative expression levels were quantified using the comparative method and calculated as 2 −ΔΔCt . The amount of expression was expressed relative to the expression level of wild-type cells grown in minimal medium (relative value = 1). Oligonucleotides used for qRT-PCR analyses are listed in S3 Table. Computational methods Homology Modeling of S. pombe Gad8. The sequence of S. pombe Gad8 (accession number: Q9P7J8) was downloaded from the UniprotKB/Swiss-Prot server [68]. Available three-dimensional structures of close homologues in the Protein Data Bank (PDB) were identified using BLAST [69]. The crystal structure of inactive (un-phosphorylated) SGK1 in complex with AMP-PNP with a resolution of 1.9Å (PDB code: 2R5T) [70] was found as the most suitable template for homology modeling. This template lacks the αC-helix region and the Cterminal extension regions, which were modeled based on the crystal structures of AKT1 kinase (PDB code: 6NPZ) [71] and PKCα (PDB code: 4RA4). Multiple sequence alignment between the target and templates were derived using CLUSTALW [72]. After visual inspection of the alignment, models were generated using the default parameters in MODELLER v. 9.23 [73]. The model was built in the presence of AMP-PNP and a Mg2 + ion. The models were ranked using DOPE energy score, which was calculated by the "evaluate model" python script in MODELLER. The highest ranked model was tested for stereochemical quality using Procheck [74] and was selected for subsequent analysis. System preparation. The mutation K263C and the phosphorylation at positions S527 and S546 were introduced into Gad8 using Schrodinger's Maestro 11.2. All the structures were prepared using the Protein Preparation Wizard as implemented in Maestro 11.2. This protocol adds missing hydrogen atoms considering a pH value of 7.0 ± 1.0, optimizes the hydrogen bond network, and performs restrained minimization. For further calculations, the ATP analogue (AMP-PNP) was replaced by ATP. Next, each construct (Gad8, Gad8-K263C and Gad8-pS527/pS546) were submerged in TIP3P water model in octahedron box with an additional 15-Å extension along each axis of the protein. Sodium and chloride ions were added to the water phase in order to neutralize the system and to obtain a salt concentration of 0.15 M. Molecular dynamics simulations. All MD simulations were performed with the GRO-MACS 5.0.6 package [75] using the Amber14 force field [76]. Force field parameters for ATP were obtained by using the ANTECHAMBER module in AMBER [77]. The partial atomic charges for ATP were derived using quantum-mechanical (QM) single point energy calculation. This calculation was performed with Jaguar [78], as implemented in Schrodinger's Maestro 11.2, using the 6-31G � basis set and the HF level of theory. The simulations were conducted in periodic boundary conditions (PBC) with particle-mesh Ewald (PME) [79] electrostatics with 8 Å cutoff for long range electrostatics. First, the simulated systems were energy-minimized with the steepest descent minimization algorithm in order to remove van der Waals clashes within the systems. Following the minimization, each one of the systems was equilibrated at two stages: (1) 2 ns of simulation in NVT ensemble (constant temperature); (2) 2 ns of simulation in NPT ensemble (constant pressure). During the equilibration stages the heavy atoms of the protein and ATP were restrained. Finally, a 50 ns the production phase was carried out for each system under constant temperature and pressure of 310K and 1 bar, respectively. A time step of 2 fs was applied. Each of the systems were simulated three times, each time starting from a different random seed. Data analysis. The resulting trajectories were visually inspected using VMD 1.9.3 software [80]. The stability of the resulting trajectories and the average mobility of all residues were tested based on the root mean square deviation (RMSD) of the backbone atoms of the protein from the initial structure and on the root mean square fluctuations (RMSF), respectively. RMSD and RMSF were calculated using the RMS and RMSF utilities of the GROMACS package, respectively. Supporting information S1 Fig. Isolation Tor1-independent alleles of gad8. A. gad8-m1/m2/m3, suppressed the temperature or osmotic stress sensitivities of tor1-L2045D or Δtor1 cells. Stress sensitivities were evaluated by growth on solid EMM media. Empty pREP81 vector or pREP81-gad8 + plasmids were used as negative control. pREP1-gad8 + was used as positive control. B. Schematic representation of the ORF of gad8 mutant alleles that were isolated in a screen for Tor1-independent alleles. Cells carrying a gad8 kinase-dead allele, gad8-K259R are sensitive to DNA damaging conditions. Stress sensitivities were evaluated by growth on solid YES media. Plates were incubated for 3 days. B. Cells carrying gad8 mutant alleles that cannot be phosphorylated by TORC2 are sensitive to genotoxic stress. Stress sensitivities were evaluated by serial dilutions as above. (TIF) S6 Fig. gad8-K263C, gad8-K263A, gad8-K263R or gad8-T260, but not gad8-S527D/S546D, confer sensitivity to DNA damaging conditions. A, Plasmids containing gad8-K263C or gad8-T260 are unable to suppress genotoxic sensitivity in TORC2 mutant cells. Stress sensitivities were evaluated by serial dilutions of cells onto EMM media B, gad8-K263A or gad8-K263R confer genotoxic stress sensitivity in wild type cells, while suppressing high temperature, osmotic stress or low-glucose sensitivities in Δtor1 cells. in wild type cells. Stress sensitivities were evaluated by serial dilutions of cells onto EMM media. C, Mutant alleles of gad8 that mimic constitutive phosphorylation at S527 and S546 suppress high temperature, osmotic stress, low glucose stress, as well as genotoxic stress. pR3C-gad8-S546D and pR3C-gad8-S527D/ S546D are a kind gift from A. Yamashita, National Institute for Basic Biology, Okazaki, Japan. (TIF) S1	2020-11-04T14:08:17.083Z	2020-11-01T00:00:00.000	{ "year": 2020, "sha1": "1f0f22ae6a3969868d2c8449afe43d0fa424a8a7", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1009196&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "365b4e108c72657530a6549cb3235327d016e4ab", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
267998736	pes2o/s2orc	v3-fos-license	Community Perceptions of the Progo River Sand and Stone Mining Activities in Yogyakarta (Study in the Communities of Sendangagung Village) This research aims to determine the perceptions of people living in mining areas regarding the impacts of sand and stone mining activities on the Progo River, Yogyakarta. This research uses a qualitative type with a case study method. Primary data collection was carried out by interviews as well as observations and supporting documents or literature for secondary data. Sand and stone mining activities in the Progo River using heavy equipment by individuals and companies under the pretext of normalizing river flow after the eruption of Mount Merapi based on Decree Number 284 of 2011. This regulation has been misunderstood by various parties, which has given rise to uncontrolled sand mining. One example of mining that caused community rejection occurred in Sendangagung Village, especially the Jomboran, Pundak Wetan and Wiyu areas of Yogyakarta. Researchers found that the community rejected mining because the community had the perception that mining companies took it with heavy equipment without paying attention to the preservation of the land and the environment, which resulted in land degradation, landslides, air pollution and water pollution. Apart from that, the community questions the legitimacy of the government in issuing non-transparent mining permits. INTRODUCTION The world, including Indonesia, is experiencing environmental damage and a serious socio-environmental crisis that is endangering the sustainability of the earth and the lives of all mankind (Wahana Lingkungan Hidup Indonesia [WALHI], 2021).Various parties accuse that the main cause of environmental damage and socio-environmental crises so far is the result of development strategies and policies that are not environmentally friendly and pro-people.National development strategies and policies prioritize achieving the economic interests of the state and capital owners (investors) rather than the interests of the environment and society.This inequality can be seen in Law no.The fundamental role of accounting is as a provider of information and a source of answers for all matters related to company finances (Jonathan, 2021).However, in the current era, accounting science is required to face various challenges and meet various demands that arise from social, technological, and economic changes.The current environmental problem is a complex crisis that has a widespread impact and influences many aspects of life, including accounting (Swastha & Irawan, 2001).The emergence of environmental accounting is a response to awareness of increasingly pressing environmental and social issues (Putri, Mimba, & Sari, 2019). The main economic source in the Special Region of Yogyakarta Province (DIY) apart from the agricultural, tourism and education sectors is sand and stone excavation or known as Sirtu.Sand mining in DIY Province is closely related to the activities of Mount Merapi.Sand mining in the Mount Merapi area began in the 1970s as a response from local residents to the abundance of sand resulting from the eruption (Umaya, Soekmadi, & Sunito, 2020).The sand mining process in the Basin River which originates at Mount Merapi has experienced a transition from traditional mining to mining with heavy equipment since 1992 (Kusmiyati, 2019). The eruption of volcanic material in the Mount Merapi area has been utilized by the local community for mining activities for mineral C.This activity has made a significant economic contribution to the surrounding community, resulting in an increase in their income (Yudhistira, Hidayat, & Hadiyarto, 2011), after the eruption of Mount Merapi.In 2010, sand mining increased significantly in the area.In response to this situation, the Regent of Sleman issued Decree (SK) Number 284 of 2011 concerning Normalization of River Flows After the Merapi Eruption (Slemankab, 2013).The aim of this normalization is to return river flows and land that has been buried by post-eruption volcanic material to a more normal condition.Research conducted by Bahtiar in 2016 showed that the river flow normalization regulations were often misunderstood by various parties.This has resulted in the uncontrolled development of sand mining in the area.Bahtiar also found that the uncontrolled use of heavy equipment in sand mining activities had a negative impact.The decline in water sources occurred due to sand being continuously excavated by heavy equipment, so that the number of water sources around the Mount Merapi area decreased by up to 50 percent. The Sleman Regent's regulations were misunderstood by various parties, which gave rise to uncontrolled sand and stone mining.Sand and stone mining activities in the Progo River using heavy equipment, both by individuals and companies, under the pretext of normalizing river flow after the eruption of Mount Merapi, have raised community concerns about the threat to residents' springs and also concerns about land collapsing due to sand mining operations close to residential areas.One of the mining areas that caused community rejection occurred in Sendangagung Village, especially the Wiyu, Pundak Wetan, and Jomboran areas of Yogyakarta.This mining conflict between residents and mining companies has been going on since 2016, the community refuses mining because they are worried about the negative impact on the surrounding environment.The existence of mining activities in the area around the Progo River, apart from destroying the natural beauty of the Progo River, has also changed social relations in the community to become divided between those who are for and against mining. Based on field observations, the sand and stone mining location in Sendangagung Village carried out by the Company is very close to residential areas.The distance between sand mining to Dukuh Jomboran is 500 m, to Dukuh Wiyu is 500 m, and to Dukuh Pundak Wetan is 400 m.From this data, it can be ascertained that sand and stone mining is very detrimental to the health and quality of the local environment.Very worrying environmental damage is not the only impact arising from mining activities.The impact of this mining activity can trigger potential conflict between local residents, or in formal terms, horizontal conflict in society. This research aims to determine the perceptions of people living in mining areas regarding the impacts of sand and stone mining activities on the Progo River, Yogyakarta.This research is also to find out the perceptions of the Yogyakarta Police and Legal Aid Institute (LBH) from a legal perspective and WALHI Yogyakarta from an environmental perspective to find out how they view sand and stone mining, including their views on the social, economic, and environmental impacts of the activity.This can help governments, mining companies, and other organizations gain insight into community concerns and expectations. Legitimacy Legitimacy in the mining context refers to the level of trust, acceptance and support of the community, government, and other stakeholders towards mining operations.The primary interest in ensuring legitimacy in mining is to ensure that mining operations are recognized as legitimate, beneficial, and fair by local communities and governments.This is extremely important as mining operations often have significant impacts on the environment, communities, and local economies. Perception Perception is a process that is preceded by sensing, namely a process that takes the form of receiving a stimulus by an individual through the sense organs or also called a sensory process (Walgito, 2010).Nevertheless, it is important to recognize that a person's perceptions can often differ from objective reality (Robbins & Coulter, 2005;Hafni, Hanum, & Hasibuan, 2021).There are several stages in the process of perception in individuals, namely the object causes a stimulus, and the stimulus hits the sensory organs or receptors (Sunaryo, 2002).This process involves individual attention, transmission of information to the brain, individual awareness of certain objects, and giving meaning to the stimuli received.Next, the individual provides a subjective assessment of the object.Therefore, people's understanding of sand and stone mining activities in the Progo River can vary because each individual experiences different cognitive processes in providing meaning and assessment of these mining objects. Environmental Accounting Environmental accounting is the identification, measurement and allocation of environmental costs and integrating these costs into business decision making and communicating the results to company stakeholders (Ikhsan, 2008).In order to maintain sustainability and minimize negative impacts on the environment, mining companies must pay attention to environmental accounting and commit to involving sustainable practices in their operations.This involves measuring, reporting, and managing environmental impacts as well as compliance with applicable regulations.Disclosure of environmental information in financial reports and sustainability reports is increasingly important in providing transparency regarding the environmental impacts of mining companies.This allows stakeholders, such as investors and the public, to better understand how companies manage environmental issues. RESEARCH METHOD This type of research is field research.In carrying out this research, researchers attempted to collect primary data obtained through direct observation of mining activities in Sendangagung Village, Minggir, Sleman. In this study, the research approach included a case study.Researchers will collect data through interviews, observations, or document analysis, and then describe in detail the characteristics, context, and dynamics of the case. The subjects in this research were Tipider Investigators from the DIY Regional Police who handled mining cases in DIY Province, Head of the Advocacy Division from the DIY Province Forum for the Environment (WALHI) who handled environmental damage due to sand and stone mining in the Progo River, Legal Expert Staff from Aid Institutions Law (LBH) Yogyakarta which handles cases of sand and stone mining in the Progo River, Yogyakarta and some local communities who live in the area around mining activities, namely the people of Sendangagung, Minggir, Sleman Villages, especially the residents of Jomboran, Wiyu and Pundak Wetan. The data collection technique used in this research uses documents obtained from public complaint reports and mass media related to research regarding the perceptions of the people of Sendangagung Village, especially the residents of Wiyu, Pundak Wetan, and Jomboran regarding sand and stone mining activities on the banks of the Progo River in Yogyakarta.In the observation stage, researchers looked directly in the field at the condition of the people of Sendangagung Village who live around the sand and stone mining activities on the banks of the Progo River in Yogyakarta.In this research, the indepth interview method was used to find out and obtain data directly from the research object regarding the perceptions of the Sendangagung Village community regarding sand and stone mining activities on the banks of the Progo River in Yogyakarta.In-depth interviews in this research will be aimed at the research subjects, namely the DIY Regional Police Tipider Investigator, Head of the Advocacy Division of WALHI Yogyakarta, Legal Expert Staff of LBH Yogyakarta, and local residents of Sendangagung Village who live around the sand and stone mining activities on the banks of the Progo River.These questions can help in exploring community views, needs and aspirations regarding mining activities.Research on community perceptions can play an important role in designing more sustainable policies and seeking to achieve a balance between economic and environmental interests by involving the opinions and concerns of local communities. Questions to investigators regarding class C mining activities based on public complaints.This question aims to gain in-depth insight into the dynamics between the mine, the local community, and the handling of the investigation.This is important to ensure that handling problems that arise can reflect the interests and needs of all parties involved.Questions to the Head of the DIY Province WALHI Advocacy Division about community perceptions regarding mining activities can help in understanding how WALHI works with local communities, exploring their views, and utilizing community perceptions in their efforts to protect the environment from destructive mining activities.Questions to the Legal Expert Staff of LBH Yogyakarta regarding sand mining activities in Sendangagung Village can help in understanding how LBH Yogyakarta's role is in dealing with mining activity problems in Sendangagung Village and how they are trying to help the affected communities.Questions to the Sendangagung Village community regarding sand and stone mining activities on the banks of the Progo River aimed to gain a deeper understanding of the dynamics between the mine and the local community.This can help companies, governments and communities work together to create solutions that are more sustainable and in line with the needs and interests of all parties involved. Overview of the Research Area Sendangagung Village is part of the Minggir District Government area, Sleman Regency, which is located in the center of Minggir District City.Geographically, Sendangagung Village can be seen from several aspects, including as follows. Business Activities of the Sendangagung Village Community Sendangagung Village is a village located on the outskirts of Sleman Regency, Yogyakarta Special Region.The village is still beautiful with rice plants that look spread out and hamlets that look green.With its vast expanse of rice fields, it is not wrong that Sendangagung Village is one of the rice barns in the Yogyakarta area.Sendangagung Village, Minggir District, Sleman Regency has been declared a food independent village.At least Sendangagung Village has 15 food barns that are still active and store rice reserves of up to one ton. Relations between the Sendangagung Village Community and the Progo River The people of Sendangagung Village, Minggir, Sleman, Yogyakarta, have a very strong relationship with the Progo River.This river has an important meaning in their daily life, culture, and beliefs.Several important aspects related to the relationship between the Sendangagung Village community and the Progo River.The Progo River is often considered a sacred place that has spiritual and religious value for the local community.They believe that this river is a source of blessings and life.The Progo River is also a source of livelihood for some people.They use this river for fishing, farming, or other economic activities.The Progo River is widely used for other purposes.In the area around the estuary, there is a lot of sand mining. Perceptions of DIY Regional Police Tipider Investigators Regarding Sand and Stone Mining Activities Investigators at mines are included as Tipider Investigators (Regional Police Development and Supervision Team) in the field of Special Criminal Investigation (Reskrimsus) in the Police covering various aspects related to law enforcement and the investigation of complex and serious criminal acts. Based on the results of interviews with sources, Tipider Investigators from the DIY Regional Police and their staff who have handled sand and stone mining cases gave the perception that there were positive and negative impacts from sand and stone mining activities in the Progo River.Perception according to AKP Bowo as Head of the Tipider Unit for the DIY Regional Police expressed as follows. "The positive impact is that the economy is running, many people get jobs, income goes to the village treasury, village roads along mining routes become better, many people work in these activities, including drivers.The negative impact when mining is carried out illegally without study can damage the environment in the future, there will be definitely impacts, perhaps the effects will be indirect.For example, if the embankment collapses during the rainy season, flooding occurs, the ecosystem habitat will be disturbed if studies are not carried out, the environment and ecosystem will be damaged." The same perception was expressed by AKP Iswahyudi as Tipider Investigator for the Kulon Progo Police that the impact of sand and stone mining has both positive and negative impacts on the community around mining activities, namely as follows. "The positive impact is that the community has employment opportunities, village areas are developed, for example by building roads.The negative impacts are damaged roads, dust, noise, dry well water." The same perception also came from Bripka Yusvi who served as a Tipider Investigator for the Gunungkidul Police, stating as follows. "The positive impact is improving community welfare and improving public facilities for the better.Meanwhile, the negative impact of life around mining is that people feel noisy due to the use of heavy equipment and air pollution which is not good for health." In accordance with the perception of Tipider Investigators both at the Yogyakarta Regional Police and their ranks, it can be concluded that mining activities can have a positive impact but behind that there are also negative impacts that have a bad effect on public health, namely air pollution caused by sand dust and noise pollution due to the use of heavy tools.Environmental damage such as landslides, erosion and depletion of water sources need to be anticipated early by miners so as not to worsen the health conditions of the community and the environment around mining activities. WALHI Yogyakarta Province's Perception of Sand and Stone Mining Activities Indonesian Forum for the Environment (WAHLI) is a non-profit organization and environmental NGO based in Indonesia.This organization was founded in 1980 and has played an important role in advocacy and activism for environmental conservation and community rights in Indonesia.WAHLI focuses on various environmental issues, including forest protection, natural resource conservation, waste management, land restoration, and other issues related to nature conservation and environmental sustainability. Based on the results of interviews with resource persons, Mr. Asegraf as Head of the WALHI Advocacy Division, DIY Province, who handles environmental damage from sand and stone mining activities in the Progo River, gave his perception that initially residents saw that the mining company would change their economic life, but the reality was beyond the community's expectations.Mr. Asegraf's explanation, namely as follows. "Actually, at first the residents saw this mine, I'm sorry, some residents saw this mine as a breath of fresh air, later there will be job opportunities, the construction of the road area will be repaired, but there are also residents who initially saw this, it is proven that in one of the mines in DIY in the Kali area There are no less than 5 people in Progo River in the Progo River who are employed in mining.Even now, before the mining ended, only 2 of them were parking attendants, not operating excavators and trucks, with payment for which I have not yet received a nominal figure, but it is just below the minimum wage.So, in my opinion, the parameters related to job creation are quite ambiguous because if this mining company only takes 1 or 2 people and claims that it has created jobs, for him that is enough, but for the community where the economy has not yet recovered after Covid, there is a stigma that it is that mining provides economic benefits, creating jobs is wrong.It is not factually proven in the field -it can be proven in the field that way.Even in terms of regional income contribution to post-mining village roads, the roads have not been repaired so they are still in a state of disrepair."And Mr. Asegraf explained in more depth about the problems that occurred in the Jomboran, Pundak Wetan and Wiyu areas providing assistance with friends from WALHI Yogyakarta.Mr. Asegraf explained that PT X as a mining company that carries out mining activities in the Progo River does not hold socialization to residents who are directly affected by the mining activities and the form of concern already exists, the community sees that mining activities have the potential -the point of the cliff or land of the residents Journal of International Conference Proceedings (JICP) Vol.6 No. 6, pp.403-415, December, 2023 P-ISSN: 2622-0989/E-ISSN: 2621-993X https://www.ejournal.aibpmjournals.com/index.php/JICP that collapsed can be seen from the explanation of Mr. Asegraf, representative of WALHI DIY Province, namely as follows. "There was a form of concern at the beginning, namely that perhaps the community saw that this mining would potentially cause landslides at first and it was proven that after 4 years of mining going on, many cliff points or people's land had collapsed, even WALHI and also the UPN Environmental Engineering Study Program.Yogyakarta Veterans of Science and Disaster Studies together conducted research related to well points, namely 32 well points in 4 padukuhans around the mine, such as Jomboran, Wiyu, Pondok Wetan and Nanggulan.At the same time, there was a decrease in water discharge, well, this has never happened before there was mining, but when there was in mining, this happens like a decrease in water discharge and landslides and for the water quality standards themselves, at several points in the 32 points that we have carried out research, there are several wells that are contaminated with oil waste and indications are that oil from excavators is proven from the polluted well points.What is polluted is that the well point is not far from the Progo River or the mining point.This could be an indication of water pollution and then the community ended there.We together with the PMKP community (Kali Progo Community Association) reported the mining activities of PT X on grounds such as impacts, violations of the law because before I went deeper into mining from PT X, in fact, there is a formal defect there, namely he manipulated the signatures of residents which he rejected due to mining so that mining could not be carried out because of a formal defect in the administration process.So, we also include excuses related to legal violations regarding mining activities because according to residents, especially PMKP, apart from being a nuisance, this mining has caused quite a lot of harm to the community." Based on the explanation from Mr. Asegraf as a representative of WALHI Yogyakarta, it can be concluded that PT X as a mining company carrying out mining activities in the Progo River does not hold socialization to residents who are directly affected from sand and stone mining activities.The local community feels disturbed and disadvantaged by these mining activities.The community formed the Kali Progo Community Association (PMKP) to carry out PT reporting.X on the basis of the impact of pollution, pollution of waste and cliffs that are landslides and the existence of violations of the law such as not conducting socialization until manipulation of citizens' signatures that clearly reject the mining. LBH Yogyakarta's Perception of Sand and Stone Mining Activities in the Progo River LBH Yogyakarta has collaborated with community groups or individuals affected by mining activities or issues arising from mining activities in Sleman.The mining activity that has drawn rejection is located on the Progo River on the border of Sleman Regency and Kulon Progo Regency.Rejection emerged from residents around the mining location who are members of the Kali Progo Community Association (PMKP).Sand and stone mining activities in the Progo River, Special Region of Yogyakarta, have received rejection from local residents because they are considered to be damaging the environment and the licensing process is not transparent. Based on the results of an interview with the resource person, Mr. Wandi, as Legal Expert Staff at LBH Yogyakarta, who accompanied the Sendangagung Village community in their action to reject mining activities on the Progo River, gave his perception that the community took action to reject it because the local community's fears about the environmental damage occurring on the banks of the Progo River were visible. in Mr. Wandi's explanation, as follows. "We have been assisting the residents since 2017 there until now and that is the mining issue.The problem with mining is that the majority reject mining there because what.Due to the environmental damage that occurs, whether from local people's wells, from local residents' surroundings, whether from mining activities that are there, it erodes irrigation and changes the flow of the river itself, the river's habitat is also lost, small children don't dare to play in it.there because of fear of its depths.The landslides continued like that, so the fears were real and they are still ongoing to this day, the impacts of which are still on the residents so that from there the residents forcibly refused and they no longer want mining there." Further explanation from Mr. Wandi, the main problem is the miners' obligations at the post-mining stage, namely to restore the environment of the mining area, which is not implemented and makes the community disappointed by taking action to reject it, as can be seen from Mr. Wandi's explanation, as follows. "Actually, compensation is different from the miners' obligations when after their permits expire, they have to carry out several conditions, including reclamation, environmental damage, it becomes an obligation and cannot be claimed as compensation.This compensation means that people's disapproval becomes a form of rupiah or money.The residents rejected this.The residents did not want to accept it and the residents agreed that reclamation was the responsibility of the miners and it could be directly supervised by the department to carry it out.Until now it turns out that the reclamation has not been carried out, that is the problem.After mining, it becomes the company's obligation." Based on the explanation from Mr. Wandi as a legal expert at LBH Yogyakarta, it can be concluded that LBH is still assisting the residents of Sendangagung Village since 2017 regarding the issue of sand and stone mining, where majority of the majority reject sand and stone mining in the Progo River due to environmental damage.This happens whether it's people's wells drying up, changes in the social environment, the emergence of pros and cons, mining activities that erode irrigation and change river flow, loss of river habitat, small children don't dare to play there because they are afraid of the depth of the Progo River, so these fears actually happen and is experienced by residents. Perception of the Sendangagung Village Community Regarding Sand and Stone Mining Activities in the Progo River Public perception is the collective view or understanding held by a group of people in a society regarding various things, including shared values, beliefs, norms, and goals.People are increasingly aware of the importance of protecting the natural environment for the sustainability of life on this planet as well as efforts to preserve the environment and reduce negative impacts on the ecosystem. Researchers interviewed 6 residents of Sendangagung Village, each represented by 2 residents from Jomboran, Pundak Wetan and Wiyu as sources who live around 100 meters to 500 meters from mining activities.From the residents' explanations, they have the same perception that the community is concerned about mining activities using heavy equipment which disrupts the residents' social environment, as can be seen from the explanation of Mrs. Yuli, a Wiyu resident who was interviewed on November 5 2023 as follows. "Yes, I lived less than 100 meters from the sand mining site when I was surveyed by the disaster service.What is clear is that the impact is negative, in the sense that it is physically disturbed, our ears are automatically uncomfortable, the sound of the machine is always loud and visible to the naked eye, we have never seen an excavator, when we see a big excavator, we are afraid.Then socially we are disturbed, in the sense that not only physically, we are disturbed socially, meaning that after we find out who gave us permission, we seem to have a conflict with them, as if there is a distance from them, and the economy is also different from the start, we know that they got it from the miners, whereas we cannot.There was no socialization and suddenly it was like we were being lied to." It is not only the social environment that is being disturbed, the impact of changes in the natural environment where they live is starting to become worrying, as explained by Mr. Marwoto as Head of the Jomboran Community Association (RW) who was interviewed on 29 October 2023 as follows. "Yes, we feel air pollution, noise and water sources are shrinking.Moreover, people living in areas near the mine, plus the dry season, the springs dry up." Likewise, the explanation of Mr. Samiyo as Head of the Pundak Wetan Neighborhood Association (RT) who was interviewed on October 29 2023 is as follows. "Yes, what is certain is that the environment is damaged, dust, noise, all of this has an impact on the people around here in the mining area." The turmoil of community rejection was increasingly felt because the miners did not carry out outreach to the affected communities.Suddenly the miners lowered their heavy equipment and started mining, explained by Mr. Iswantoro, a resident of Jomboran who was interviewed on 22 October 2023 as follows. "Until now, we do not know what the official permit is like because it is all an acknowledgment from the company that he already has a permit.Officially, the company does not want to show it to the public, but we tried to find it and we found it.The socialization should be in other hamlets, not in our place.The socialization should be for affected residents who want to mine, but it should be outside the hamlet or in other places." The residents' disappointment deepened after the miners did not carry out their obligations, namely environmental restoration or reclamation.This was explained by Mr. Sutrisno as a resident of Pundak Wetan who was interviewed on 29 October 2023 as follows. "For the last company (Limited Company (PT)) reclamation, reclamation should be the restoration of the mining environment, he said, those who took part in the socialization, the PT set aside funds from the PT to the relevant agencies later for reclamation costs, land restoration, but if it was land restoration, it might be something like If to prevent landslides, we plant something that is strong enough to withstand landslides and erosion.Now, if it is just a pile of land which is said to be God's creation, it can slide, what is more, if it is just a machine-made pile, if it is hit by the current of a river, it will still landslide.The first is that it erodes little by little and then collapses.In my opinion, the PT did not carry out full reclamation, only if I asked the PT, the reclamation was like that.Put it this way, now the land filling is said to be reclamation and not yet 100% finished.Now it's a pile of soil and stones where grass is planted, the grass's roots grow as long as they can't hold the soil." CONCLUSION Based on the results of interviews and observations conducted by researchers, the community's perception of sand and stone mining activities in the Progo River resulted in the following conclusions.Firstly, residents of Jomboran, Pundak Wetan and Wiyu live close to the sand and stone mining area on the Progo River, an average distance of 100 meters to 500 meters.And sand and stone mining activities in the Progo River affect their daily lives, namely pollution and noise problems as well as the drying up of spring wells.Secondly, mining operations in the Jomboran, Pundak Wetan and Wiyu areas do not receive full support from the local community.There are several residents who are promining, but only a few, between 2 and 5 people.There are Village Heads and Hamlet Heads who support mining activities.But almost all residents reject the existence of sand and stone mining activities in the Progo River.Thirdly, according to information from the people of Jomboran, Pundak Wetan and Wiyu, the process of obtaining mining permits did not comply with procedures because there was no outreach to affected residents and there were legal and administrative defects regarding mining permits.Fourthly, according to statements from Jomboran residents, Pundak Wetan and Wiyu, they felt disturbed by the mining activities.They feel that air pollution is caused by dust from mining activities, air pollution from the noise of heavy equipment machines and dump trucks and the shrinking of water sources in residents' wells which makes it difficult for residents to obtain clean water.Lastly, according to a statement from the people of Jomboran, Pundak Wetan and Wiyu hope that mining activities will not resume in their area.They want to be free from mining activities, which according to them, mining activities change the natural environment around them and the social environment between them, which triggers conflicts for and against mining between them. Based on data obtained from research related to community perceptions of sand and stone mining activities in the Progo River, the suggestions were as follows. Revoke All Mining Business Permit Areas (WIUP) that Use Heavy Equipment Along the Progo River This decision was taken by considering important aspects related to environmental preservation and the balance of the river ecosystem.This withdrawal aims to protect and maintain water quality, flora, fauna, and the sustainability of river ecosystems which have a strategic role in maintaining environmental balance. Revise the Spatial and Regional Planning (RTRW) Policy so that the Progo River is Excluded from Areas that Can Be Mined Using Heavy Equipment This revision is based on in-depth considerations regarding environmental impacts, hydrological sustainability, and biodiversity conservation directly related to the Progo River.This exception aims to ensure that the Progo River area remains protected from mining activities that could harm its natural integrity. Ask the Sleman Regency Government to Provide Recommendations that Support the Struggle of Residents Who Benefit from the Source of the Progo River By providing positive recommendations, the Sleman Regency Government has effectively expressed its support for the community's efforts to protect and utilize the Progo River Source in a sustainable manner and in accordance with environmental conservation principles.This can include sustainable water management, natural resource conservation, and environmental monitoring to ensure that human activities do not harm river ecosystems and their benefits to society. LIMITATION In conducting research on the Development of Strategic Management and Application of Accounting on Community Perceptions of the Progo River Sand and Stone Mining Activities in Yogyakarta (Study in the Community of Sendangagung Village, Minggir, Sleman Yogyakarta), there were limitations faced by researchers regarding access to information, especially information on mining company data which is difficult to obtain becomes an obstacle in accurately describing the implementation of strategic management and accounting practices of mining companies because mining companies are not willing to provide data. 3 of 2020 concerning Amendments to Law no. 4 of 2009 concerning Mineral and Coal Mining (The Audit Board of Indonesia, 2020).Several articles that have the potential to harm society and the environment are contained in Article 8, Article 96, Article 162, and Article 169 A of Law no. 3 of 2020 concerning Mineral and Coal Mining.	2024-02-27T16:52:34.068Z	2024-01-10T00:00:00.000	{ "year": 2024, "sha1": "fcda71bec0fb58acab8ed02aa0b931bec64df008", "oa_license": "CCBYNCSA", "oa_url": "https://ejournal.aibpmjournals.com/index.php/JICP/article/download/2850/2321", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "8e00cc3a11d19eed2cae5ab8d244b97f7d350003", "s2fieldsofstudy": [ "Environmental Science", "Geography" ], "extfieldsofstudy": [] }
253554225	pes2o/s2orc	v3-fos-license	Maternal mortality in eight European countries with enhanced surveillance systems: descriptive population based study Abstract Objective To compare maternal mortality in eight countries with enhanced surveillance systems. Design Descriptive multicountry population based study. Setting Eight countries with permanent surveillance systems using enhanced methods to identify, document, and review maternal deaths. The most recent available aggregated maternal mortality data were collected for three year periods for France, Italy, and the UK and for five year periods for Denmark, Finland, the Netherlands, Norway, and Slovakia. Population 297 835 live births in Denmark (2013-17), 301 169 in Finland (2008-12), 2 435 583 in France (2013-15), 1 281 986 in Italy (2013-15), 856 572 in the Netherlands (2014-18), 292 315 in Norway (2014-18), 283 930 in Slovakia (2014-18), and 2 261 090 in the UK (2016-18). Outcome measures Maternal mortality ratios from enhanced systems were calculated and compared with those obtained from each country’s office of vital statistics. Age specific maternal mortality ratios; maternal mortality ratios according to women’s origin, citizenship, or ethnicity; and cause specific maternal mortality ratios were also calculated. Results Methods for identifying and classifying maternal deaths up to 42 days were very similar across countries (except for the Netherlands). Maternal mortality ratios up to 42 days after end of pregnancy varied by a multiplicative factor of four from 2.7 and 3.4 per 100 000 live births in Norway and Denmark to 9.6 in the UK and 10.9 in Slovakia. Vital statistics offices underestimated maternal mortality by 36% or more everywhere but Denmark. Age specific maternal mortality ratios were higher for the youngest and oldest mothers (pooled relative risk 2.17 (95% confidence interval 1.38 to 3.34) for women aged <20 years, 2.10 (1.54 to 2.86) for those aged 35-39, and 3.95 (3.01 to 5.19) for those aged ≥40, compared with women aged 20-29 years). Except in Norway, maternal mortality ratios were ≥50% higher in women born abroad or of minoritised ethnicity, defined variously in different countries. Cardiovascular diseases and suicides were leading causes of maternal deaths in each country. Some other conditions were also major contributors to maternal mortality in only one or two countries: venous thromboembolism in the UK and the Netherlands, hypertensive disorders in the Netherlands, amniotic fluid embolism in France, haemorrhage in Italy, and stroke in Slovakia. Only two countries, France and the UK, had enhanced methods for studying late maternal deaths, those occurring between 43 and 365 days after the end of pregnancy. Conclusions Variations in maternal mortality ratios exist between high income European countries with enhanced surveillance systems. In-depth analyses of differences in the quality of care and health system performance at national levels are needed to reduce maternal mortality further by learning from best practices and each other. Cardiovascular diseases and mental health in women during and after pregnancy must be prioritised in all countries. Introduction Maternal mortality, a key indicator of the quality of maternal healthcare, is at historic lows in high income countries. 1 Most of these countries seem to be in stage IV or V of the obstetric transition, in which maternal deaths are mostly due to extra-obstetrical conditions. 2 International comparisons of maternal mortality are important as they can highlight national similarities or differences, both of which are valuable for determining priorities in maternal health. Cause specific profiles of For numbered affiliations see end of the article Correspondence to: C Deneux-Tharaux catherine.deneux-tharaux@ inserm.fr (or @Epope_Inserm on Twitter; ORCID 0000-0002-1176-0991) Additional material is published online only. To view please visit the journal online. maternal mortality provide warnings about the quality of maternal care in particular conditions. They can lead to the identification, analysis, and remediation of inadequate practices, as they have for postpartum haemorrhage in France and Italy and for venous thromboembolism in the UK. [3][4][5] Historically, cross country comparisons of maternal mortality have been based on data from vital statistics offices, based on death certificates. Several studies, however, showed that vital statistics underestimate maternal mortality and provide inaccurate information about the cause of death, thus preventing meaningful international comparisons. [6][7][8][9][10] Previous reports highlighted the resulting inability to use these data for informative comparisons between European countries, as many of the variations observed reflect differences in measurement methods rather than actual differences in maternal mortality. [11][12][13] For example, because countries with the least advanced case ascertainment methods are likely to miss the most cases, they may paradoxically seem to have the lowest maternity mortality ratios. 6 7 In response to these major limitations, several countries have implemented enhanced surveillance systems for maternal mortality to optimise and standardise the successive steps of identifying, documenting, classifying, and learning lessons from every maternal death. 14 We hypothesised that these enhanced systems ensure that intercountry variability of maternal mortality is not due to differences in data collection methods and that they provide reliable and comparable data. The International Network of Obstetric Survey Systems (INOSS), an international collaboration of researchers and clinicians collecting population based data on severe maternal complications and rare diseases in pregnancy, offered the opportunity to gather and compare such data. 15 Our aim was to compare maternal mortality patterns between countries with national data obtained through enhanced maternal mortality surveillance systems. Methods study design and data sources Eight countries collaborating in the INOSS and using enhanced surveillance systems for maternal mortality participated in this descriptive population based study: Denmark, Finland, France, Italy, the Netherlands, Norway, Slovakia, and the UK. 15 We defined enhanced surveillance systems as those with specific prerequisites for the three essential steps of this process: the identification, documentation, and review of maternal deaths. For the first step, completeness of case identification must be ensured by at least one type of linkage between the death register and one of the following: birth register, pregnancy loss register, and hospital discharge database. We considered the Dutch system to be enhanced despite the lack of linkage for the identification of women who died (prevented by data privacy regulations). Because the Netherlands is a relatively small country where reporting of maternal deaths up to one year after the end of pregnancy is mandatory, we considered its maternity mortality surveillance system "possibly enhanced" and included its data in this analysis. The second step, documentation of each case, requires going beyond the content of the death certificate and including data from the birth certificate, birth register, medical records, hospital discharge database, or a confidential inquiry. Finally, the third step-case review-must be conducted by a committee auditing all maternal deaths. Table 1 describes the main features of the enhanced surveillance system in each participating country. Full protocols are published elsewhere. 5 16-21 collected data We collected the most recent aggregated and complete dataset available at the time of the study design from a time period that varied according to the number of births in each country: a three year period for France, Italy, and the UK and a five year period for Denmark, Finland, the Netherlands, Norway, and Slovakia. The Finnish data cover deaths from 2008 to 2012, and the other countries provided data from 2013 and after (table 2). We included deaths at any time during pregnancy and up to one year after the end of pregnancy, regardless of pregnancy duration and/or site. 14 Late deaths were those occurring between 43 days and one year after the end of pregnancy. Pregnancy associated deaths were those occurring within one year of the end of pregnancy, regardless of their cause. Maternal deaths were those with a cause related to or aggravated by the pregnancy or its management, but not from an accidental or coincidental cause, as per the World Health Organization's application of ICD-10 (international classification of diseases, 10th revision) to deaths during pregnancy, childbirth, and puerperium (ICD-MM), used by all countries. 22 We requested the following aggregated numbers from enhanced systems in each country: pregnancy associated deaths, maternal deaths, and maternal deaths by age group (<20, 20-24, 25-29, 30-34, 35-39, ≥40 years) and by migrant status or ethnicity defined by the categorisations available in each country (current citizenship, country of birth, or ethnicity). We also collected the number of maternal deaths by underlying cause, as classified by the national committees and according to the following groups, also based on ICD-MM but with further developments for some groups relevant for high income settings: pregnancies with abortive outcomes (ectopic pregnancy, post-abortum haemorrhage, post-abortum infection or pulmonary embolism, other abortive outcomes), obstetric haemorrhage (uterine atony, morbidly adherent placenta, placenta previa, uterine rupture, placenta abruption and other obstetric haemorrhage), pregnancy related infection, hypertensive disorders of pregnancy (pre-eclampsia, eclampsia, HELLP syndrome), venous thrombosis and thromboembolism (cerebral pulmonary or unspecified), amniotic fluid embolism, anaesthesia complications, other direct causes, cardiovascular causes (peripartum cardiomyopathy, ischaemic cardiomyopathy, other cardiomyopathy, other cardiac, aortic dissection, arterial rupture), stroke (arterial, haemorrhagic, ischaemic), indirect sepsis (influenza, pneumonia, meningitis, urinary sepsis, other indirect sepsis), malignancy (trophoblastic or other), other indirect causes, suicide, illicit drug or alcohol misuse, and unascertained. 22 Finally, we obtained the number of maternal deaths according to the country's office of vital statistics during the same time periods (official numbers provided by death registers and not reviewed by a committee). We collected the numbers of live births and maternities (see each country's definition of a live birth and maternity in appendix 1) over the same specified time period, overall, as well as by age and origin. analysis We calculated maternal mortality ratios, as defined by WHO, as the number of maternal deaths during the study period per 100 000 live births during the same time period (number of maternities was unavailable for Italy). 23 We calculated the maternal mortality ratios up to 42 days after the end of pregnancy, with their 95% confidence intervals, according to the enhanced surveillance systems and compared them with those obtained from vital statistics in each country. To quantify between country variation in maternal mortality ratios up to 42 days, we divided the highest maternal mortality ratio by the lowest one and also calculated the maximum absolute difference in maternal mortality ratios by subtracting the smallest maternal mortality ratio from the highest one, with its 95% confidence interval. We also calculated maternal mortality ratios up to one year for countries where enhanced surveillance of maternal mortality was conducted for late deaths. We calculated age specific maternal mortality ratios and those for women with migrant or minoritised ethnic backgrounds (based on current citizenship, country of birth, or ethnicity) for each country. For age, we also calculated the pooled relative risks and 95% confidence intervals of death by maternal age group, including data from all the countries that used linkage for case identification, with women aged 20-29 as the reference group, using a random effects meta-analysis model with inverse variance weighting and the DerSimonian and Laird method. 24 Finally, in each country, we calculated the proportion of each cause among all maternal deaths, as well as the cause specific maternal mortality ratios per 100 000 live births. We did this for maternal mortality up to 42 days in all countries and for maternal mortality up to one year in countries where enhanced surveillance took place for late deaths. We used Stata v13 for analyses. Data availability Data on the citizenship of women who died and the number of live births according to citizenship were available for five countries: Finland, France, Italy, Norway, and Slovakia (appendix 2). The country of birth of women who died and the number of live births according to country of birth were available for six countries: Denmark, Finland, France, Norway, Slovakia, and the UK. Maternal ethnicity of women who died and the number of live births were available for two countries: the Netherlands (ethnicity reported by healthcare providers) and the UK (self-reported ethnicity). Patient and public involvement No patients were involved in setting the research question, as we thought that discussing the topic of maternal mortality with mothers may be difficult. Being involved in the maternal mortality surveillance systems rather than directly speaking to patients inspired this research. However, although there was no direct patient involvement in this paper, members of the public have read our manuscript. All agreed on the importance of the topic and of the specific findings of this study. results The maternal mortality ratios up to 42 days varied by a multiplicative factor of four between countries (table 2; fig 1). The lowest maternal mortality ratios were in Norway (2.7, 95% confidence interval 1.2 to 5.4) and Denmark (3.4, 1.6 to 6.2) and the highest in the UK (9.6, 8.4 to 11.0) and Slovakia (10.9, 7.4 to 15.5), all per 100 000 live births, with a maximum absolute difference of 8.2 (3.9 to 12.5) per 100 000 live births. Compared with the enhanced systems, vital statistics offices underestimated by 36% or more the number of maternal deaths and the corresponding maternal mortality ratio in every country except Denmark (table 2; fig 1). Enhanced methods for identification and/or review of late maternal deaths (between 43 days and one year after the end of pregnancy) were not available for Denmark, Finland, Italy, the Netherlands, Norway, or Slovakia. In the two countries where enhanced surveillance of maternal mortality extended up to one year, maternal mortality ratios up to one year varied from 10.8 (9.5 to 12.1) in France to 19.1 (17.0 to 21.0) in the UK, per 100 000 live births. The contribution of late deaths to total maternal mortality up to one year was 25% in France and 50% for the UK (fig 1). Age specific maternal mortality ratios were highest for the oldest mothers in all countries except the Netherlands (fig 2; appendix 3). In the pooled analysis, using data from all countries except the Netherlands, women aged under 20 years had a higher risk of maternal mortality than did those aged 20-29 (pooled relative risk 2.17, 95% confidence interval 1.38 to 3.43), as did women aged 35 In Finland and Slovakia, all deaths occurred in women who were both citizens of and born in that country. For the other countries, we observed year after end of pregnancy, in countries with enhanced surveillance systems (listed from lowest to highest maternal mortality ratio up to 42 days). error bars represent 95% confidence intervals. Pregnancy associated deaths are those occurring within 1 year of end of pregnancy, regardless of their cause. maternal deaths are those with cause related to or aggravated by pregnancy or its management but not from accidental or coincidental cause. in norway, Finland, and italy, pregnancy associated deaths are identified up to 1 year after end of pregnancy, but cases occurring between 43 days and 1 year are not reviewed, which is why late maternal deaths were unavailable. in Denmark and slovakia, pregnancy associated deaths are identified only up to 42 days after end of pregnancy; accordingly, pregnancy associated deaths up to 1 year and late maternal deaths were unavailable for these two countries. in the netherlands, results up to 1 year after pregnancy end were considered not reliable as linkage is not used to identify deaths, and they therefore not included. maternal deaths between 43 and 365 days after end of pregnancy disparities in maternal mortality according to where the women were from regardless of the categorisation used, with maternal mortality ratios ≥50% higher in minoritised compared with reference subgroups, except in Norway (fig 3; appendix 4). Among the three countries with data available for citizenship, maternal mortality ratios were higher for women with a foreign nationality in both France and Italy but not in Norway. Women born abroad had a higher maternal mortality ratio than those born in Denmark and in France; this was not the case in either the UK or Norway. In both countries collecting ethnicity data, women who were not white had higher maternal mortality ratios. Major contributors to maternal deaths in all countries were cardiovascular diseases (for maternal mortality up to 42 days and up to one year) (table 3; table 4; fig 4) and suicide (for maternal mortality up to one year) (appendix 5 and 6). In addition, specific conditions contributed notably more to maternal mortality in some countries than in others. These include venous thromboembolism in the UK and the Netherlands, hypertensive disorders in the Netherlands, amniotic fluid embolism in France, haemorrhage in Italy, and stroke in Slovakia. discussion This comparison of maternal mortality data from enhanced national surveillance systems in eight European countries shows that maternal mortality ratios up to 42 days after the end of pregnancy varied by a multiplicative factor of four-from 2.7 and 3.4 per 100 000 live births in Norway and Denmark to 9.6 in the UK and 10.9 in Slovakia. The analysis showed similarities between countries in the subgroups of women at high risk (older and with migrant or minoritised ethnic backgrounds) and with cardiovascular disease and suicide as leading causes of death, but also disparities between countries for other important causes of maternal mortality. importance of enhanced systems Our analysis confirms that national vital statistics registers are not a valid source for surveillance of maternal mortality in high income countries, as previously reported with older data from some of these countries and others. 6 8-10 The substantial underestimation of maternal mortality from vital statistics alone observed in our study underpins that previous reports on national maternal mortality levels worldwide using this source of data for high income countries are not informative for this category of countries. 25 26 This strongly supports the current WHO initiative to integrate data from enhanced surveillance systems in countries where they exist when reporting international comparisons of these deaths. 1 12 The extent of this underestimation may vary between countries, as vital statistics in some countries, such as Finland, did not include suicides as maternal deaths when these data were collected, whereas those in other countries, such as France and the UK, did. Our detailed description of enhanced systems also highlights the major importance of data linkage for maternal death case finding; it is a key component of an enhanced maternal mortality surveillance system. We hypothesise that the singular maternal mortality pattern found in the Netherlands (the only participating country not using linkage) might be explained by the absence of linkage to birth records, which might have resulted in missing some deaths, especially those in the late postpartum period or from non-obstetric causes. Keeping the Dutch data in our analysis illustrates our point on the importance of linkage and shows how some strict data privacy rules (barrier to the implementation of data linkage in this country) might prevent the ascertainment of maternal deaths and the calculation of valid maternal mortality ratios. Receiving official support from national health authorities might help enhanced systems to highlight the specificities of maternal mortality measurement and of the methods needed; as table 1 shows, the two countries with extended enhanced surveillance up to one year after the end of pregnancy are receiving specific funding from national health agencies to run the system. Government support, beyond the visibility it brings, is certainly a guarantee of the quality of the system's operation. Other interesting findings were the heterogeneity between countries in the methods used to study late maternal deaths (those occurring between 43 days and one year after the end of pregnancy), both in terms of case identification or review-not conducted for these late deaths in most countries-and of obviously heterogeneous classification rules, which make interpreting national variations in maternal mortality up to one year difficult. Differences of classification were particularly striking for late deaths from cancer. Deaths from or after trophoblastic tumours were considered maternal in all countries. In France, maternal deaths also included those from hormone sensitive cancers or any cancer for which the pregnancy delayed diagnosis or treatment, whereas the UK's definition of maternal deaths due to cancer ‡Maternal mortality ratios per 100 000 live births (95% confidence interval or one sided 97.5% confidence interval). §Ectopic pregnancy, post-abortion (spontaneous, elective, and medically indicated) haemorrhage, post-abortion infection, post-abortion pulmonary embolism, and other abortive outcomes. ¶Haemorrhage by uterine atony, morbidly adherent placenta, placenta praevia, uterine rupture, placenta abruption, and other obstetric haemorrhage. *Pre-eclampsia, eclampsia, and HELLP syndrome. † †Peripartum cardiomyopathy, ischaemic cardiomyopathy, other cardiomyopathy, other cardiac, aortic dissection, and arterial rupture. ‡ ‡Influenza, pneumonia, meningitis, urinary sepsis, and other indirect sepsis. § §In all countries, deaths after trophoblastic tumours were considered maternal. For other tumours, definitions of maternal death varied between countries. In Denmark, only pregnancy associated deaths (PADs) from breast cancers were considered maternal. In Finland, no formal rules were used to classify PAD as maternal, and cases were discussed individually. In France, maternal deaths were PADs from all hormone sensitive cancers or any other type of cancer for which pregnancy delayed diagnosis or treatment. In Italy, all PADs from cancers of breast, ovary, cervix, skin, blood, or brain were considered maternal. specified cancers of the breast, ovaries, or uterus. In Italy, all deaths from cancers of the breast, ovaries, cervix, skin, blood, or brain were considered maternal. This heterogeneity is likely due to the lack of precision in the ICD-MM guidelines regarding the classification of late maternal deaths. Further standardisation of classification is urgently needed to allow relevant international comparisons of those late deaths and of maternal mortality up to one year to be conducted. 27 All lessons learnt about the importance of enhanced surveillance of maternal mortality in high income countries may also be valuable for low and middle income countries. Differences in maternal mortality profiles between countries We showed that maternal mortality up to 42 days after the end of pregnancy varied by a factor of four among the participating countries. As methods for identifying and classifying maternal deaths up to 42 days were very similar across countries (except for the Netherlands), we believe that the differences in the level and profile of causes of maternal mortality in this time frame are not at all or very little related to measurement variations. Several possible explanations for these variations exist. Firstly, differences could exist in baseline characteristics of pregnant women between countries. Wide variations exist in the proportions, among all births, of younger and older mothers and of mothers with a migrant or minoritised ethnic background (see appendix 7 and 8 for distribution in the different countries). These may partially explain the differences in maternal mortality ratios, as those characteristics were associated with higher risks of maternal death. A standardisation of the sociodemographic variables collected for each maternal death and each birth, not only on geographical origin but also other dimensions of the social status, would help to further explore this hypothesis. Other characteristics not reported in our study, such as body mass index, also vary between countries, and this may be matched with variations in cardiovascular maternal mortality in our study. 28 Alternatively, variations in maternal mortality ratio up to 42 days may reflect differences in the quality of healthcare provided or performance of healthcare systems between countries. Differences in maternal mortality from causes that are not strongly related to individual characteristics, such as haemorrhage or amniotic fluid embolism, may suggest explanatory hypotheses related to care. Finally, organisation of care in large countries with high numbers of births and of maternity units is probably more challenging than for countries with a smaller perinatal community. Future research on individual data is needed to disentangle these non-exclusive explanatory hypotheses. Between country variation of maternal mortality was the key focus of our study, but we acknowledge that national rates may mask within country variations, as previously highlighted in France and in the Netherlands. 29 30 Although the characteristics of women may vary from one region to another, a meaningful national profile of pregnant women exists, and the organisation of care and recommendations for good practice are defined according to a national standard. Furthermore, the appropriate regional categorisation may vary from one country to another; thus, in the two studies cited above, France isolated the overseas territories and the Netherlands the urban regions, in relation to each national question of healthcare organisation. Sticking to the national scale in our analysis therefore seemed justified. similarities in causes of maternal mortality between countries Although differences in the main causes of death were highlighted in some countries, we also noted similarities. In particular, cardiovascular diseases and suicides were leading causes of maternal death in most countries. Although the increasing importance of cardiovascular causes and suicides within the maternal deaths has already been highlighted in individual country reports, the major contribution of our analysis is to compare these country patterns with homogenised data. For example, cardiovascular mortality is the first cause of maternal mortality up to 42 days in France and in the UK, but the specific maternal mortality ratio due to this cause is twice as important in the UK (2.2 v 1.2 per 100 000 live births); the same observation can be made concerning maternal suicide mortality up to one year between these two countries (2.9 v 1.4 per 100 000 live births). This shared pattern underlines the importance of women's mental and cardiovascular health and the need to develop strategies before, during, and after pregnancy to prevent the morbidity and mortality these problems can cause. [31][32][33][34][35][36][37][38] This is a substantial challenge, as the management of these conditions implies an extension of maternal care to coordinate various medical disciplines and levels of care, from preconception to postpartum. Most high income countries are in stage IV or even V of the obstetric transition. 2 In stage IV, indirect causes of maternal mortality become more important, especially those related to non-communicable diseases. Improved quality of care and the elimination of health system delays are needed to reduce maternal mortality further. Stage V is the ultimate goal: the avoidance of all avoidable maternal deaths. At that point, the main concerns would be real progress against structural violence (for example, gender inequality), effective care of vulnerable populations (for example, displaced people, immigrants, and disadvantaged racial, ethnic, and sexual minorities), and finally successfully sustaining this quality of care over the long term. conclusion We report variations in maternal mortality ratios up to 42 days between European countries with enhanced surveillance systems that minimise measurement variability. Although these variations may result from differences in the sociodemographic characteristics of pregnant women between countries, they also raise questions about differential quality of healthcare provided and the performance of health systems. To further reduce maternal mortality by learning from best practices and each other, in-depth analyses of differences in quality of care and health system performance at national levels are needed. Cardiovascular diseases and mental health in women during and after pregnancy need to be prioritised in all countries. authOr aFFiliatiOns 1 National Perinatal Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, UK	2022-11-17T14:05:33.255Z	2022-11-16T00:00:00.000	{ "year": 2022, "sha1": "71401292a23095df75567e8deabdd860eeed62b8", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Highwire", "pdf_hash": "71401292a23095df75567e8deabdd860eeed62b8", "s2fieldsofstudy": [ "Medicine", "Political Science" ], "extfieldsofstudy": [ "Medicine" ] }
21702832	pes2o/s2orc	v3-fos-license	An Empiric Risk Score to Guide PrEP Targeting Among MSM in Coastal Kenya Men who have sex with men (MSM), who have heterogeneous HIV-acquisition risks are not specifically targeted in Kenyan pre-exposure prophylaxis (PrEP) guidelines. We used data from an open cohort, which followed 753 initially HIV-negative MSM participants for more than 1378.5 person-years, to develop an empiric risk score for targeting PrEP delivery. Independent predictors of incident HIV-1 infection in this cohort were an age of 18–24 years, having only male sex partners, having receptive anal intercourse, having any unprotected sex, and having group sex. Poisson model coefficients were used to assign a numeric score to each statistically significant predictor. A risk score of ≥ 1 corresponded to an HIV-1 incidence of ≥ 2.2 [95% confidence interval (CI) 1.2–4.1] and identified 81.3% of the cohort participants as being at high risk for HIV-1 acquisition. The area under the receiver operating characteristic curve was 0.76 (95% CI 0.71–0.80). This empiric risk score may help Kenyan health care providers to assess HIV-1 acquisition risk and encourage PrEP uptake by high-risk MSM. Introduction Pre-exposure prophylaxis (PrEP) is effective in reducing the risk of HIV-1 acquisition [1], and has been recommended by the World Health Organization for populations that have an HIV-1 incidence of 3 per 100 person-years (PY) or higher [2]. In 2016, the Kenyan guidelines for use of antiretroviral drugs for treating and preventing HIV infection recommended PrEP for individuals who have significant risk for acquiring HIV-1 infection [3]. Although "significant risk" in the Kenyan PrEP guidelines is defined as reporting a wide range of risk behaviors, anal intercourse is not specifically mentioned. In May 2017, Kenya became the second country in Africa-the first was South Africa-to roll out PrEP nationally through the National AIDS & STI Control Programme (NASCOP) [4,5], thereby making PrEP available to individuals who meet eligibility criteria in the published guidelines. The Kenya PrEP program targets individuals who report any of the following: a sexual partner who is HIV-1 positive or has unknown HIV-1 status, transactional sex, a recent sexually transmitted infection (STI), recurrent use of postexposure prophylaxis, having sex while under the influence of alcohol, inconsistent condom use, and injection drug use during which needles and syringes are shared [3]. Although many men who have sex with men (MSM) may meet these eligibility criteria, the PrEP guidelines do not specifically target known risk factors for HIV-1 acquisition among MSM, including condomless anal intercourse, group sex, and the biological sex of sexual partners. High HIV-1 and STI risks have been documented among adult male sex workers and MSM in Kenya [6][7][8][9][10], with estimates of HIV-1 incidence as high as 35.2 [95% confidence interval (CI) 23.8-52.1] per 100 PY among MSM who have sex with men only [7]. Receptive anal intercourse (RAI) 1 3 and group sex have also been identified as independent predictors of HIV-1 acquisition in this population. The risk of acquiring HIV-1 was eightfold to tenfold higher among MSM who reported having RAI, compared with those who did not [7,9], and was twofold higher among MSM who reported having group sex, compared with those who did not [7]. Targeting PrEP initiation among MSM at highest risk for HIV-1 acquisition would optimize any impacts on HIV-1 transmission [11,12], if these men can be reached and supported. In a secondary analysis of the iPrEX trial, MSM and transgender women who practiced RAI without a condom in the 3 months before enrollment accounted for 64% of new infections in this high-risk cohort [13]. Several screening tools have been developed to identify those at high risk for HIV-1 acquisition, including serodiscordant couples [14,15], young women [16], sexually active men and women [17], and pregnant and postpartum women [18] in Africa. While tools to assess HIV-acquisition risk among MSM have been developed for use in well-resourced settings [19][20][21][22][23], no risk scoring tool is presently available to guide PrEP uptake by MSM in sub-Saharan Africa. As current PrEP eligibility criteria in Kenya's NASCOP guidelines have not been validated among this population, we set out to develop an empiric risk score to assess HIV-1 acquisition risk among MSM in coastal Kenya. We envisaged that this risk score could help Kenyan health care providers to target PrEP initiation among high-risk MSM. Study Design, Setting, and Participants Since July 2005, individuals residing on the Kenyan coast who are at high risk for HIV-1 acquisition have been prospectively recruited for an open cohort study on HIV-1 vaccine trial feasibility. Participants were identified for recruitment into the study by 10-15 trained peer mobilizers who approached individuals through personal networks and at venues where they meet to establish contact with sexual partners and clients [7]. Men aged 18-49 years were eligible if they reported having had anal sex with a man in the 3 months prior to enrollment [7]. Study Procedures Detailed cohort procedures are described elsewhere [7]. In brief, enrollment and follow-up included a face-to-face interview using a standardized risk-behavior questionnaire, HIV-1 testing and counseling, risk-reduction counseling, medical history, and physical examination at each visit. Through 2015, follow-up was quarterly for most participants and monthly for men who reported having RAI. Beginning in 2016, all participants had monthly follow-up visits. Treatment for genital symptoms suggestive of STIs and minor illnesses was provided free of charge, and hepatitis B vaccination was provided from 2009 onward [24]. Patients who had genital symptoms were treated syndromically, and laboratory-diagnosed infections were treated according to Kenya Ministry of Health recommendations. Since 2016, participants who had a gonorrhea infection were given directly observed treatment with oral cefixime (400 mg) and azithromycin (2 mg). Free condoms and water-based lubricants were also provided at each visit. Laboratory Methods HIV-1 testing was performed at each study visit using two rapid antibody test kits (Determine, Abbott Laboratories; Unigold, Trinity Biotech) in parallel. Discordant rapid HIV-1 test results were resolved using an enzyme-linked immunosorbent assay (ELISA) (Genetic System HIV-1/2 plus O EIA, Bio-Rad Laboratories). All HIV-1 negative samples were tested for p24 antigen (Vironostika HIV-1 p24 ELISA, Biomérieux) through 2015, or for HIV-1 RNA (Xpert ® HIV-1 Qual, Cepheid) since 2016. Pre-and post-seroconversion samples were tested for HIV-1 RNA level (Amplicor Monitor 1.5, Roche). Gonococcal infection among participants who reported urethral or rectal symptoms was defined as the detection of gram-negative, intracellular diplococci consistent with Neisseria gonorrhoeae in urethral or rectal secretions [7]. Prevalent syphilis infection was diagnosed using a positive rapid plasma reagin (RPR, tested annually) titre confirmed by Treponema pallidum hemagglutination assay (TPHA). Incident syphilis was defined as a fourfold increase in RPR titre confirmed by TPHA [7]. Measures Four risk behaviors that had been previously associated with HIV-1 acquisition among this population were the primary predictors [7]. The first-sexual activity and condom use in the past week-was categorized in three groups: "no activity," when no sex was reported in the past week; "all protected," when the number of sexual acts (either vaginal or anal intercourse) for which a condom was used was equal to the number of sexual acts reported; and "any unprotected," when the number of sexual acts for which a condom was used was lower than the number of acts reported. For the second variable, men were asked at each visit to report whether they had been sexually active with men, women, or both men and women in the past 3 months or since their last visit. This variable was categorized as "men only," "both men and women," and "women only." The third and fourth variables-having RAI and having group sex (defined as sex 1 3 with more than one person at the same time)-were assessed for the past 3 months, or since the last visit, and categorized as either yes or no. Other variables evaluated as potential predictors of HIV-1 acquisition included sociodemographic data collected at enrollment (age, education level, religion, marital and employment status) and time-updated data collected at each study visit (specific sexual behaviors, transactional sex [2 variables, defined as either paying for or being paid for sex with cash, living expenses, or goods], alcohol use, having sex after alcohol use, intravenous drug use, sharing needles among intravenous drug users, use of post-exposure prophylaxis, medical injections, urethral discharge or dysuria, rectal discharge, circumcision status, and self-reported genital sores). Data Analysis and Statistical Methods Descriptive statistics were used to summarize baseline sociodemographic and behavioral characteristics of MSM enrolled in the study. Data for each participant were censored at the end of 2016, the last visit for those who became lost to follow-up, or, for those who acquired HIV-1 infection during follow-up, the last seronegative and HIV-1 RNA-negative visit. Total observation time was obtained by adding up separate observation times for all participants in the study, and expressed in terms of PY. HIV-1 incidence rates were calculated as the number of HIV-1 incidence cases divided by PY of follow-up, and expressed as incidence per 100 PY. All potential predictors were assessed for association with incidence of HIV-1 infection using Poisson models with robust standard errors, to obtain population-averaged incidencerate ratios that accounted for correlation due to repeated measurements of the same subject over time. Variables significant at P ≤ 0.1 in bivariable analysis were included in an initial multivariable model of potential predictors for HIV-1 acquisition. Variables with P > 0.1 in the initial multivariable model were then dropped to produce a final multivariable model. Development of Empiric Risk Score Predictors in the final multivariable model were used to develop an empiric, model-based risk score, after excluding religion and insertive anal intercourse (IAI) (both of which were negatively associated with HIV-1 acquisition and would have led to negative risk scores for some men). A risk score was assigned to each statistically significant predictor for HIV-1 acquisition, based on its coefficient in the risk score model, and rounded to the nearest integer. Risk scores were then summed to generate predictor scores at a given visit for each participant. Crude HIV-1 incidence was calculated by different risk score cutoff values (e.g., ≥ 0, ≥ 1) to determine which risk score cutoff value correlated most closely with the HIV-1 incidence of ≥ 3 per 100 PY recommended for PrEP initiation. To calculate the proportion of MSM who should be targeted for PrEP based on this risk score cutoff, we assessed risk over the previous 3 months at the last follow-up visit, as this would best reflect current risk behavior. We assessed whether higher risk scores correlated with higher HIV-1 incidence using a nonparametric test for trend. We evaluated risk score performance by assessing sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). P values were 2-sided, and significance was set at P ≤ 0.05. Data were cleaned, recoded, and analyzed using Stata 15.0 (StataCorp LLC, College Station, Texas, USA). Ethical Considerations The Kenya Medical Research Institute Ethics Review Committee approved this study. All participants provided written informed consent. Study Population and Follow-up Duration A total of 753 HIV-1 negative MSM had at least 1 follow-up visit and contributed 1378.5 PY (median 14.3, interquartile range 5.6-31.8 months). Table 1 presents the sociodemographic characteristics of the study population. At enrollment, the majority were 18-24 years of age and had never been married. At this visit, 314 (41.7%) reported having only male sex partners, 451 (59.9%) reported having any unprotected sex in the past week, 523 (69.5%) reported having RAI, 523 (69.5%) reported having IAI, and 121 (16.1%) reported having group sex in the past 3 months. Thirty percent had paid for sex in the past 3 months, and nearly 70% had received payment for sex. Almost two-thirds had used alcoholic beverages in the past month, and more than half had had sex after using alcohol. The vast majority (94.2%) were circumcised, 92 (12.2%) reported urethral discharge or dysuria, 37 (4.9%) reported rectal discharge, and 11 (1.5%) had a prevalent gonorrhea infection diagnosed at enrollment. HIV-1 Incidence Estimates and Risk Factors for HIV-1 Acquisition Ninety-seven MSM acquired HIV-1 during follow-up, for an estimated HIV-1 incidence rate of 7.0 (95% CI 5.8-8.6) per 100 PY. In bivariable analysis, HIV-1 acquisition was associated with age (18-34 years), never being married, not being Muslim, and with reporting at a study visit having only male sex partners, having any unprotected sex, having RAI, having Table 3 presents the risk score model, which excluded religion and IAI for reasons explained above. A risk score of 1, based on coefficients, was applied for each of these independent predictors of HIV-1 infection: younger age (18-24 years), having only male sex partners, having RAI, having any unprotected sex, and having group sex (Table 3). While the maximum score was 5, only a very small proportion (~ 1.0%) of MSM visits were in this category (data not shown). We combined visits that had risk scores of 4 and 5, to avoid wide confidence intervals due to sparse data. A risk score cutoff of ≥ 1 corresponded to an HIV-1 incidence of ≥ 2.2 (95% CI 1.2-4.1) per 100 PY (Table 4), and had a sensitivity of ≤ 97.9% and a specificity of ≤ 16.9% for detecting the visit at which a participant had acquired HIV-1. If this cutoff is applied to predictors assessed at the last visit of men participating in the cohort, approximately 81.3% of MSM would be identified as high-risk individuals who should be targeted for PrEP initiation. The AUC for predictive ability of the risk score was 0.76 (95% CI 0.71-0.80), indicating fair to good performance in identifying men who eventually seroconverted. A unit increase in risk score strongly correlated with an increase in observed HIV-1 incidence (P < 0.001, test for trend) (Fig. 1). Discussion We developed an empiric risk score for PrEP targeting of MSM based on 5 predictors of HIV-1 acquisition derived from our MSM cohort. Adding 1 point for each of the predictors present-having only male sex partners, RAI, any unprotected sex, group sex, and young age (18-24 years)-led to a score that strongly correlated with increased HIV-1 incidence and had a fair to good predictive ability. By applying this risk score to MSM at their last cohort visit, we identified more than 80% of MSM as being at high risk for HIV-1 acquisition, with scores corresponding to an HIV-1 incidence as high as 4.1 per 100 PY (upper limit of our confidence interval). Importantly, this tool identified 86 men (11.4% of the cohort) as being at high risk for HIV-1 acquisition yet they did not meet PrEP eligibility criteria per Kenyan guidelines. In Kenya, PrEP guidelines do not specifically target individuals who report having RAI, only male sex partners, or group sex-predictors that were strongly associated with HIV-1 acquisition among MSM in coastal Kenya [7]. While many MSM do qualify for PrEP based on other risk behaviors, the application of our risk score, which includes young age and sexual behaviors that increase vulnerability among MSM, will assist providers in identifying those who are likely to benefit from PrEP initiation; therefore, our model should be beneficial to HIV-1 prevention programming in Kenya. In Kenya, homosexuality is illegal and punishable by law [25]. MSM are stigmatized and face challenges in accessing health care services [26]. Although efforts have been made to train health care workers on the needs of MSM, trained providers themselves have reported feeling stigmatized by community members and other health care staff [27]. Despite these challenges, more than 1200 health care providers have taken an MSM sensitivity training that is freely available online (www.marps -afric a.org) [28], and various programs engage MSM for PrEP programming in Kisumu, Nairobi, and coastal Kenya. Unfortunately, stigma surrounding male-male sex and RAI may have led to the omission of these factors as risk criteria in the Kenyan guidelines. The empiric risk scoring tool had fair to good performance (AUC 0.76) in detecting HIV-1 among MSM in our cohort. Although the scoring tool has not been validated elsewhere, our aim was to develop a tool that could guide health care providers in both targeting MSM at highest risk for HIV-1 acquisition and discussing the risks that should prompt PrEP initiation. This scoring tool could potentially be used by clinicians and counselors to reevaluate PrEP eligibility among MSM already on PrEP and guide a discussion on continuation or discontinuation. Outside the clinical Our study confirmed that having RAI, any unprotected sex, and group sex remained independent predictors for HIV-1 acquisition among our cohort, similar to the period of 2005-2011 [7]. We found a twofold higher incidence of HIV-1 acquisition among men who reported having only male sex partners, compared with men who reported both male and female partners, which was consistent with our previous reports [10]. MSM aged 18-24 years had a fourfold higher incidence of HIV-1 acquisition, compared with men older than 34 years. Elsewhere, in a Bangkok cohort, young MSM (aged < 21 years) had an increased risk for HIV-1 acquisition [29,30]. Men who reported having IAI were less likely to acquire HIV-1 infection than men who did not report IAI (5.5 vs 9.0 per 100 PY) in our study (Table 2). Although we did not specifically assess whether condoms were used for IAI, with whom IAI was practiced, or the frequency of IAI versus RAI, a protective association for IAI remained after adjusting for other risk factors for HIV-1 acquisition among our cohort. This finding is consistent with a lower per-act risk for HIV-1 transmission via IAI, compared with the risk via RAI [31], and may also result from a protective effect of circumcision during IAI [32]. We found that MSM who were Muslim had a lower HIV-1 incidence than MSM who were Christian (4.2 vs 8.7 per 100 PY, Table 2), which has not been reported previously [7]. Why MSM who are Muslim have lower HIV-1 acquisition risks is not clear. In our risk score model, we did not assign a negative risk score for MSM who reported having IAI or for MSM who were Muslim, as both groups already had substantial HIV-1 acquisition risks. As national programs target key populations for PrEP initiation in Kenya, our risk score model may guide health care CI confidence interval, IRR incidence rate ratio, aIRR adjusted incidence rate ratio a "Sharing needles among intravenous drug users," "use of post-exposure prophylaxis," and "syphilis infection" excluded, as no HIV-1 acquisitions occurred in the exposed categories b Only factors significant at P ≤ 0.1 in the bivariable analysis were included in the initial multivariable model c Only factors significant at P ≤ 0.1 in the initial multivariable model were retained in the final multivariable model providers in discussing specific behaviors that put MSM at increased risk for HIV-1 acquisition and are not included in the current NASCOP criteria for PrEP initiation. MSM with a risk score of ≥ 1 should be made aware of their elevated risk for HIV-1 acquisition and encouraged to consider PrEP as an additional risk reduction strategy. Additionally, they should be evaluated for symptoms of acute HIV-1 infection before PrEP initiation [33,34], in accordance with Kenyan national guidelines [3]. MSM who have an empiric risk score of < 1 may be willing to take PrEP and may qualify for PrEP according to Kenyan guidelines for other factors (e.g., an ongoing relationship with an HIV-1-positive partner, transactional sex, or injection drug use with a needle-sharing partner) [3]. Our risk score model is intended as a supplement to the Kenya guidelines, not as a replacement for them. Our study had several limitations. First, we did not collect data on partner characteristics, duration of partnerships, partner's HIV-1 status, or condom use by the participant or partner specifically during anal sex. Second, self-reported sexual behavior is subject to recall and social desirability biases, and may have led to underreporting of risky sexual behaviors during risk assessment. Third, our STI assessment was restricted to symptomatic patients (having urethral discharge or dysuria or having rectal discharge) only, so we were unable to detect asymptomatic infections. Fourth, the time-updated PrEP eligibility score reflects risks assessed over the previous 3 months. As risk is not static, PrEP eligibility should be assessed and periodically updated over time. Fifth, while we have identified factors associated with risk among this cohort, their importance and relative Conflict of interest The authors declare that their research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. This paper has not been submitted elsewhere. Preliminary results were presented at the Conference on Retroviruses and Opportunistic Infections (CROI) 2017 (#1601). Ethical Approval 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. Informed Consent Informed consent was obtained from all individual participants included in the study. 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.	2018-05-21T21:28:04.077Z	2018-05-16T00:00:00.000	{ "year": 2018, "sha1": "c3d385beb0e38a624184a50f666d062976fe9ee9", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10461-018-2141-2.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "a269435f862d9ef8add93f42dd9046eb0f72aed8", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
247156130	pes2o/s2orc	v3-fos-license	Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter. Supplementary Information The online version contains supplementary material available at 10.1007/s10577-021-09679-w. Introduction Eukaryotic replication origins are defined by origin recognition complex (ORC)-dependent loading of the Mcm2-7 helicase complex onto chromatin in the G1 phase (Bleichert et al. 2017). After loading onto chromatin, Mcm2-7 complexes are significantly redistributed across chromosomes and cover a substantial part of the genome with gaps within active transcription zones (Powell et al. 2015). Each potential replication initiation origin has certain efficiency and characteristic activation time. The origin activation time and efficiency are not related directly. Some later-activated origins are efficient, and others are not. Some origins are inefficient due to their proximity to earlier origins, while others are inefficient by themselves (Raghuraman et al. 2001;Weinreich et al. 2004). Origin efficiency in yeast varies widely and can reach 90% (Raghuraman et al. 2001;Heichinger et al. 2006). In metazoans, origins are much less efficient. Wellcharacterized origins fire in 5-20% of cells (Lebofsky et al. 2006;Hamlin et al. 2008). This low efficiency indicates that replication initiation events are probabilistic, and that their distribution may vary between different cell types and among subsequent cell cycles (Méchali 2010;Rhind et al. 2010;Herrick 2011;Wang et al. 2021). In recent years, several powerful approaches emerged that allowed genome-wide analysis of the probabilistic nature of replication initiation (Löb et al. 2016;Dileep and Gilbert 2018;Wang et al. 2021;Massey and Koren 2021). It is assumed that almost any site of the genome can initiate replication, but ORC assembly sites are considered the most efficient potential replication initiation sites (Wu and Nurse 2009;Borowiec and Schildkraut 2011;Gros et al. 2015;Powell et al. 2015;Miotto et al. 2016;Petryk et al. 2016). Usually, potential replication origins cluster to form so-called replication initiation zones. In humans, their median size is ~40 kbp (Tao et al. 2000;Dijkwel et al. 2002;Anglana et al. 2003;Hamlin et al. 2008;Borowiec and Schildkraut 2011;Lubelsky et al. 2011;Mesner et al. 2011;Bechhoefer and Rhind 2012;Besnard et al. 2012;Demczuk et al. 2012;Mesner et al. 2013;Petryk et al. 2016;Wang et al. 2021). When the first origin is activated within a cluster, neighboring origins become inactivated via interference with an extension of up to 100 kbp (Lebofsky et al. 2006). Drosophila salivary gland polytene chromosomes are composed of 2 × 1024 DNA strands representing both homologues. All strands are cohesively aligned in parallel. The visible band/interband pattern reflects the genome organization (Zykova et al. 2018). Interbands are the most decondensed regions with a specific protein composition conserved across tissues (Demakov et al. 2011, Zhimulev et al. 2014). According to their morphology, bands can be classified into gray and black. The most compact "black" bands, which look uniformly dense, even in sections imaged by electron microscopy (Fig. 1a, b), show a chromatin condensation ratio of 1:200 (Spierer and Spierer 1984;Kozlova et al. 1994;Vatolina et al. 2011;Zhimulev et al. 2014). After 4′,6-diamidino-2-phenylindole (DAPI) staining, these bands appear as the brightest ones ( Fig. 1c) and correspond to clusters of tissuespecific genes. Besides, they contain silent chromatin and are almost devoid of ORC-binding sites (Zhimulev et al. 2014;Kolesnikova et al. 2018). The 4-chromatin-states model of Zhimulev et al. (2014) was established based on the bioinformatic analysis of the distribution of interband-specific chromatin proteins in cell cultures (modENCODE Consortium et al. 2010). The Drosophila genome has been divided into four chromatin types formerly referred to as cyan, blue, green, and magenta (Zhimulev et al. 2014). Later, these four chromatin types were renamed as follows: aquamarine (cyan), lazurite (blue), malachite (green), and ruby (magenta). The genomic distribution of the four chromatin types is closely associated with the polytene chromosome morphology and allows to predict the genomic coordinates of polytene chromosome band boundaries with high accuracy because 1l experimentally characterized interbands coincide with aquamarine chromatin. Different combinations of the three other chromatin types correspond to different classes of bands (Zhimulev et al., 2014, Boldyreva et al. 2017, Demakova et al., 2020. The presence of ruby chromatin indicates a compact black band. Bands that do not contain ruby chromatin are referred to as gray bands . The chromosomal regions between rb-bands are the alternation of thinner and less compact so-called gray bands and mainly decondensed interbands (Fig. 1b), which together form intervals (INTs) ). INTs represent "open" chromatin and are enriched in genes expressed in different tissues. The promoters of these genes lie mainly within interbands, but the coding sequences within gray bands. According to cytological studies, the first replication marks during earliest S phase appear in INTs Lakhotia 1979, 1981;Mishra and Lakhotia 1982;Kolesnikova et al. 2018). More than 90% of ORC2-binding sites are situated in aquamarine chromatin, primarily in INTs (Sher et al. 2012;Zhimulev et al. 2014;Kolesnikova et al. 2018). It seems that the ORC2-binding sites are markers of potential replication origins, although it is assumed that some replication initiation events may occur outside the ORCbinding sites in Drosophila and human (Eaton et al. 2011;Gros et al. 2015;Powell et al. 2015;Miotto et al. 2016;Petryk et al. 2016). Via immunostaining, the presence of the prereplication complex component DUP/Cdt1 was shown in all INTs analyzed along all polytene chromosomes (Belyaeva et al. 2012). Thus, all observations suggest that the vast majority of potential replication origins are located in INTs. The visualization of replication in Drosophila polytene chromosomes by conventional wide-field (WF) microscopy indicates a well reproducible set of labeling patterns (Fig. 1f) (reviewed by Zhimulev 1999) reflecting the spatiotemporal organization of replication . At the beginning of the S phase, replication occurs in INTs. Then, continuous replication progresses until all chromosomes, except the middle parts of the thickest rb-bands, are labeled. Afterwards, an inversion of the pattern occurs: all rb-bands are marked but not the INTs. Finally, the rb-bands finish the replication. The more DNA an rb-band contains, the later is replication completed (Fig. 1f) (Zhimulev et al. 2003a;Kolesnikova et al. 2018). Intercalary heterochromatin bands and pericentromeric heterochromatin continue to replicate until the end of the S phase and remain under-replicated in salivary gland polytene chromosomes (Lakhotia 1974;Zhimulev et al. 1982Zhimulev et al. , 2003b. Proliferating cell nuclear antigen (PCNA) is a DNA clamp acting in eukaryotic cells and essential for replication (Moldovan et al. 2007). On the basis of PCNA immunostaining, Kolesnikova et al. (2018) analyzed the replication schedule of the entire chromosome arm 2R by WF microscopy. Additionally, an algorithm for mapping rb-bands to Drosophila genomic coordinates was developed . This helped to compare replication timing between polytene chromosomes of salivary glands and chromosomes from cultured diploid cell lines. Substantial similarities in the global replication patterns were observed between the two tissues. In that paper, we proposed the following model. In general, the spatiotemporal replication process is closely related to the genome organization into two types of domains corresponding well to the polytene chromosome structures: rb-bands and the INTs in between of them. INTs correspond to early replication initiation zones ( Fig. 1e-g). The activation of replication in these zones occurs in time and space stochastically. On each individual chromatin strand, every interband belonging to an INT contains a potential replication origin. But only one of these potential origins (randomly chosen) activates replication in each cell cycle ). This means that in polytene chromosomes inside one INT, different DNA strands initiate replication at different positions. We have indirect evidence for such a scenario: (1) In nuclei of cultured cells, INTs have an average replication profile corresponding to replication initiation zones in which replication is probabilistic in every replication cycle . (2) The characteristic replicon size is ~100 kbp, which is more than the average size of INTs (~30 kbp). This observation indicates that INTs cannot initiate replication on average more than once per cell cycle. (3) INTs have several interbands containing potential replication origins. That is, they correspond to the replication initiation zones, where (4) spatial replication asynchrony between DNA strands was observed in partially digested polytene chromosomes (Lakhotia and Sinha 1983). To test this replication scenario directly in polytene chromosomes, here we used spatial super-resolution structured illumination microscopy (3D-SIM) as an efficient approach allowing multicolor detection of replication sites. In mammalian nuclei, the replication sites detected by 3D-SIM represent individual replicons (Schermelleh et al. 2008;Baddeley et al. 2010;Chagin et al. 2016). It is challenging to analyze replication initiation in polytene chromosomes of wild-type nuclei because they replicate asynchronously. Therefore, we synchronized salivary glands by inducing S phases via ectopic expression of cyclin E by using a D. melanogaster line carrying the hsp70-CycE transgene (Knoblich et al. 1994;Duronio and O'Farrell 1995;Su and O'Farrell 1998). By means of induced-S-phase analysis, 5-ethynyl-2′-deoxyuridine (EdU) replication detection, and super-resolution analysis, in the present study, we confirm the hypothesis of stochastic replication initiation in salivary glands at the ultrastructural level. We demonstrate that the multifilament nature of polytene chromosomes provides unique opportunities for visualizing stochastic processes within chromatin. Flies Flies were reared on enriched semolina (36 g/l) medium with raisins at 18 °C. The Oregon R (Bloomington Stock Center) stock served as a control. The Vol.: (0123456789) hsp70-CycE line was kindly provided by C. Lehner (University of Zurich, Switzerland) and by P. O'Farrell (UCSF, USA). Additionally, we constructed the hsp70-CycE; SuUR line. In contrast to wild-type chromosomes, intercalary and some regions of pericentromeric heterochromatin remain not under-replicated in SuUR mutants (Belyaeva et al. 1998). EdU incorporation and detection Actively moving wild-type third instar larvae were employed for replication analysis because they have a higher percentage of nuclei in S-phase stages. To analyze replication in induced S phase, larvae shortly before pupation were used, when the last larval S phase is already finished (Zhimulev et al. 2003a;Kolesnikova et al. 2013) and many nuclei enter the additional induced S phase. This approach provided an additional round of replication. Salivary glands were dissected and stored in 1 × PBS (137 mM NaCl, 3 mM KCl, 8 mM NaH 2 PO 4 , 2 mM KH 2 PO 4 ). EdU incorporation was carried out in a 4 μM EdU solution in 1 × PBS for 10 min. Salivary glands were placed into 1 × PBS supplemented with 0.1% of Tween 20 (1 × PBST) for 2 min incubation, transferred to a formaldehyde-based fixative (2% NP40 and 2% formaldehyde in 1 × PBS) for 2 min incubation, incubated in an acetic acid-formaldehyde mixture (45% acetic acid, 3.2% formaldehyde) for 1.5 min, and squashed in 45% acetic acid. The squashes were snap-frozen in liquid nitrogen, and the coverslips were removed. The slides were stored in 70% ethanol at −20 °C. For EdU detection, the Click-iT™ EdU Alexa Fluor™ 555 Imaging Kit (Thermo Fisher Scientific) was used. The slides were washed first in 1 × PBS for 20 min, incubated in PBST with 0.1 BSA for 30 min, and then treated with a reaction cocktail at room temperature for 30 min (40 μl/slide). After three 5-min washes in 1 × PBST, the slides were air-dried by means of a rubber syringe, and the squashes were mounted in the VectaShield (Vector Laboratories) medium containing 1.5 µg/ml DAPI. Salivary glands of third instar hsp70-CycE; SuUR larvae were dissected in 1 × PBS solution 70-90 min after 30 min heat shock, incubated with EdU, and Fig. 1 The spatiotemporal organization of replication in polytene chromosomes is closely related to the two main chromatin domain types present in polytene chromosomes: rb-bands and intervals (INTs) between them. a The classic photographic map of polytene chromosome 2R, on which dark "black" bands are clearly visible after aceto-orcein staining and phase contrast microscopy (Lefevre 1976). b A segment of chromosome 2R [an electron micrograph from the classic map of polytene chromosomes from Saura (1986); available in FlyBase: https:// wiki. flyba se. org/ wiki/ FlyBa se: Maps]. Between the black bands are regions where decondensed gray bands (red arrows) and interbands (black arrows) alternate, together forming intervals (INTs). For two black bands and one INT between them, the sizes are indicated below according to genomic coordinates of these structures . c When stained with fluorescent dyes, black bands appear as the brightest regions on the polytene chromosomes (DAPI staining, 3D-SIM, this study). Gray band/interband structures in the INTs are faintly visible when stained with DAPI. d The Zhimulev et al. (2014) model of four states of chromatin (ruby, aquamarine, malachite, and lazurite) makes it possible to predict the localization of black bands in the genome based on the distribution of chromatin characteristics for black bands (chromatin called "ruby": rb-bands). All interbands are associated with aquamarine chromatin, but not all aquamarine regions are interbands (Zhimulev et al. 2014;Boldyreva et al. 2017). The presented region corresponds to that in b. e A scheme of a polytene chromosome fragment bearing three black bands. Median lengths (in kilobase pairs) of black bands are shown, as are the intervals between them according to Kolesnikova et al. (2018) and the median sizes of replicons and replication initiation zones according to Lakhotia and Sinha (1983) and Schwaiger et al. (2009). f The scheme of replication patterns in polytene chromosomes and a model explaining the progression of these patterns [adapted from ]. Consecutive changes in PCNA-binding patterns (red) during the S phase. Three substages of the S phase are presented. From top to bottom: (i) early S phase, when INTs are labeled exclusively; (ii) middle S phase, when all rb-bands are labeled; and (iii) late S phase, when only the thickest rb-bands are labeled. g The scheme of replication fork locations and the respective PCNA/EdU patterns in the INT zone of alternating gray bands and interbands and in an rb-band (black). The arrows indicate the directions of replication fork progression ◂ placed in a fixative (96% ethanol: acetic acid, 3:1) for 4-5 h. Slides were squashed in 45% acetic acid, frozen in liquid nitrogen, the coverslips were removed, and the slides were stored in 70% ethanol at −20 °C. Slides were treated with RNAse A. For this, slides were washed in 2 × SSC (0.3 M NaCl, 0.03 M sodium citrate) 3-times for 5 min and incubated with RNAse A (100 μg/ml in 2 × SSC, 30 μl/slide) for 1 h at 37 °C and washed in 2 × SSC for 5 min. Then, slides were incubated in 2 × SSC for 1 h at 60 °C, denatured in 0.07 M NaOH, passed through a series of ethanol (70, 90, 96%) for 5 min, and air-dried. Afterwards, to detect EdU, slides were treated as described above. After the final washing, the hybridization mixture was added to the slides. To prepare this mixture, 4 μl of a labeled probe, 5 μl of water, and 1 μl of a calf thymus DNA solution were mixed in a tube and heated at 95 °C for 5 min, then cooled and centrifuged. To the same tube, 20 μl of warm hybridization solution (50% formamide, 2 × SSC, 10% dextran sulfate) was added, and then 30 μl of this mixture applied per slide. The hybridization was performed for 1.5-2 days in a humid box at 37°C. The unbound probe was removed by three 15-min washes in 0.2 × SSC with a gradual increase in temperature (42-60 °C). Slides were mounted in VectaShield (Vector Laboratories) containing 1.5 μg/ml DAPI. Indirect immunostaining For polytene chromosome immunostaining, salivary glands (genotypes are specified in the text) were dissected in 1 × PBS supplemented with 0.1% of Tween 20. The glands were then transferred into a formaldehyde-based fixative (0.1 M NaCl, 2 mM KCl, 10 mM NaH 2 PO 4 , 2% of NP40, 2% of formaldehyde) for 1 min incubation. Next, the salivary glands were placed in an acetic acid-formaldehyde mix (45% acetic acid, 3.2% formaldehyde) for 1 min incubation and squashed in 45% acetic acid. For combined immunostaining and EdU labeling, the glands were first labeled with EdU as described above, then the preparations were first stained with antibodies, and afterwards, EdU was detected as described above. Super-resolution microscopy To analyze the ultrastructural organization of replication beyond the classic Abbe-Rayleigh limit of ~250 nm, 3D-SIM was performed to achieve a lateral resolution of ~140 nm (super-resolution, attained with a 561 nm laser). We used an Elyra PS.1 microscope system equipped with a Plan-Apochromat 63×/1.4 oil objective and the ZENBlack software (Carl Zeiss GmbH). Image stacks were captured separately for each fluorochrome by means of 405 nm (DAPI), 488 nm (Alexa Fluor 488), and 561 nm (Alexa Fluor 555) laser lines for excitation with appropriate emission filters (Weisshart et al. 2016). Zoom-in sections are presented as single slices to detect the subnuclear chromatin structures at the super-resolution level. Movies were prepared based on 3D-SIM image stacks using the Imaris 9.7 (Bitplane) software. To investigate the spatial chromatin ultrastructure, 3D-SIM was performed on ~30 optical sections from ~3 μm thick chromosomes. By significantly increasing the resolution, 3D-SIM showed clearly more structural details in comparison with WF microscopy, as demonstrated by imaging of different replication patterns during S-phase progression ( Supplementary Fig. 1a, b). Orthogonal projections indicated that the polytene chromosomes preserve their cylindrical shape after the fixation applied. The EdU signals were conspicuous within the chromosomes, both in decondensed regions (the pattern characteristic for early replication) and inside compact bands (the pattern characteristic for late S phase; Supplementary Fig. 1c). Thus, to analyze the replication patterns in more detail, we employed exclusively 3D-SIM in this work. S-phase induction To induce S phases in salivary glands, cyclin E was expressed ectopically (Knoblich et al. 1994;Duronio and O'Farrell 1995;Su and O'Farrell 1998) in hsp70-CycE; SuUR third instar larvae. To obtain more simultaneously induced S phases in salivary glands, the hsp70-CycE; SuUR larvae were heat shocked at the end of the 3rd instar, a period with a minimum of S-phase cells (Kolesnikova et al. 2013). First, we followed previous protocols (Duronio and O'Farrell 1995;Su and O'Farrell 1998) and applied a heat shock at 37 °C for 30 min. Ca. 30 larvae were analyzed by incorporating EdU into the salivary glands isolated 50 min-6 h after the heat shock. After 70 min, we found in some preparations a slight enrichment of nuclei at very early S phase. In glands prepared 2-3 h after heat shock, the proportion of labeled nuclei reached 100%. However, due to the non-simultaneous entry of the cells into S phase, no high synchronization could be achieved. It was published that after the 37 °C heat shock, the cells do not recover synchronously after the transcription block, but at 35 °C, transcription becomes restored at the end of the heat shock (Kutskova and Mamon 1995;Gong and Golic 2006). After the 35 °C (for 30 min) heat shock, all analyzed salivary glands isolated 70 min after heat shock showed stable groups of nuclei at very early S phase. Ninety minutes after heat shock, all nuclei were labeled, what never occurred in glands without heat shock. In total, more than 70 slides 70-90 min after 35°C heat shock were analyzed by WF microscopy. Besides, we analyzed by WF microscopy slides after 10 min EdU incorporation into the salivary glands isolated 2, 3, 4, 5, 6, 7, and 8 h after heat shock (about 10 slides per moment) from hsp70-CycE; SuUR larvae. Up to 3 h after heat shock, the induced S-phase dynamics could be observed. The nuclei with induced S phase were distinguished from the others by the predominance of them at the same early S-phase stages (early and early-middle). When analyzing glands isolated 3 or more hours after heat shock, we found an enrichment of stages where all compact bands of the polytene chromosomes were stained (middle S phase). But we could not identify nuclei related to induced and non-induced S phase. In addition, the high percentage of nuclei at middle S phase in larvae 7-8 h after heat shock suggested that the S phase is inhibited, as this pattern is similar to the pattern after hydroxyurea treatment (Kolesnikova et al. 2013). Thus, we showed that our approach is suitable for studying early S-phase stages, but not for analyzing the S-phase timing more than 2-3 h after S-phase initiation. To investigate the reproducibility of early replication patterns after S-phase induction, the EdU signal distributions were analyzed in 24 nuclei from three preparations of hsp70-CycE; SuUR larvae (90 min after heat shock) compared to wild-type. We compared several selected regions on three preparations ( Supplementary Figs. 2, 3a, b, and 4a). Supplementary Fig. 4a illustrates very early replication in the 10A-11A region of chromosome X. On slides 1 and 2, replication was detected by the EdU incorporation assay, and on slide 4, by PCNA immunostaining. A region of early replication lies between late-replicating rb-bands 10B and 11A (Belyaeva et al. 2012). The chromatin morphology (after DAPI labeling) was well reproducible, but small differences in the stretching degree of the chromosomes due to squashing were noticeable. EdU and PCNA signals were similar. Consequently, we conclude that for early replication detection, both markers are suitable for 3D-SIM analysis. Although the signals were reproducible overall, there were some differences between and within the slides. The intraslide differences were primarily related to incomplete synchronization of the cells entry into the S phase. On different slides, there were various chromosome stretching degrees and differences in the appearance of all signals. The scheme in Supplementary Fig. 2d presents a hypothetical explanation of the effect of chromosome stretching on the visible replication pattern. The analysis of the dynamics of replication timing showed that it is most appropriate to use nuclei from the same preparation, because then, the chromosome stretching is comparatively uniform and the stages of development are identical. Genomic mapping of polytene chromosome bands: a comparison of cytological and molecular data To map cytological regions to genomic coordinates, we applied the approach of Kolesnikova et al. (2018). All the genomic coordinates used in this work are available in the D. melanogaster Release 5 assembly. The UCSC Genome Browser (Meyer et al. 2013) was employed for data visualization. Genomic positions of rb-bands were identified using the coordinates of four chromatin types established by Zhimulev et al. (2014). The CHRIZ protein distribution in cell cultures (modENCODE_275, modENCODE_277, modEN-CODE_278, and modENCODE_276), SUUR (Maksimov et al. 2014) and H3K27me3 (Posukh et al. 2017) protein distributions in salivary glands, and data from in situ hybridization (available from the FlyBase database) were used for more detailed mapping of band edges. Additionally, we utilized photographs (kindly provided by Todd Laverty) from in situ hybridization assays of P inserts from the BDGP project (Spradling et al. 1999). Insert or target gene names served as queries for retrieving the genomic coordinates of the inserts from FlyBase. Hi-C data for salivary glands (Eagen et al. 2015) were analyzed using accession GSE72512 in Gene Expression Omnibus. Computer simulation of replication The replication simulation for a polytene chromosome fragment was carried out by a computer program written in the Delphi Pascal language (Supplementary Text 1). We regarded a polytene chromosome as a number (N) of similar threads. In each thread, replication was assumed to be initiated stochastically. Given that the threads are exact copies of each other and are precisely aligned, we assumed that in all the threads, positions of potential replication initiation sites and the probability of replication initiation are identical. Therefore, the distribution of replication initiation sites on N DNA strands should reflect the distribution of initiation probabilities for one strand. We examined N = 1024 threads, which is the number of DNA strands in one homolog of a polytene chromosome (late third instar larvae). The rate of movement of replication forks along a DNA strand was considered constant. Accordingly, replication durations along the chromosome axis in interbands, gray and rb-bands depended on the DNA content. This assumption is a simplification because the actual speed in late-replicating bands is ~10 times lower as that was shown for D. nasuta polytene chromosomes (Lakhotia and Sinha 1983). Our evaluation of the replication fork movement speed was based on the following assumptions. In D. melanogaster, the mean replication fork rate is ~0.24 kbp/min for late replicons (Kolesnikova et al. 2009). If we suppose that during the very early S phase, the speed is ten times higher, then we get ~2 kbp/min. Visible thickness of the bands and interbands along the chromosome axis depends on the stretching degree of the chromatin. DNA packing ratios in interbands are 3-15, in gray bands 54-63, and in black bands >200 (Spierer and Spierer 1984;Kozlova et al. 1994;Vatolina et al. 2011;Zhimulev et al. 2014). When modeling, we depicted gray bands and interbands with the same thickness for simplification. According to the four-color chromatin model, all interbands correspond to aquamarine chromatin, with average and median sizes of 2.7 and 1.8 kbp, respectively (Zhimulev et al. 2014;Boldyreva et al. 2017), consistently with the Beermann (1972) estimate of 2 kbp DNA per interband. Thus, we chose an interband size of 2 kbp. The thickness of polytene chromosome structures cytologically revealed as gray bands varies considerably and ranges from 2 kbp (the minimum band visible by electron microscopy in semithin sections) to tens of kilobase pairs Demakova et al. 2020;Khoroshko et al. 2020). In the present work, we estimated the size of the 21C6 gray band to be ~60 kbp. Thick and thin gray bands were found to differ significantly in morphology. The thinnest ones are detectable only by electron microscopy and on the very best preparations, while the bands containing more chromatin are sometimes very similar to black bands. Therefore, we introduced two types of gray bands into the model. The first type corresponds to 5 kbp of DNA and is only 2.5-fold more compact than the interbands, while the second type is 20 kbp long and 10-times compacter. Accordingly, we simulated an INT of 6 × 2 kbp + 4 × 5 kbp + 20 kbp = 52 kbp. This is close to the average size and 1.5-fold larger than the median size of INTs. The INTs sizes vary from 0.5 to 300 kbp on chromosome 2R . S-phase induction by cyclin E is reliable to analyze the early replication dynamics in polytene chromosomes Previously, it was demonstrated that in actively moving wild-type larvae, when grown at 18 °C, early S-phase stages in the salivary glands occur with a frequency of up to 50% (Kolesnikova et al. 2013). A small proportion of the nuclei is at a very early stage when replication starts only in several decondensed regions representing INTs between compact bands as well as in several puffs. Local band-shaped and more diffuse patterns were found (Fig. 2). The INT between bands 94D1-2 and 94A1-4 of chromosome 3R is an example of a bright local signal pattern. The EdU signals lie in the loose chromatin that does not form a clear band, and the signals are concentrated in a small region of the INT (Fig. 2a). A similar situation was observed in the region of the thin gray bands to the right of the compact band 63A1-2 (Fig. 2b). Here, the signals were seen as two band pairs. Movie 1) showed that the signals are evenly distributed within the entire INTs. This pattern is consistent with our assumption that INTs match replication initiation zones emerging in different strands and origins. It is challenging to analyze replication initiation in polytene chromosomes of wild-type larvae because the nuclei in a salivary gland do not replicate synchronously. Therefore, all conclusions about S-phase progression were based on circumstantial evidence. To obtain more information about the initiation of replication in polytene chromosomes and to reveal temporal dynamics of the early S phase, we synchronized salivary gland cells by inducing the S phase via ectopic expression of cyclin E. For this purpose, we used a D. melanogaster line carrying the hsp70-CycE transgene (Knoblich et al. 1994;Duronio and O'Farrell 1995;Su and O'Farrell 1998). Additionally, to obtain a better chromosome morphology, we introduced the SuUR mutation into the line. The SuUR mutation affects the progression of replication forks in silent chromatin regions, primarily in the regions of pericentromeric and intercalary heterochromatin. This leads to suppression of under-replication but does not affect replication initiation (Sher et al. 2012). Previously, we reported that this mutation, which influences the latereplication pattern, does not affect the very beginning of the S phase (Kolesnikova et al. 2013). Late third instar hsp70-CycE; SuUR larvae and 0 h prepupae were subjected to a 35°C heat shock (see "Materials and Methods" for details). Then, we kept the larvae at room temperature and isolated the salivary glands after different time intervals, incubated them with 4 μM EdU for 7-10 min and fixed them immediately. Seventy minutes after the heat shock, the first induced S phases became visible. After another 20 min, the proportion of labeled nuclei reached 100%. This result suggests that the cells enter the S phase with a temporal shift of ~20 min. The induction at this stage of development allowed to obtain a high proportion of nuclei at very early S-phase stages, which is normally very low (Zhimulev et al. 2003a;Kolesnikova et al. 2013). Besides, we obtained an additional round of polytenization, leading to larger and better-structured polytene chromosomes ( Supplementary Fig. 3a, b). In diploid cells, S-phase activation by cyclin E impairs the distribution of replication initiation sites. This effect may cause conflicts between replication and transcription and induce carcinogenesis (Teixeira and Reed 2017; Macheret and Halazonetis 2018). Therefore, we checked whether S-phase induction via cyclin E overexpression altered the early replication patterns in polytene chromosomes. We compared the early patterns of normal and induced S phases. No differences were detectable as exemplified by region 56A-57B of chromosome 2R in Supplementary Fig. 3c. The pattern of middle replication was also similar to the control, as visible in the specimen prepared 180 min after S-phase induction. Our approach did not allow analyzing the induced S phases at later stages, but the patterns of late replication observed in nuclei that were in S phase before heat shock were normal. We conclude that CycE overexpression does not led to qualitative changes in replication patterns in preparations made 1-3 h after heat shock induction. In mammalian cells, forced expression of cyclin E abridges G1 phase, resulting in premature S-phase entry with prereplication complexes still present at the 3′-end of long genes. Normally, prereplication complexes become removed by active transcription during G1 (Macheret and Halazonetis 2018). To check whether there is an ectopic initiation of replication within highly expressed long genes, we analyzed the localization of loci of highly efficient early initiation simultaneously with the detection of active transcription. Supplementary Fig. 5 shows the earliest replication pattern of a nucleus at a stage when less than 30 bright EdU signals can be clearly identified, but the rest is still not yet activated. Some of these signals are close to bright transcriptional signals, others not. Many transcriptionally active regions do not show early, highly efficient replication signals. We chose locus 47B for a detailed analysis. Here, we found early replication and active transcription signals close together. The rb-bands 47A1-2 and 47B4-5 were mapped on the genomic map earlier ). According to ModEncode project data in salivary glands, the most actively transcribed gene within this locus is the long (~50 kb) gene lola. Multiple ORC2 sites are located at the 3′ and 5′ ends of this gene (Sher et al. 2012). Besides, the 5′ region has the highest peak of "early origins" in the Kc cell culture. We assumed that the upstream intergenic region of the lola gene induces the bright replication signal, while the lola gene is responsible for the active transcription. To prove this, we performed FISH with probes corresponding to the 5′ and 3′ gene regions. The probes localized at both sides of the gray band, i.e., the band contains a transcribed part of the lola gene. Simultaneous FISH and RNAPIIser2ph detection, and FISH and EdU incorporation demonstrated that the bright signals of early replication lie right to the transcription signals, partially colocalize with the 5′ probe, but are clearly separated from the 3′ probe. Thus, we conclude that the replication initiation is confined to the non-coding region near to the active gene promoter. Due to the small number of early signals, we consider them as specific. We conclude that S-phase induction is a reliable technique to analyze early replication dynamics in polytene chromosomes. At the very beginning of the S phase, replication is initiated differently in various INTs but similarly in salivary glands and diploid cells While analyzing very early replication patterns, we found that the signals are distributed unevenly in different chromosomal regions. To demonstrate this observation, we chose the distal region of chromosome 2L as an example (Fig. 3, Supplementary Movie 2). This polytene chromosome region has not yet been mapped to genomic coordinates. Therefore, we applied the algorithm of Kolesnikova et al. (2018) to determine the position of big bands. Four large bands enriched with "ruby" chromatin (Zhimulev et al. 2014) were predicted. Relative positions and sizes of these bands exactly match bands 21C1-2, D1-2, E1-2, and 22A1-2 ( Supplementary Fig. 6). Consequently, these four bands can be assigned to rb-bands. Between rb-bands 21C1-2 and D1-2, two thinner and less compact bands (21C4 and 21C6) are evident. The analysis of the EdU distribution corresponding to the very early S phase in the 2L chromosome region presented in Fig. 3 shows mostly the absence of EdU signals in rb-bands. All INTs contain EdU signatures, but intensity levels and distributions are significantly different (Supplementary Movie 2). The INT to the left of 21C1-2 is characterized by a bright signal pair (in Fig. 3 on the left) with relatively bright diffuse signals toward the telomere and rare diffuse signals in the rest of the chromosome region. Obviously, in this INT, the initiation does not occur uniformly. There is a zone where one or more highly efficient origins are localized. In INTs 21D1-2/21E1-2 and INT 21E1-2/22A1-2, the EdU signals are homogeneously distributed within the whole INT volumes (Fig. 3; Supplementary Movies 2, 4, and 5), while in INT 21D1-2/21E1-2, the signals seem to be more clustered and are brighter and larger, possibly indicating a replicon grouping. In INT 21E1-2/22A1-2 of the Fig. 3, the signals appear as 102 dots. This number means that at this time, less than 10% of the DNA strands initiated replication, suggesting that the initiation process proceeds gradually, which is consistent with data from autoradiographic analysis of replication (Lakhotia and Sinha 1983). In INT 21E1-2/22A1-2, thin bands are well discernible, which indicates that the DNA is precisely aligned along the chromosome axis. In contrast, the EdU signals appear to be distributed homogeneously. This distribution is in good agreement with the hypothesis that INTs act as replication initiation zones where any interband can initiate replication. Both INTs of chromosome 2L showing the bright band-shaped EdU signals correspond to peaks of newly synthesized DNA in the presence of hydroxyurea presumably relevant to "early origins" in S2 cells. These signals probably represent early and efficient origins acting in both salivary glands (our data) and S2 cells [according to MacAlpine et al. (2010)]. The other three INTs also have peaks of "early origins," but they are significantly lower than the peak in the INTs between 21С1-2 and 21D1-2. The concentration of EdU signals in the INTs correlates with the peak heights in S2 cells (Fig. 3). A similar correlation between the highest peak of "early origins" and the strongest signal of early replication in salivary glands is present in region 47A-B ( Supplementary Fig. 5). In short, it can be concluded that different INTs initiate replication differently and that the origin efficiency is similar between salivary glands and diploid cells. Early replication is highly dynamic EdU signal intensity varies significantly among the nuclei prepared 90 min after heat shock (Fig. 4). Considering that the S-phase induction is shifted in different nuclei, a comparison of the patterns allows to make a conclusion about the temporal dynamics of replication during the first 20 min of the S phase. In Fig. 4a, two chromosomes of neighboring nuclei are presented. They differ in total EdU signal intensity. The left nucleus has an EdU pattern typical for the specimens prepared 70 min after heat shock (data not shown). The overall signal intensity is relatively low, only a few band-shaped signals are clearly visible. In the right nucleus, the signal intensity is much higher. Such nuclei do not occur 70 min after heat shock. Accordingly, we assume that this nucleus exhibits a later pattern. For a detailed analysis of the dynamics of replication in the first 20 min of the S phase, we focused on region 4F-6A of chromosome X (boxed in Fig. 4a). The comparison of three consecutive patterns revealed that the paired signals in regions Schwaiger et al. (2009). The corresponding optical crosssection and maximum intensity projection (MIP) of a 3D-SIM image stack below the scheme illustrate the very early replication pattern via EdU incorporation. The two variants of representation, differing in brightness, are presented to show all details of weak and bright signals. The red brackets indicate the sections of the chromosome matching to the INTs. The specimen was prepared from Hsp70-CycE; SuUR larvae, 90 min after heat shock 4F9-10/5A1-2 and 5C1-2/5D develop sequentially from weaker diffuse signals emerging at the very beginning of the S phase ( Fig. 4b-d). At later stages, the signals become brighter and larger and accumulate as thin bands. This finding clearly shows that the replication initiation in different chromatids takes place gradually in these regions. In most nuclei, symmetric bands of double signals are characteristic replication patterns in the early but not the earliest replication stage (Figs. 4 and 5a,Supplementary Figs. 7 and 8). Two hypothetical scenarios may be responsible for this pattern. First, double bands may reflect bidirectional replication forks moving in opposite directions from efficient origins in between. Namely, the signals look like a clear band perpendicular to the chromosome axis at the origin site and generate two distinct replication bands moving apart from each other. Second, replication initiation may also arise at various origins on different DNA strands but because of rapid replication fork movement followed by deceleration inside the compact bands, the replication signals become concentrated at the rim of highly condensed chromatin (rb-bands) and thereby appear paired (Fig. 5b). Consequently, both scenarios may result in similar paired signals. To clarify which scenario occurs predominantly in polytene chromosomes, we analyzed such double signals during progression in several individual regions by 3D-SIM. The increased resolution allowed detecting many EdU signals scattered in the INT of region 5C/5D of the top chromosome X in Fig. 5c. Note, that the distribution of signals predominates at the left side of the INT, enriched in ORC2-binding sites mapped Fig. 4 S-phase induction helps to study replication dynamics within the first minutes after induction. a Chromosomes from two nuclei in different early S-phase stages (two arrows). The left-hand chromosome shows the earliest pattern where rare bright signals lie in puffs and loose bands. The righthand nucleus features a later pattern. b-d The temporal dynamics of early replication with an example of X chromosome region 4F-6A from the same preparation as in a; d is the framed region in a. The specimen was prepared from Hsp70-CycE; SuUR larvae, 90 min after heat shock. Given that the first nuclei enter the S phase 70 min after the shock, the temporal distance between the patterns is ~20 min by chromatin immunoprecipitation (data from Sher et al. 2012). On the middle X chromosome, the entire INT is labeled, and the highest signal intensities levels correspond to the mirrored signals outside. On the bottom chromosome, the mirrored signals are mainly concentrated at the edges. Obviously, the three chromosomes represent the progression of replication forks initiated inside the INT toward the edges of boundary bands. Similar patterns were evident in regions 47D-48C and 41F-43B1-2 ( Supplementary Fig. 8), i.e., during earlier stages, the signals are distributed uniformly within the INTs, but at later stages, they concentrate at the edges. At locus 3C, we found a very thin bright signal at the very beginning of S phase. This is an example of a scenario with one efficient origin rather than a broad initiation zone (Fig. 5d). This early replication signal colocalizes with the Sgs4 gene, forming a puff at the end of the third instar (Korge 1975;Supplementary Fig. 7c). Besides, at this locus, also a group of short genes occur, which are highly expressed in salivary glands ( Supplementary Fig. 7d). All of them become activated by the hormone ecdysone, and their products are responsible for the secretion of the salivary gland (data from FlyBase database). That is, it is a group of tissue-specific genes. The distribution of ORC2-binding sites (Sher et al. 2012) shows that a small group of tissue-specific origins lie near this group of genes. The highest ORC2 peak corresponds to the Sgs4 gene. Supplementary Fig. 7c demonstrates that the Sgs4 FISH signal is flanked by EdU signals at the very early replication stage (paired signals occur). Thus, locus 3C demonstrates an example of scenario 1 (Fig. 5b). But even in this case, a cluster of several potential origins is evident. In general, the second scenario is much more common within the chromosomes. In short, we draw the following conclusions. At the very beginning of the S phase, replication is not uniform at different sites. There are regions with distinct or diffuse EdU signals. Moreover, the number of diffuse signals varies among different INTs. The signal number per chromatid and signal size increase with time, indicating gradual replication initiation of different origins within the INT. Replication is initiated spatially stochastically inside the INTs. The activated replication forks pass quickly through the entire INTs in both directions to accumulate at INT borders. This finding is in good agreement with the notion that INTs match replication initiation zones with multiple potential origins. Replication spreads from the INTs into rb-bands devoid of ORC2 sites Three hours after heat shock, most EdU signals accumulate at the borders of compact bands where characteristic distortions of the band contours emerge ( Fig. 6a and Supplementary Fig. 9). This phenomenon is especially obvious in bands 70C and 10A (Fig. 6a). Supplementary Fig. 4b shows bands 10A and 10B from six nuclei from four preparations demonstrating earlier replication patterns. In none of them, such edge abnormalities occur. Thus, the distortions seem to be caused by the replication process. In region 48D-49A, a group of pronounced rbbands is located ( Supplementary Fig. 9). The bands also represent mainly ruby chromatin and lack ORC2 sites. They form well-pronounced but smaller TADs in the salivary glands. The biggest (48E1-2) is ~100 kbp. Not all rb-bands concentrate the EdU signal along their surfaces. In some preparations, band 50D appears as a distinct band, while on others, it occurs as swollen chromatin (a puff) implying high transcription intensity. Band 50D contains a low amount of ruby chromatin, and DAPI staining showed that it is significantly less condensed and is completely labeled by EdU throughout the entire volume ( Supplementary Fig. 9). Hence, band 50D is not a typical rb-band. In terms of replication, it behaves differently from most rb-bands. Two scenarios may induce similar replication signal pairs at rb-band edges during early replication. a Multiple EdU signal pairs of similar intensity are present along the polytene chromosomes. b Two hypothetical scenarios may cause these patterns. Paired signals may derive from replication forks diverging from very efficient origins, at which most DNA strands initiate replication synchronously (scenario 1). Replication initiation may also occur on different DNA strands at various origins, but due to the rapid movement of replication forks along the chromosome axis inside of the INTs, and owing to a sharp slowdown of the movement of replication forks along the chromosome axis inside the compact bands, the replication signals become concentrated on both edges of the INTs and thus appear also paired (scenario 2). c INT 5C1-2/5D illustrates scenario 2. The INT contains multiple ORC2-binding sites that predominate on the left side of the INT. The earliest detectable EdU pattern in this INT represents diffuse signals dominant in the left half of the INT (top). The two bottom patterns reflect the gradual accumulation of signals at the edges of the boundary rb-bands 5C1-2 and 5D. d The 3C locus illustrates scenario 1 (see Supplementary Figure 7 for details). The specimen was prepared from Hsp70-CycE; SuUR larvae, 90 min after heat shock ◂ Overall, it can be concluded that uniformly compact rb-bands devoid of ORC2 sites are replicated from edges to the middle and that bands with complex organization and internal ORC2 sites can initiate replication inside. The analysis of nuclei during late replication on preparations of wild-type and hsp70-CycE; SuUR larvae without heat shock revealed that the signals lie in the middle of very thick intercalary heterochromatin bands (Fig. 6b). Supplementary Figure 10 represents two examples of replication progression within thick bands at successive stages of middle and late S phase. Computer simulation confirms that dissimilar initiation rates may explain the different early replication patterns To test the idea that different replication initiation rates are the main reason for the dissimilar earlier replication patterns observed in different INTs of Fig. 6 In the middle and late S phase, replication spreads from INTs into rb-bands. a Representative EdU labeling 3 h after heat shock (~100 min after S-phase initiation). At this middle-S-phase stage, most signals are concentrated along the borders of compact bands as seen in regions of chromosomes X (10A1-2 and B1-2), 3L (70A1-5 and C1-2), and 2R (44C1-2, D1-2, F1-2, and 45A1-2). The majority of EdU signals cover compact bands, but many signals are still visible between the rbbands. At 10A1-2 borders, characteristic distortions of the band contour are noticeable. b During late-S-phase replication (not the induced S phase, Hsp70-CycE; SuUR larvae, without heat shock), the EdU signals are mainly concentrated inside the thickest compact rb-bands. In chromosome 2R region 56F-57B, PCNA staining is present inside intercalary-heterochromatin regions 56F1-7, 57A1-2, 57B1-2, and B4-6 (left). EdU signals occur in intercalary-heterochromatin bands 11A1-2 (middle), 50A1-4, and 50C1-4 (right) polytene chromosomes, we performed the computer simulation of replication in a model chromosome fragment consisting of one INT limited by two rbbands (Fig. 7, see "2" for details). According to the model, a single replication origin activates in each chromatin fiber within interbands at a random position and gives rise to two replication forks, which are represented by red dots moving away from the origin along the chromosome axis at a rate constant for each type of structure. In Fig. 7, we consider four replication initiation scenarios: (1) all 1024 origins of the INT fire simultaneously; (2) the origins are activated gradually at a rate of ~10 origins per minute, that is, all 1024 origins are activated within 10 min; (3) 1024 origins are activated within 30 min; and (4) 1024 origins are activated within 60 min. The scenarios reflect the various experimentally observed replication initiation patterns in different INTs. Replication fork speed was assumed to be 2 kbp/ min. Therefore, the replication rate (the rate of the red point movement) along the interband was V = 1 interband/min. The relative speed was V/5 in a gray band, V/20 in the middle gray band, and V/50 in a black band. The distribution of red points in the INT after 4 Fig. 7 A computer simulation of replication progression in a 52 kbp INT localized between two rb-bands and composed of alternating interbands and gray bands of different compactness. a The INT includes six interbands and five gray bands. All bands were drawn to be of equal width along the chromosome axis but contain different DNA amounts. b In each of the 1024 DNA fibers, only one single replication origin activates at a random position within an interband. Then, each origin generates two replication forks running in opposite directions (visualized as red points) at a speed of 2 kbp/min. Four initialization scenarios were simulated (from left to right): One origin per chromatid may become activated in all chromatids simultaneously, within 10, 30, or 60 min. The replication fork distribution progresses from INTs toward rb-bands in both directions and is shown after 4 s to 120 min (from top to bottom) Vol:. (1234567890) s and 1, 5, 20, 30, 60, and 120 min is shown from top to bottom in Fig. 7. We noticed the following trends: With synchronous initiation of replication, within 1 min, all signals gravitate toward the bands. There are bright signals in the form of stripes from the very beginning because the signals from the interbands are concentrated in the bands that contain more DNA and the probability of encountering a signal there is higher. After 30 min, an INT with a length of ~50 kbp completes replication, and the signals become appreciably concentrated at the edges of the condensed thick bands. When the initiation of replication is prolonged, we see fewer ordered signals at all stages. A high concentration of signals is present in the more compact middle gray band and at the edges of the black bands, i.e., in the bands with more DNA per unit of chromosome length. The longer replication initiation takes, the longer are diffusely scattered signals present throughout the INT. Thus, different rates of replication initiation explain very well the dissimilar replication patterns in the different INTs of polytene chromosomes. Discussion Drosophila polytene chromosomes are well-suited for deciphering replication In this work, we demonstrated that polytene chromosomes are well-suited to investigate the very early replication parameters of Drosophila that are probabilistic. In these chromosomes, more than 1000 DNA filaments are arranged in parallel with a distinct pattern of thick and thin bands denoting TADs (Zhimulev et al. 2014;Eagen et al. 2015;Ulianov et al. 2016;Stadler et al. 2017;Kolesnikova 2018). Highly stretched chromosomes allow the direct visualization of differently compacted chromatin along the chromosome axis. At the beginning of the S phase, the distribution and density of replication signals reflect the probability of replication initiation. The replication initiation zones correspond to INTs, and EdU signal density within the INTs reflects their efficiency. Moreover, the efficiency here is not only the probability of activation during the cell cycle but also the probability of activation per time unit during early S phase. The distribution of early origins in cultured cells exposed to hydroxyurea (MacAlpine et al. 2010) is in good agreement with our reasoning about the dissimilar origin efficiency grades among different INTs. We showed that ectopic S-phase induction is useful for investigating early replication in Drosophila polytene chromosomes. The analysis of replication in salivary glands of wild-type larvae grown under standard conditions (24-25 °C) did not permit identifying very early replication patterns because the stages preceding continuous labeling take several minutes. Other authors used temperature reduction, the Giant mutation, and an analysis of other Drosophila species to find a model system that would allow more detailed research on the earliest S-phase patterns Lakhotia 1979, 1981;Mishra and Lakhotia 1982). The synchronization with FdU induced the accumulation of later S-phase stages in salivary gland cells (Achary et al. 1981). To study the stages in detail, which are normally too short in D. melanogaster, we developed a system based on ectopic S-phase induction. The use of the hsp70-CycE transgene (Knoblich et al. 1994) helped to induce S phases in many salivary gland nuclei with a time shift of no more than 20 min. We demonstrated that the replication patterns are identical between the induced and normal S phases. The S phases caused by ectopic cyclin E expression differ from those without induction in diploid cells. Here, due to the transcription/replication conflict, the initiation events occur at the wrong sites (Teixeira and Reed 2017;Macheret and Halazonetis 2018). One would expect similar differences between the induced S phase and the normal one in polytene chromosomes, but we did not find examples of ectopic initiation of replication at the beginning of the S phase. The correct early replication initiation during ectopic S phase in salivary gland may occur due to the specificity of the endocycle regulation, because the amount of MSM2-7 complex components is lower than in diploid cells (Maqbool et al. 2010). This could prevent covering all chromosomes with MSM2-7, and thus preventing initiation at wrong places. Despite the fact that the early S phase does not differ from the normal one during the induction of replication in our system, the middle and late S phases appear to be impaired. The reason may be that in contrast to diploid cells, the E2F (transcription regulator of many genes involved in the S-phase progression) acts upstream of cyclin E. In particular, the synthesis of ribonucleotide reductase is under control of E2F. It can be assumed that the ectopic expression of cyclin E triggers S phase without activating some genes necessary for the complete passage of the S phase (Edgar et al. 2014;Kim et al. 2021;Dimova and Dyson 2005). Possibly, that the cells enter the induced S phase with a limited amount of nucleotides. Replication initiation is stochastic in polytene chromosomes During the 1970s, DNA replication studies on Drosophila polytene chromosomes were conducted via 3 H-thymidine incorporation. This method allowed researchers to draw quantitative conclusions about the intensity of label inclusion after silver grain counting. Various authors have identified up to seven categories of labeling patterns and arranged them chronologically (Rodman 1968;Kalisch and Hägele 1973;Lakhotia 1979, 1981;Achary et al. 1981;Mishra and Lakhotia 1982). The labels have been detected in decondensed areas (called interbands in the cited papers, corresponding to our INTs) in three early stages representing low, medium, and heavy interband patterns. During these stages, a sequential increase in the silver grain number occured. It has turned out that the regions differ in the rate of 3 H thymidine incorporation. There are regions in which many silver grains arose already during the first minutes of the S phase. Circa 40 such areas have been observed. After these stages of early discrete labeling, stages of continuous labeling followed. Besides, the stages differed from each other in labeling intensity, showing medium or heavy the continuous labeling. In the last three stages, the discrete labeling decreased in number and signal intensity (heavy, medium, and low discontinuous labeling), indicating the exit of most replicons from replication (Mishra and Lakhotia 1982). Our results completely match these earlier findings, namely, that during the first hour after S-phase induction, new replicons are switched on, as revealed by a signal intensity increase. Additionally, various chromosomal regions manifest different dynamics of replication initiation. The detection of a "continuous labeling" stage was due to the low resolution of autoradiography (~1 μm), which is determined by the size of the emulsion grain and the path length of β particles after tritium decay. Even in larvae with a delayed S phase, those authors have observed a "continuous" coverage of chromosomes with a signal present already 10 min after the S-phase beginning. The use of fluorescent labeling in combination with WF microscopy allows to identify signal gaps in the thickest bands at the stage of continuous labeling, but the maximum resolution does not exceed thick band/ INT sizes ). In the present work, we applied super-resolution microscopy and reached for the first time a resolution visualizing single signals representing multiple replication patterns. The model of stochastic replication initiation in polytene chromosomes was first proposed by Lakhotia and Sinha (1983). After an analysis of fibrils in partially lysed chromosomes, they concluded that initiation on different DNA strands is asynchronous. Our results fully support this model. Stochastic regulation of replication kinetics is a fundamental feature of eukaryotes and is conserved from yeast to humans (Wang et al. 2021). Origin efficiency usually refers to the probability of activation during the cell cycle. Nevertheless, there is accumulating evidence that this parameter is more complex. It can be regarded as the competition between origins for activation, where more efficient origins have a higher activation probability at the S-phase start. In contrast, after the release of limiting factors, a new pool of origins becomes competitive. The observation that on average, 9% of late-initiating origins initiate replication in the early S phase led to the idea that origin activation probability determines all replication timing (Wang et al. 2021). The results of our work are in good agreement with these new ideas about the gradual initiation of replication across the genome. Replication in Drosophila and mammals: similarities and differences The general genome organization differs between Drosophila and mammals. The Drosophila genome is almost an order of magnitude more compact. While the mammalian genome contains megabase scale chromatin domains and TADs well coinciding with replication domains, the alternation of shorter domains is present in the Drosophila genome. These are open chromatin domains with a median size of ~30 kbp attracting more than 90% of ORC-binding sites. Closed-chromatin domains contain predominantly silent tissue-specific genes and almost no ORC-binding sites (Zhimulev et al. 2014;Kolesnikova et al. 2018). INTs representing the replication initiation zones in Drosophila match those in mammals very well in size and many properties. An important difference is that the replication initiation zones in mammals lie predominantly in long intergenic regions (Lebofsky et al. 2006;Petryk et al. 2016). Instead, in Drosophila, they are situated in a set of short intergenic regions alternating with housekeeping genes . In mammals, the characteristic replicon size is >100 kbp (Edenberg and Huberman 1975;Berezney et al. 2000;Lebofsky et al. 2006). In Drosophila, this size is similar, but almost coincide with the median size of silent domains. Apparently, these silent domains of chromosome arms (corresponding to rb-bands of polytene chromosomes) are replicated mostly passively, that is, by replication forks coming from neighboring INTs . We suppose that the patterns of replication initiation that we observed in polytene chromosomes are universal because they are consistent with the recent finding that replication initiation occurs stochastically in space and time, and that replication initiation events are distributed across broad initiation zones consisting of many initiation sites, whereas the dynamics is heterogeneous (Su et al. 2020;Wang et al. 2021).	2022-03-01T06:23:10.877Z	2022-02-28T00:00:00.000	{ "year": 2022, "sha1": "56e8e2993102baf1555d88d32dfa03d60f719b2c", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s10577-021-09679-w.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "e9ae9a4228ec657097b434739160d99429faf27d", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
214306235	pes2o/s2orc	v3-fos-license	Embedding Stock Tracking Module into Electronic Fiscal Device Machine and its Management System to Reduce Tax Evasion: A case of Tanzania — The Electronic Fiscal Device (EFD) Machines have been operating in Tanzania since the year 2010 for the purpose of helping the Tanzania Revenue Authority (TRA) to increase revenues from tax collection. Regard-less of years of its existence, there are still reported cases of tax evasion, and this study was conducted to review the current tax collection system and analyze requirements for the development of Stock Tracking Module (STM) to be embedded in the current tax collection sys-tem. This paper earmarked some problems relating to Electronic Fiscal Device Machine Management System (EFDMS) and EFD machine. Data collection was done in Kilimanjaro and Arusha, the two regions of Tanzania that involved tax officers and Information Technology (IT) personnel from TRA and drug traders. Data collection process involved both qualitative and quantitative meth-ods to gather data for the development of the system Stock Tracking Module (STM) such as interview, questionnaire, role-playing and observation. The major findings of the study: The efficiency of the EFDMS is at I. INTRODUCTION Tax evasion is considered to be the major cause of financial fatigue that most governments are facing nowadays (1). With the increase of the Information Communication Technology (ICT) use in recent years, significant number of nations have adopted ICT tools to combat the problem of tax evasion in different areas of trading whereby Accounting Systems (AS) and e-government systems are designed and/or integrated for the purpose of collecting, storing and auditing of tax-related information (2). Electronic Fiscal Devices (popularly known as EFD machine in Tanzania), Electronic Cash Register (ECR), computer-based point of sales systems (POS), or those that are tablet or smartphone-based have proven to be efficient ICT tools to many countries in improving tax collection that include Income, Corporate, Service levy, and Value Added Taxes (3) According to the Organization for Economic Cooperation and Development (3) whose its 27 member countries are using such systems, revenues from tax had increased. Particularly in Hungary, Australia, Belgium, Canada, and Sweden, revenues from tax has increased at an average of 8% per annum. The use of EFD machines to combat tax evasion among East African countries that include Tanzania, Kenya, Rwanda, Uganda, and Burundi proved to be successful such that in Rwanda between year 2013 and 2015 revenues had increased by 20% annually(3), while in Kenya between year 2000 and 2005 tax collections had increased at rate of 5% per annum (4). System In 2010, Tanzanian government geared a parliamentary act and finance act that fostered to the adoption of EFD machines by businessmen across the country for the purpose of dealing with the problem of tax evasion in the trading arena (5). Upon the introduction of the devices, it was observed that the operations of EFD machines had eventually increased the number of government revenues raised from Income, Corporate, Service Levy and Value Added tax (6). Although TRA has presented EFD machines to traders that eventually improved tax collections, but falsification of taxation still exists due to difficulties of monitoring the use of those machines whereby various techniques that appear to popularly being used by some traders being; trading without issuing receipts, issuing underpriced receipts, and as well, the existence of fake EFDMS (7). Even though falsification of information that leads to underpayment of tax is an offense that results in imprisonment or fine but still there are reported cases of tax evasion (8). It is clear that the use of EFD machines to Tanzania governments is not a single fix solution to tax evasion, but nations that were able to implement it effectively, substantial achievements speaks for themselves. A case in point, in Tanzania, the current EFDMS can neither detect nor identify traders who trade without issuing a receipt, falsifying sales value nor did issue forged receipts. While EFD machines and EFDMS can be incorporated with STM to keep track of stock flow from manufacturers to whole sellers and trickle it down all the way to retailers so that TRA can have information about the amount of stock owned and sold by a trader, it is not yet that way. Therefore, this paper unleashes an on-going study that aims at incorporating STM to both EFD machines and EFDMS for the purpose of detecting tax evaders. The aim of this study is to analyze the requirements for the development of STM that will alert TRA of the falsification of sales value, forged receipts and transactions took place without issuing a receipt. The paper consists of six sections: Section One outlines the problem background and problem statement. Section Two entails the review of the related works. Section Three presents data collection methods, sample size, and sampling techniques. Section Four describes the current system, proposed system, database schema, and EFD App design. On the other hand, section Five discusses the findings of the research while section Six contains conclusion and future work. II. RELATED WORKS Following the adoption of EFD machines as a tool for tax collection around the global, significant number of governments have increased their revenue collections raised from taxes. The main goal of adopting EFD machines by revenue authorities is to have proper business records keeping, easing the process of tax estimation, and improving information flow from traders to revenue authorities and vice versa. (9) Presented a study that analyzes challenges that hinder EFD machine implementation in Tanzania. The chal-lenges include: lack of education, system breakdown, and commodities underpricing. The study further discussed solutions that could be implemented by the authority to reduce the grief, in such ways as stakeholder's awareness and friendly operating environment; however, the researcher didn't discuss the technological weakness of the system and solutions to bring down the case. The study done by (6) on the effectiveness of EFD implementation in Tanzania, shows that the problem of tax evasion exist among EFD machine users whereby the scholar identifies the high cost of buying EFD device as the main reason for the lack of support from business operators. Furthermore, the researchers suggest that educational programs and increase of EFD machine rollouts in the country was a good ways for pursuing the reduction of tax evasion, but however, the study does not address the EFD Management System and EFD machine gaps that encourage traders to evade tax without being detected. According to (10) in a publication on challenges facing the adoption of EFD machines in Tanzania, indicates that, TRA is facing communication constraints, claiming of false VAT returns, lacking culture of requesting financial receipts, issue of monetary receipt to fewer deals than actual, and opposing the procurement and utilization of EFD machines as the major challenges. In spite of the study being among the few studies backing up the revenue authority, but EFD Management System weaknesses were not discussed. In Ethiopia, EFD machines were adopted by Ethiopia Revenue and Customs Authority (ERCA) since 2008 for business management and revenue collection. According to (11) the implementation of EFD automated system increased the number of revenue collections and so easies the process of preparing tax returns. Results from the study signals challenges of implementing EFD machines being the purchasing and maintenance cost of the devices, inexperienced customer support employees and the inability of users to correct errors. The researcher is, however, focusing only on challenges that business operators are facing rather than the Revenue Authority. The study done by (12) investigated factors influencing implementation and utilization of EFD machine in Kenya by Small and Medium Enterprises. The study revealed that higher purchasing, installation, maintenance and annual checkup cost, poor quality of service offered by EFD suppliers and inconsistency in business classification influenced traders to evade tax. It was moreover evidenced that, EFD cumbersome when dealing with small transactions. To promote compliance and hence reduce the problem of tax evasion, the study proposes regular training and enforcement of tax law among traders to be increased. Thus, from the solutions proposed by the scholar, none of them touches technological improvement of the EFD machine and/or EFD Management System. In March 2014, Malawi Revenue Authority (MRA) rolled out EFD system to replace the manual receipt system. As in other countries, MRA is facing various challenges in the implementation and administration of the EFD system. A paper done by (13) uncovers a number of challenges that hinder smooth deployment, in such ways as resistance from taxpayers, few taxpayers using the device, falsification of sales value, and insufficient number of EFD supply. The researcher furthermore proposes sanctions, penalties, stronger legal standings to offenders of tax evasion and improvement of ICT infrastructures as measures to combat tax evasion. Though the scholar suggests improvements in ICT infrastructure but didn't specify where and how should the ICT infrastructures be improved. It was earmarked that, most of the works of literature are far more addressing challenges that EFD users are facing than challenges that revenue authorities are encountering. While the few that favors revenue authorities such that of (11) and (13) are proposing a solution that touch policy, law, education to users, purchasing price and maintenance cost of the machines but none of them are talking about the weakness of the EFD machines and EFD Management System. According to IMF working paper prepared by (14), fiscal devices alone cannot stand as a "Silver Bullet" of tax administration; rather other technological improvements need to be considered. Therefore, this study suggests developing and embedding Stock Tracking Module into EFD Management System that will enhance the comparison between business stock records and sales return so as to identify tax evaders. III. MATERIAL AND METHODS The study was conducted in Kilimanjaro and Arusha, the two regions of Tanzania. Both regions are located in the northern part of Tanzania. The regions conduct many businesses but human drug trading was selected. Therefore, the target groups were TRA systems analysts, EFD unit officers, and drug traders. The motive for selecting drug business is the fact that human health is considered to be a priority; therefore, the same system could later be used by other regulatory authorities such as Tanzania Bureau of Standards (TBS) and Tanzania Food and Drugs Authority (TFDA) to track expired and fake drugs in the market. Kilimanjaro and Arusha regions were selected because they have a large number of drug traders who own EFD machines. A. Sample Size and Sampling Technique The study used a total number of 47 respondents who were clustered into three groups namely: drug traders, systems analysts and tax officers. To increase the degree of participation, 36 respondents from the first group were randomly selected from 18 different drug shops based on the factor that the pharmaceutical shop uses both an IT system to store drug records and EFD machines to print a receipt. Drug traders were involved to get information on drug acquisition, storage, and selling procedures. In the second and third group, a purposeful sampling technique was used in selecting a sample of system analysts and tax officers. System analysts were involved in the study to understand how the current system operates while tax officers to learn procedures used in tax collection and estimation process, and how to deal with tax evaders. Since each region of Tanzania contains 2 system analysts and 4 tax officers, 4 system analysts were selected in the second group and 7 tax officers out of 8 from the third group were available during the study. B. Data Collection Methods Data collection in the stipulated areas of study was done in the period of February to April 15, 2019. The process involved several data collection techniques in such as questionnaire, interview, observation, and roleplaying. Questionnaire: Questionnaires were used to gather data from drug traders. They were structured into multiple choices questions, close-ended questions, ranking, scaled, and open-ended questions. Questionnaire for drug traders was set to acquire information about stock storage, the flow of drug stock from manufacturers to retailers and also their experience in using EFD machines. Structured interview: Guided interview questions were posed to systems analysts and tax officers to collect information about their experience on how they manage and operate EFDMS, and how data exchange between EFD machines to EFDMS is done. Furthermore, the study also investigated whether embedding STM into EFD machines and EFDMS will improve tax collection. Role-playing & Observation: On data collection process we participated in selling and buying several items. The main aim was to understand inside-out how EFD machines work in daily business transactions and as well as how information exchange takes place between EFD machines and EFDMS. A. Description of the Current System The interviews and questionnaires administered to TRA officials and traders were done to get knowledge on how EFD machines and EFDMS are currently working for the purpose of developing an improved one. The ongoing study uncovers numerous system weaknesses that are used by traders to evade tax. It was observed that using the current EFDMS traders are able to evade tax without being detected in such ways as under declaration of sales, avoid using EFD machine, use fake EFD machine, overestimate of expenses, division of business and conducting business in unknown areas. Fig.1. indicates the techniques used by traders to evade tax such that 37.8% of the respondents identified underdeclaration of sales as the main technique followed by avoidance of EFD machine at 21.6%, meaning that a lot of revenues are lost due to underpriced sales and avoidance of EFD machine. It was further noticed that division of business and the use of unknown areas are the two emerging techniques which are not yet popular as they smash 10.8% and 5.4% respectively on the figure. It was further revealed that TRA had no control over traders stock, therefore tax estimation is done by refer System to Reduce ring to sales reports submitted by traders whose their loyalty is questionable. Fig.1. Techniques for Tax Evasion Following the weaknesses pointed out by TRA respondents, 54.5% ranked the efficiency of the current system in collecting tax to be between 50% -75% which confirm that the current system needs some improvement. Fig.2. provides more details on such. Fig.2. EFDMS Efficiency in Collecting Tax Majority of the respondents confirmed that by embedding STM into EFD machines and EFDMS will have a positive impact on mitigating tax evasion in various ways as indicated in Fig. 3. B. Proposed System This study outlines functional and non-functional requirements for the development of improved Electronic FDMS with STM embedded. Functional requirements will present the contribution of STM to EFDMS while non-functional requirements will describe the qualities of the proposed system. C. Functional Requirements a) The embedded module must be able to capture stock source b) The module must track stock flow between traders c) The module must set an estimated price per unit item d) The proposed module must produce a report on traders stock e) Produce a report on traders daily sales f) Generate a comparison summary between traders stock records and sales g) Issue alert on possible tax evaders h) Generate projection on the amount of tax to be collected i) Produce a summary report on total tax collected and evaded j) A trader must be able to view cleared and outstanding amount of tax Receive stock information Tax officer will be able to retrieve initial stock amount entered the market 3 View stock-sales report The module will generate a summary report showing the comparison between stock flow and sales report submitted 4 Receive alert The module will alert tax officer of possible tax evader 5 View outstanding taxes The system will produce tax information for each trader based on the stock sold 6 Set directive price Manager will be able to set directive price each stock item. This price will be used to compute the amount of tax to be paid by a trader 7 Confirm tax evader Based on the comparison between stock information and sales report manager will be able to confirm tax evaders 8 Confirm stock amount The module will enable the tax officer to confirm the amount of stock received and sold in a particular range of period 9 Forecast tax collections The manager will be able to forecast the amount of tax to be collected in certain business area 10 View tax evaders The system will generate a list of possible tax evaders System to Reduce D. Non-functional Requirements System Scalability: While reviewing the existing system and gathering requirements for the development of the stock tracking module we have earmarked several points of benefit to other regulatory organs if their systems will be linked with the proposed system. The system can appropriately enable Tanzania Food and Drugs Authority (TFDA) to identify and locate traders owning fake and expired drugs or food products in the market as among of the attributes captured by the module being the manufacturing and expiry date of the stock. Not only that but, the system can be linked with the Tanzania Bureau of Standards (TBS) database to tell the existence of unapproved products in the market. System Security: Since stock details are a crucial attribute for the tax calculation, the security of stock tracking module will carefully be considered during development. Each user of the system will be provided with login credentials whereby sessions will be generated for every successful login and terminated upon logout. To make the security more robust, the system will have different access levels. Again in each access level, the system will be implemented in a role-based fashion, such that, a user of a certain access level can perform only the assigned tasks of that level. Fig.6. represent database schema of the system to be developed. Database tables are categorized into: User and Role management tablesthe tables that contain details for user login, access levels, personal information, roles and privileges; Stock and Sales tablesthe tables that contain information about stock flow and sales return; Traders, EFD machine and Tax tablesthe tables that store information concerning registration of traders, EFD machines, financial years and various types of taxes charged by TRA. Fig.8, Fig.9, and Fig.10, are some of EFD machine application layouts where by: fig.7, is the EFD Machine application main screen designshowing six operation modes of the EFD machine, fig.8, is the Registration Mode layout that provide room for trader to sale an item, fig.9, is one of the Programming Mode layouts that provide interface for fiscalization of the device, while fig.10, is also a Programming mode layout that provides room for a trader to register business items in the EFD machine. The findings of this study entail several weaknesses that the current EFDMS is having, which includes inability to identify underpriced sales, overstatement of expenses, division of a business, the use of unknown areas, forged receipts and transactions that receipts were not issued at all. Tax estimation is done by looking at the sales report submitted by traders; however, EFDMS cannot verify the correctness of such reports which results in loss of government revenues. Furthermore, the study observed that neither stock information recorded nor directive price is set by the system which is important parameters to verify sales report submitted. E. Database Schema and App Design Therefore, to deal with the pointed weaknesses this study proposes the development of the Stock Tracking Module which will be embedded into the current EFDMS. The system will capture initial stock from importer or manufacturer (stock origins) and sale to suppliers or whole sellers who may also sell to fellow wholesellers/suppliers and finally to retailers who will finally sale to consumers. On selling, the system will decrement the seller's stock and increments buyer's stock and the process will keep on repeating along the business chain such that every commodity sold will be seen on the system. The system will also provide room for a systems administrator to set directive price (estimate price) for each commodity such that the system will now be able to compare sales return submitted by traders and identify whether there is under-priced commodities or sales done without issuing a receipt. Results of the study suggest that by embedding the module will have an impact on mitigating tax evasion in such a way that avoidance of EFD machine, underpriced and forged transactions will be detected by the system automatically. The process will also give TRA knowledge on where and when the stock was acquired and whether is depleted or otherwise. Not only that but it will reduce the workload to a tax officer who visits one business after another for tax inspection. With the forecasted solution the system will generate various reports to both traders and TRA officials. To ac-complish the proposed solution the researcher will design, develop and embed STM into current EFDMS whereby various software, programming languages, and tools will be involved. VI. CONCLUSION This paper has reviewed both Electronic Fiscal Device Management System, EFD machine and then analyzed requirements for the development of Stock Tracking Module for the purpose of mitigating tax evasion to drug traders in Tanzania. Various techniques used by traders to evade tax was uncovered by the study such as; Underpricing of sales, Avoid using EFD machines, Use fake EFD machines to print business receipts, Division of business, Overestimate of expenses and running a business in unknown areas. The study also confirms that the proposed solution if adopted by TRA will increase EFDMS efficiency and hence increase revenue collections. Moreover, the proposed solution will help to reduce manual work and number of tax officers needed for tax inspection and lastly the process of tax estimation will be more open and accurate hence reducing complains among traders and government at large. However in future, more research is open to integrate EFDMS database with Tanzania Food and Drugs Authority (TFDA) and Tanzania Bureau of Standards (TBS) databases to identify and locate traders owning fake and expired drugs or food products in the market. Not only that but this study opens the door to other scholars to research on incorporating bar code reader into the EFD machine to easy the process of filling in commodity information during selling and printing receipts.	2019-12-19T09:09:39.291Z	2019-09-08T00:00:00.000	{ "year": 2019, "sha1": "171cf9baa928534f7310dd1c5e23c2150f85261e", "oa_license": null, "oa_url": "http://www.mecs-press.org/ijieeb/ijieeb-v11-n5/IJIEEB-V11-N5-4.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "73b6a3f50ade605d1b8289efaed7899393b504f9", "s2fieldsofstudy": [ "Computer Science", "Business" ], "extfieldsofstudy": [ "Business" ] }
28384731	pes2o/s2orc	v3-fos-license	Magnetite-Containing Sulfonated Polyacrylamide as a Nanocatalyst for the Preparation of Biscoumarins Magnetite-containing sulfonated polyacrylamide was easily prepared through polymerization of the corresponding monomers followed by the reaction with Fe3O4 nanoparticles. The characterization of the obtained catalyst was performed by Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). The acidic SO3H moiety was found to be 1.1 mmol per gram of the obtained polymer. The catalytic activity of the polymer was examined for the synthesis of biscoumarin derivatives by two-component one-pot domino Knoevenagel-type condensation/Michael reaction between aldehydes and 4-hydroxycoumarin. Biscoumarins were obtained in high to excellent yields in short time. The work-up procedure of this reaction was very simple. The catalyst is stable (as a bench top catalyst) with easy-handling and it can be used again. Introduction Due to the unique characteristics of magnetite Fe 3 O 4 nanoparticles, they have attracted great attention in the field of biology, medicine, electronics, and catalytic processes. 1However, Fe 3 O 4 nanoparticles respond to external stimuli such as the pH value and temperature, due to the pH-and temperature-sensitive properties.Also, they are unstable in the presence of oxygen at light and coagulation of nanoparticles is usually unavoidable during their use.Therefore, there has been a considerable research effort to stabilize Fe 3 O 4 nanoparticles through coating the surface of such nanoparticles with carbon, precious metals, silica, and polymers.][5][6] Recently, magnetic nanomaterials have emerged as a promising catalysts for various organic and inorganic reactions because of their large surface-to-volume ratio relative to bulk materials and hence the large ratio of atoms available at the surface. 2 These types of catalysts can be easily separated using an external magnet and their catalytic activity remains high even after several reaction cycles.Magnetite catalysts were used as efficient catalytic systems in many chemical transformations including asymmetric hydrogenation of aromatic ketones, 7 the desymmetrization of racemic 1,2-diols through asymmetric benzoylation, 8 synthesis of 14-arylor alkyl-14H-dibenzo[a,j]xanthenes and 1,8-dioxooctahydroxanthene derivatives, 9,10 Sonogashira cross-coupling reaction, 11 synthesis of tetrahydrobenzo[a]xanthen-11-ones, 12 C-N bond formation via aza-Michael addition, 6 and preparation of functionalized tricarboxamide derivatives. 135][16] Among various derivatives of coumarin, biscoumarins have aroused considerable interest.These important compounds were usually prepared from the reaction of aldehydes with 4-hydroxycoumarin.Several types of catalysts were introduced previously for this reaction, such as piperidine, 17 tetrabutylammonium bromide, 18 I 2 , 19 sodium dodecyl sulfate, 20 1buthyl-3-methylimidazolium tetrafluoroborate, 21 1-ethyl-3-(3-sulfopropyl)-benzimidazolium trifluoromethanesulfonate, 22 para-dodecylbenzenesulfonic acid/piperidine, 23 3-methyl-1-(4-sulfonic acid)butylimidazolium hydrogen sulfate, 24 B(HSO 4 ) 3 , 25 polyvinylpyrrolidone-supported nickel nanoparticles, 26 poly(4-vinylpyridine)-supported ionic liquids, 27,28 and cellulose sulfonic acid. 29Also, biscoumarin derivatives have been prepared by the condensation of 1,2-diols and 4-hydroxycoumarin using Pb(OAc) 4 . 30owever, many of these methods have some drawbacks, such as a requirement for either a long reaction time or harsh reaction conditions, provide low yields, include laborious work-up procedures, inefficiency of method when aliphatic aldehydes are used in the reaction, and the use of unrecyclable, hazardous or difficult to handle catalysts. 1. Materials and Methods All chemicals were either prepared in our laboratory or were purchased from Merck and Fluka.Reaction monitoring and purity determination of the products were accomplished by GLC or TLC on silica-gel polygram SILG/UV 254 plates.Gas chromatography was carried out on Shimadzu GC 14-A.IR spectra were obtained by a Shimadzu model 8300 FT-IR spectrophotometer. 1 H NMR spectra were recorded on 400 MHz spectrometer in CDCl 3 .TGA was carried out on a Stanton Redcraft STA-780 with a 20 °C/min heating rate.Melting points were determined on a Fisher-Jones melting-point apparatus.XRD patterns were recorded by a Phillips X-ray diffractometer using graphite monochromatized Cu Kα radiation.A morphological study of the synthesized products was carried out directly by a Hitachi S4160 field emission scanning electron microscope (FESEM).Room temperature magnetic properties were investigated by Lakeshore device in an applied magnetic field sweeping between ±8000 Oe. Preparation of Fe 3 O 4 Nanoparticles To a solution of ferrous chloride (FeCl 2 , 1 M), Na-OH (4 M) was added dropwise with vigorous stirring to produce a black solid product (magnetite Fe 3 O 4 nanoparticles) when the reaction media reaches pH 12.The resulting nanoparticles were carefully decanted and washed repeatedly with doubly distilled water and absolute ethanol and then dried under vacuum at room temperature. 3. Synthesis of (Poly(AMPS-co-AA) @Fe 3 O 4 ) First, in a round bottomed flask (50 mL) equipped with a reflux condenser, 0.051 g (0.214 mmol) of Bz 2 O 2 was added to a solution of acrylamide (15 mmol, 1.066 g), AMPS (6.42 mmol, 1.330 g), and ethanol (25 mL) and the mixture was refluxed for 5 h.Afterwards, Fe 3 O 4 nanoparticles (12 mmol, 2.78 g) were added to the mixture and refluxed for 1 h.Then, the obtained solid was easily separated from the reaction mixture by an external magnet and washed with deionized water and ethanol three times and dried overnight in vacuum at 80 °C. The same procedure applied for the preparation of poly(AMPS-co-AA)@Fe 3 O 4 was also repeated for the preparation of poly(AMPS-co-AA) without addition of Fe 3 O 4 nanoparticles.In this case, the polymer formed in the first step was collected by filtration, washed three times with ethanol, and dried overnight in vacuum at 60 °C. 1. Synthesis of Poly(AMPS-co-AA)@Fe 3 O 4 Poly(AMPS-co-AA)@Fe 3 O 4 was synthesized by free radical polymerization of AA and AMPS monomers Boroujeni et al.: Magnetite-Containing Sulfonated Polyacrylamide ... in the presence of Bz 2 O 2 initiator followed by the reaction with Fe 3 O 4 nanoparticles (Scheme 1).The acidic sites loading in poly(AMPS-co-AA)@Fe 3 O 4 obtained by means of acid-base titration was found to be 1.1 mmol/g. 27,28For comparison purposes, poly(AMPS-co-AA) was also prepared via the same procedure applied for the preparation of poly(AMPS-co-AA)@Fe 3 O 4 without addition of Fe 3 O 4 . 2. Characterization of Poly (AMPS-co-AA)@Fe 3 O 4 Figure 1 shows the FT-IR spectra of Fe 3 O 4 , poly(AMPS-co-AA), and poly(AMPS-co-AA)@Fe 3 O 4 .The IR spectral data of Fe 3 O 4 show two characteristic peaks at 425 and 567 cm -1 , which are due to Fe-O stretching vibrations (Figure 3a). 31As shown in Figure 3b, the IR spectrum of poly(AMPS-co-AA) exhibits peaks at 3346 and 3428 cm -1 (N-H vibrations of amide groups), 1663 cm -1 (C=O vibrations of carbonyl groups), and 1208 cm -1 (S=O vibrations of sulfonic groups). 27,28Also, the IR spectrum of poly(AMPS-co-AA)@Fe 3 O 4 displayed peaks at 3422, 1658, and 1185 cm -1 which are assigned to stretc-hing vibrations of NH and NH 2 , C=O, and S=O, respectively (Figure 3b).In IR spectrum of poly(AMPS-co-AA)@Fe 3 O 4 the appearance of peaks at 429 and 574 cm -1 , attributed to Fe-O stretching vibrations, indicates that Fe 3 O 4 nanoparticles were attached to the polymer chains. Thermal data obtained from TGA analysis are presented in Figure 2 Figure 3 shows the SEM images of synthesized samples.Figure 3a indicates that the most of Fe 3 O 4 nanoparticles are monodisperse and have a spherical crystal morphology with a diameter range between 40-50 nm.With comparison of SEM micrographs of the magnetic poly(AMPS-co-AA) (Figure 3c) with poly(AMPS-co-AA) (Figure 3b), it can be deduced that the polymer chains clearly loaded on Fe 3 O 4 nanoparticles. EDS analyses of poly(AMPS-co-AA) and poly(AMPS-co-AA)@Fe 3 O 4 are shown in Figure 4. EDS of poly(AMPS-co-AA)@Fe 3 O 4 shows that there are no impurities in this catalyst and confirms the presence of S and Fe elements (Figure 4b). the agreement with the card no.88-0315, it seems the resultant particles are pure magnetite.The crystal size of the particles was calculated by line broadening from the XRD pattern using the Debye-Scherrer formula and they were estimated to be between 20-25 nm.The weaker diffraction lines of poly(AMPS-co-AA)@Fe 3 O 4 (Figure 5b) compared with Fe 3 O 4 nanoparticles indicate that the Fe 3 O 4 nanoparticles were covered by amorphous polymer. Magnetic properties of the samples were also studied.Hysteresis loops of Fe 3 O 4 nanoparticles and poly(AMPS-co-AA)@Fe 3 O 4 are depicted in Figure 6.The Fe 3 O 4 nanoparticles exhibited ferromagnetic behavior in saturation magnetization of 63.53 emu/g and a coactivity of 1.76 Oe at room temperature (Figure 6a). The results showed that the saturation magnetization value of poly(AMPS-co-AA)@Fe 3 O 4 (Figure 6b, 48 emu/g) was lower than Fe 3 O 4 nanoparticles due to the interaction of polymer and Fe 3 O 4 nanoparticles. Catalytic Application of Poly(AMPS-co-AA)@Fe 3 O 4 in the Synthesis of Biscoumarins After synthesis of poly(AMPS-co-AA)@Fe 3 O 4 we tried to convert aldehydes to the corresponding biscoumarins in the presence of this catalyst.To optimize the reaction conditions, initially, the condensation reactions of benzaldehyde (1 mmol) with 4-hydroxycoumarin (2 mmol) in the presence of different molar ratios of poly(AMPS-co-AA)@Fe 3 O 4 in various solvents and also under solvent free conditions were studied.The results indicate that this reaction goes well in toluene and the product was obtained in excellent yield in the presence of 0.04 mmol of the catalyst at 90 °C.After establishing the optimal conditions, in order to prove the generality of this method, a variety of aromatic aldehydes bearing electrondeficient or electron-rich substituents on the aromatic ring (Table 1, entries 1-7), heteroaromatic aldehydes such as 2-thienyl and 2-furanyl carbaldehyde (entries 8, 9), and aliphatic aldehydes (entries 10-12) reacted with 4hydroxycoumarin to give the corresponding biscoumarin products in excellent yields. 1 To determine the extent of sulfuric acid groups leaching from poly(AMPS-co-AA)@Fe 3 O 4 one test was performed.Poly(AMPS-co-AA)@Fe 3 O 4 was added to toluene and the mixture was stirred for 2 h at 90 °C.Then, the catalyst was removed by using magnetic field and the filtrate was analyzed for its acid content, which showed a negligible release of the acidic sites.The filtrate was found to be inactive in the reaction of aldehydes with 4-hydroxycoumarin.Thus, this leaching test suggested that the poly(AMPS-co-AA)@Fe 3 O 4 catalyst was a true heterogeneous catalyst without significant acid moieties leaching. Scheme 2 shows a possible mechanism for the preparation of biscoumarins using poly(AMPS-co-AA)@-Fe 3 O 4 .First, the acidic nature of catalyst may facilitate the enolization step of 4-hydroxycoumarin.Second, nucleophilic addition of 4-hydroxycoumarin to the activated aldehyde followed by loss of H 2 O generates intermediate I, which is further activated by poly(AMPS-co-AA)@-Fe 3 O 4 .Then, the 1,4-nucleophilic addition of a second molecule of 4-hydroxycoumarin on the activated intermediate I, in the Michael addition fashion, affords the biscoumarin product.Based on this mechanism, it is clear that in the cases of electron withdrawing groups present on the aromatic aldehyde (Table 1, entries 6, 7), the 1,4nucleophilic addition of a second molecule of 4-hydroxycoumarin on the activated intermediate I is more favored which can finally cause the decrease of reaction time for these types of aldehydes when reacting with 4-hydroxycoumarin. 27,33We believe that the catalytic efficiency of poly(AMPS-co-AA)@Fe 3 O 4 may be attributed to the acidic hydrogen (SO 3 H) as well as Lewis acid properties of Fe 3 O 4 nanoparticles. Very recently, we have introduced carbon nanotubesupported butyl 1-sulfonic acid groups as a heterogeneous catalyst for the synthesis of 1,8-dioxo-octahydroxanthenes. 32Along this line and based on the above encouraging results, we tried to examine the applicability of poly(AMPS-co-AA)@Fe 3 O 4 for the synthesis of 1,8-dioxo-octahydroxanthenes and 12-aryl-9,9-dimethyl-8,9,10,12-tetrahydrobenzo[a]xanthene-11-ones through condensation of aromatic aldehydes with dimedone and β-naphthol.We observed that 1,8-dioxo-octahydroxanthe- [34][35][36] Finally, we investigated the reusability of the poly(AMPS-co-AA)@Fe 3 O 4 catalyst, and it was found that the catalyst could be completely recovered and used again at least six times without any noticeable loss of catalytic activity (Figure 8). In comparison with selected previously known protocols employed for the synthesis of biscoumarins, poly(AMPS-co-AA)@Fe 3 O 4 showed that in addition to having the general advantages attributed to the solid catalysts it has a much higher activity evident in the terms of high yields reached after short reaction times and at mild reaction conditions (Table 2). Conclusion In this study the coating of Fe 3 O 4 nanoparticles with sulfonated polyacrylamide has been described.Magnetization measurements showed that the obtained poly(AMPS-co-AA)@Fe 3 O 4 particles have paramagnetic properties.Poly(AMPS-co-AA)@Fe 3 O 4 showed good catalytic activity in a one-pot domino Knoevenagel-type condensation/Michael reaction between aldehydes and 4hydroxycoumarin, aromatic aldehydes with dimedone, and aromatic aldehydes with dimedone and β-naphthol.Work-up of these reactions is a "green" process because the catalyst was easily separated from the reaction media by the application of an external magnetic source.The high thermal and chemical stabilities of the catalyst, easy preparation, handling, and recycling of the catalyst, high yields achieved, and short reaction times needed are the other obvious advantages of the present method. Fe 2 O 3 , the anchoring amount of Fe 3 O 4 in poly(AMPS-co-AA)@Fe 3 O 4 is about 20 wt.%.The relatively high char yield of poly(AMPS-co-AA)@Fe 3 O 4 also indicates that the incorporation of Fe 3 O 4 into the polymer chains im-parts significant thermal stability to the resulting poly(AMPS-co-AA)@Fe 3 O 4 . Table 1 : Synthesis of biscoumarin derivatives catalyzed by poly(AMPS-co-AA)@Fe 3 O 4 .Isolated yields.b All products are known compounds and were identified by comparison of their physical and spectral data with those of the authentic samples. Table 2 : Comparison results of poly(AMPS-co-AA)@Fe 3 O 4 with other catalysts reported in the literature in the condensation of benzaldehyde with two equivalents of 4-hydroxycoumarin.and 12-aryl-9,9-dimethyl-8,9,10,12-tetrahydrobenzo[a]xanthene-11-ones were obtained in high yields in ethanol at room temperature in the presence of 0.05 mmol of poly(AMPS-co-AA)@Fe 3 O 4 (Scheme 3).It is worth noting that, to the best of our knowledge, relatively few examples were reported on the synthesis of xanthene derivatives at room temperature.Usually, these reactions need high temperatures, long time or an additional source of energy (ultrasound or microwave irradiation).	2018-04-03T01:11:52.269Z	2017-09-02T00:00:00.000	{ "year": 2017, "sha1": "bcbd484991c8274018895cd7aad61d4e5cb42385", "oa_license": "CCBY", "oa_url": "https://journals.matheo.si/index.php/ACSi/article/download/3437/1423", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "bcbd484991c8274018895cd7aad61d4e5cb42385", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
7630930	pes2o/s2orc	v3-fos-license	The Effect of Injury-Related Characteristics on Changes in Marital Status after Spinal Cord Injury. BACKGROUND Spinal cord injury (SCI) imposes a significant burden on the social and marital life. Here, we assessed the divorce rate and changes in marital status among a sample of Iranian individuals with SCI. METHODS Referred patients to Brain and Spinal Cord Injury Research Center were invited to participate in this cross-sectional investigation. The Main exclusion criteria were coincidental brain injury, history of chronic diseases before SCI and substance use. Demographic characteristics (including age, gender, educational level, marital status before and after injury and duration of marriage) and Injury characteristics (level of the injury, American spinal injury association (ASIA) scale and Spinal cord independence measure III (SCIM)) were collected. RESULTS Total of 241 subjects with SCI participated in this investigation (164 (68%) male and 77 (32%) female). Among men, 16.5% [95% CI: 10.81%-22.18%] and among women 18.2% [95% CI: 9.58%-26.81%] got divorced after injury. Duration of marriage before injury was significantly related to lower divorce rate (P< 0.001 and 0.016 in men and women, respectively). Injury characteristics had no relationship with marital longevity. Age was a protective factor against marital dissolution only in men (P< 0.004). CONCLUSION Our study revealed the divorce rate of 17% [95% CI: 13%-20.9%] after SCI in a sample of Iranian population. The protective influence of age in maintenance of marriage was only detected in men, which proposes existence of a sexual polymorphism in the role of age. Divorce rate was similar between two genders and injury characteristics were not related to divorce rate as well. Introduction Spinal cord injury (SCI) has a noticeable effect on medical, social, psychological and economic conditions among affected individuals (1,2). Higher rate of divorce has been demonstrated after SCI (3) which may affect patients' mental health as well. The association between mental health and marital status has already been described (4,5). However, while some investigations have de-scribed the mental health advantages of being married (5) other studies do not support the relationship between marital status and overall wellbeing (6,7). Marriage has a positive impact on health because of greater social support in the context of disability (8). Being married among patients with SCI has been reported to be associated with higher life satisfaction and quality of life and better adjustment (9,10). The estimation divorce rate after SCI has been reported to be 1.5-2.5 times higher than that of the general population (11). However the factors that are associated with divorce rate are poorly categorized. Higher divorce rate occurs mostly in the first three years after SCI (12,13) which illustrates the effect of post injury duration. Other factors such as gender may affect patients' mental health after marital transitions since it has been demonstrated that dissolution of marriage after SCI may be experiences differently by men and women (14). Previous studies have tried to describe the factors that are associated with divorce after SCI. In this regard, demographic characteristics (including younger age at marriage, being female and lower education) and injury characteristics (indicative of more severe injuries) have been shown to be related to higher divorce rate (3). However social and cultural issues and the conditions that divorced couples should go through after divorce vary among different nations and may affect the probability of divorcing. Because of these cultural differences, the changes on marital status after SCI and its related variables should be evaluated in each nation separately. In this study, we tried to assess the changes in marital status among Iranian men and females after SCI. Our purpose was to identify the factors that may affect divorce rate and marital longevity. To our knowledge, this is the first study which investigates changes in marital status and its related factors in a sample of Iranian population with disability. Study Design and Participants In this cross-sectional study, data were collected from men and women with traumatic SCI referred to Brain and Spinal Cord Injury Research Center (Tehran, Iran) which is tertiary hospital-based rehabilitation center. Total of 241 patients were recruited. Inclusion criteria were traumatic spinal cord injury and age above 18 years old. Exclusion criteria included coincidental brain injury, non-traumatic SCI, previous history of severe chronic diseases before injury occurrence (cancer, mental disorders, renal failure, liver dysfunction etc.). Those with history of addiction to illegal drugs or alcohol before injury were excluded as well. Procedure of sampling was performed based on patient selection according to inclusion and exclusion criteria. According to previous study (15), the prevalence of SCI is 1.2-11.4 per 10,000 people and by considering the population of Tehran to be 8 million people, the estimated population size of patients with SCI is 970 and the calculated sample size with 5% error will be 275. However, in this investigation, 241 patients could be recruited. Data were collected by interview from March 2014 to August 2014. There were some concerns about patients' responsiveness when addressing marital and familial issues. To increase responsiveness, patients were assured about the confidentiality of their information. Informed consent has been obtained from each individual before enrollment. Participation in this investigation was voluntarily. This study was approved by Ethics Committee of Tehran University of Medical Sciences. Demographic characteristics and Marriage features Demographic characteristics including age, gender, educational level of the patients and their spouses were collected during interviews with direct questions. Educational level was classified as: illiterate, primary school, high school and collegiate educations. Marital status before and after injury, and duration of marriage were asked and were indexed into pre-prepared forms. Patients were asked to describe if they have got divorce after injury or they are living separately from their spouses without official divorce. Being widow/widower has also been indexed in pre-prepared forms. Injury characteristics including time since injury occurrence were also indexed in these forms. Neurological assessment Completeness of injury was classified as either complete (no preserved sensory or motor function) or incomplete (variable motor function pre-served below the neurological level of injury) (16). Level of injury was assessed with clinical examinations and magnetic resonance imaging (MRI) and was confirmed by expert neurologists. Patients were also classified according to American Spinal Cord Injury Association (ASIA) impairment scale (17) determined based on clinical examination. In this classification, ASIA-A indicates complete injury with no preserved motor or sensory function below the neurological level. ASIA-B describes incomplete injury in which only sensory function is preserved below the neurological level. ASIA-C illustrates preserved motor function in which more than half of key muscles below the neurological level have a muscle grade less than 3. ASIA-D indicates preserved motor function in which at least half of key muscles below the neurological level have a muscle grade of 3 or more and finally ASIA-E represents normal motor and sensory function. Only ASIA-A represents complete injury (18). Excellent intrarater reliability (0.9) and Criterion Validity (0.8) of this measurement tool have been demonstrated (19). Spinal cord independence measure III (SCIM) was used to evaluate patients' independency and ability in conventional daily activities (20). This instrument consists of three subscales: self-care (0-20), mobility (0-40 scores) and respiration and sphincter management (0-40 scores) which comes to a maximum score of 100. Feeding, bathing, dressing and grooming are evaluated in self-care domain. Respiration, bladder and bowel management along with ability of use toilet are assessed in the domain of respiration and sphincter management. Indoor and outdoor tasks are evaluated in the domain of mobility. Higher scores illustrate more independency (21). This measurement tool has been shown to have an acceptable reliability with Cronbach's alpha above 0.7 and validity with correlation coefficient of 0.9 (22) Statistical Analysis All statistical analysis was performed using SPSS software, Version 18.0 (SPSS Inc., Chicago IL, USA). Categorical data were expressed by percentages and continuous quantitative values were expressed by mean± standard deviation (SD). Pear-son Chi square test was used to compare categorical data. Comparison of means was performed using one-way analysis of variance (ANOVA). Correlation test was used to identify the relationship between continuous quantitative variables. Confidence interval (CI) of the prevalence of divorce between subgroups as categorical variables (classified based on gender, injury level (cervical, thoracic and lumbar)), ASIA scale (A, B, C, D) and completeness of the injury) was measured using Kay's excel spreadsheet of Confidence Intervals for Proportions. CI in continuous variables was measured using one-sample t-test. The factors that may affect marital status are social interactions, income, previous familial problems and having children which were not evaluated in this study and therefore no adjustments for these confounders was exerted. Partial correlation with adjustment for age was performed to assess the effect of duration of marriage on divorce rate. Results Total of 241 subjects with SCI participated in this investigation (164 (68%) male and 77 (32%) female). There was no significant difference in the mean age between two genders (P<0.16). Similarly, educational level was not significantly different between men and women (P<0.58). Times since injury, the level of injury and ASIA scores were also similar between male and female individuals (P< 0.77, 0.11 and 0.08, respectively). Table 1 illustrates the demographic and injury-related characteristics in patients with SCI who participated in this investigation. Completeness of injury was also similar between men and women (P< 0.33). Mean SCIM score was 47.58± 23.78 in males (95% CI: 43.40-51.76) and 43.52±25.32 in females (95% CI: 37.44-49.61) and showed no significant difference between two genders (P< 0.26). Among men with SCI, 113 (68.9%) subjects were married before injury and 51 (31.1%) were single. After injury 27 patients got divorce and in two patients their spouses reported to be dead. However, three cases of marriage occurred in males patients after injury. All these patients, who had marriage after injury, had incomplete injury with ASIA C and SCIM score higher than 70. Marital status after injury in men was married in 87 (53%) and non-married in 77 (47%) of patients (48 single, 27 divorced and 2 widowers). Divorce rate among Iranian men with SCI was 16.5% (95% CI: 10.81-22.18). Among those men who were married before injury and remained being married after injury, the duration of marriage before occurrence of accident was 13.84± 10.29 years while duration of marriage among those who were divorced after injury was 5.94±8.21 years. Longer marriage duration before injury was significantly related to marriage maintenance (P<0.001). However level of injury and its completeness showed to have no relationship with marriage maintenance (P<0.53 and 0.28, respectively). Similarly, divorce rate was not associated with injury level, completeness and ASIA score (P<0.56, 0.70 and 0.12, respectively). Older ages was related with higher probability in maintaining marriage (P< 0.004). In fact, those men who were older and had a longer duration of marriage be-fore injury had higher chances to keep their marriage but divorce probability was higher among those with younger age and shorter marriage duration before accident. Table 2 illustrates the factors related with marriage maintenance after SCI. Among women, 56 (72.7%) subjects were married before injury and 21 (27.3%) were single. After accident 13 patients (16.9%) got divorced, one had marital separation (without official divorce) and five women lost their spouses due to the death of their husbands (6.5%). One case of marriage occurred after injury in a 32 year old woman with post injury duration of 17 years who had incomplete injury at L2 level with ASIA C and SCIM of 74. So, the number of married women after injury reached 38 (49.4%) and the number of non-married females (single, divorced, separated or widowed) reached 39 (50.6%). Divorce rate among Iranian women after SCI was 18.2% (95% CI: 9.58-26.81). Similar with men, higher marriage duration before injury was associated with higher probability of marriage maintenance in women with SCI (13.59±8.4 years among those who continued being married after injury and 7.25±6.98 among those who had a terminated marriage, P<0.016). Unlike men, age was not a factor affecting the maintenance of marriage in women (Table 2). Discussion The largest proportions of patients before injury were married in our investigation in Iranian population whereas Krause et al. (23) showed larger number of single individuals in the United States. One reason for this difference is that in modern countries like U.S, couples may live together without official marriage but in Islamic countries registered marriage is an obligation. Our study revealed 'age' as a factor positively related with marriage longevity which is in line with Karana-Zebari et al.'s report (24). Higher ages were associated with lower divorce probability in general population (25) and our study illustrated the similar influence of age on marriage longevity. This is maybe because of a better adjustment with environment and a more stable life in older ages. However here we detected this protective effect of age only among males. To our knowledge, our study is the first showing a sexual polymorphism in the role of age in marriage maintenance among patients with SCI. To understand the reasons of more stability of marriages in older ages when male partner is spinal cord injured requires investigating the cultural issues and familial roles of partners. Further investigation with consideration of social and familial relationships should be performed to clarify this sexual polymorphism. Higher educational level was associated with lower risk of divorce but we found no relationship between educational level and probability of marriage maintenance in Iranian population with SCI (24). Moreover, similar to previous study (24), our investigation found no influence of injury level, ASIA score and independency (SCIM score) on marriage longevity. The insignificant effect of all these factors may be explained when considering the social concerns that divorced individuals may encounter in Iranian culture. Further investigation with adjustment for these social factors should be performed to clarify the effect of educational level and injury characteristics on marital status after SCI. Divorce rate of general population in Iran in 2013 has been reported to be one in each five marriages (20%) (26). Here, we have shown a divorce rate of 17% among patients with SCI which shows comparable and approximately similar rate between general population and patients with SCI. Karana-Zebari et al. (24) also showed longer duration of pre-injury marriage as a protective factor against marital dissolution which is in consistency with our results. While DeVivo et al. (12) reported higher divorce rate among those patients with SCI with lower educational level, we could not identify educational level as a protective factor against divorce. When comparing the results on the effect of educational level, it should be considered that a large proportion of Iranian population with SCI are illiterate or only have finished primary school (49.4% in men and 48.1% in women). This significant difference in the percentages of people with low educational level between Iran and modern countries may cause such controversies between our results and previous literatures (12). DeVivo et al. (12) also showed lower divorce rate among those with low injury level (at lumbosacral injuries) which contradicts with our findings, since we detected no association between injury level and marital longevity. Here, we have concluded that the negative image of divorce which is imposed through cultural issues has resulted in higher resistance and adjustment in Iranian families. In this study we did not asked the participants about the reasons for their divorce (or similarly for not getting a divorce) and further investigations are required to clarify the social and cultural aspects of this controversy. In our study, four patients married after injury (three men and one woman). As Crewe and Krause (27) have already described, those patients with SCI who marry after injury are measurably different in several respects from those who remained single. In our study all these four patients had incomplete injury at lumbosacral level (ASIA C) with acceptable independency (SCIM>70). The probability of marriage for those single patients with SCI who have complete injury at higher levels is noticeably low. Those marriages that occur after injury compared with pre-injury marriages have greater satisfaction with their sex lives, living arrangements and social lives even when the severity and the duration of injury were similar (28). In our study, injury characteristics were not major predictors of divorce. Previous literatures have supported that other factors including lower social interactions and fewer activities (29) may play a major role the success of their marriage rather than injury characteristics. Here, we did not evaluate the level social integration and activities in these patients but our study have similarly illustrated the non-significant role of injury characteristics in after SCI. One source of bias in our study was existence of some concerns about patients' responsiveness. However, patients were assured about the confidentiality of their information at the time of enrollment to increase patients' responsiveness. Since our research center is a referral center and patients from all over the country are referred to this center, our sample can present the Iranian population with SCI. The number of participants (241) by considering the prevalence of SCI in Iran is also admissible. The results of our investigation can be further generalized to the Iranian population with SCI. The major practical implication of our study is to identify the major factors that may affect marital status and to implement supportive strategies in the sensitive groups. Our study showed that younger individuals with shorter marriage duration are more susceptible to marriage dissolution and may require more support. Study Limitations In this cross-sectional study, no follow-up visits were planed and the recorded marital statuses of patients were indexed at the time of interview. Further investigations with long term follow-up are needed to clarify the role of post injury duration on marriage by calculating the divorce rate in each pre-defined time intervals after injury. Cohort studies to compare the incidence of divorce between patients with SCI and general population may enlighten the impact of SCI on changes in marital status. Conclusion This study evaluates the factors affecting marital status after SCI. Our results show that age (only in males) and duration of marriage before occurrence of injury are protective factors against divorce. Divorce rate was rather similar between men (23.8%) and women (23.2%). Injury characteristics including injury level, completeness and patients' independency (SCIM score) were not associated with divorce probability. Ethical considerations Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.	2018-04-03T05:20:51.595Z	2015-10-01T00:00:00.000	{ "year": 2015, "sha1": "96da0a4fda78c435a83109205989606910a6835f", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "96da0a4fda78c435a83109205989606910a6835f", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
7169038	pes2o/s2orc	v3-fos-license	Smoking Use and Cessation Among People with Serious Mental Illness Smoking rates in people with serious mental illness (SMI) are disproportionately high compared to the general population. It is a leading contributor to the early mortality in this population. Smoking cessation rates are low in this group, though patients are motivated to quit. Unfortunately, health care providers do not always prioritize smoking cessation for this population. This review provides an overview of prevalence rates, biological effects that maintain smoking, and evidence-based treatments for smoking cessation in SMI. In addition, objective and qualitative data from a chart review of 78 patients with SMI prescribed smoking cessation treatment at one community mental health center are described. Of these, 30 (38.5 percent) were found to either quit (16/78) or reduce (14/78) smoking. Varenicline appeared to be particularly effective. Review of the literature and results of this study suggest that smoking cessation pharmacotherapies are effective for SMI patients and should be offered to those who smoke. INTRODUCTION Since the 1964 landmark U.S. Surgeon General report outlining evidence of harmful health effects of smoking, comprehensive programs and public campaigns have greatly reduced smoking prevalence. Nonetheless, smoking continues to be the leading cause of preventable disease and death in the United States [1]. Currently, the prevalence rate of cigarette smoking for women and men is 20.5 percent and 15.3 percent, respectively [2]. The prevalence of smoking in people with mental illness is much higher [3,4], and people with mental illness comprise a large majority of cigarette smokers [5]. Smoking rates in this group have declined much less than in those without mental illness [6]. Serious mental illness (SMI) is defined as a diagnosable mental disorder of sufficient duration to meet diagnostic criteria specified within the Diagnostic and Statistical Manual (DSM), resulting in substantial functional impairment [7]. A meta-analysis of mortality in mental disorders showed a significantly higher rate of mortality than in the comparison population, and a total of 67.3 percent of deaths were from medical causes [8]. People with SMI have a reduced lifespan of at least 10 years [8,9]. Smoking, along with obesity, contributes to the health risks in this population [10,11]. There is a perception that patients with mental illness are not interested in quitting [12], and few mental health providers offer smoking cessation treatment. However, there are effective evidence-based interventions for this population. This review summarizes the literature on smoking and smoking cessation in patients with SMI. The paper also describes the experience of patients with SMI who received pharmacotherapy for smoking cessation at one community mental health center. PREVALENCE The U.S. 2009-2011 national survey of substance use and health estimated a prevalence of cigarette smoking at 36.1 percent in those with any behavioral disorder [13]. The Substance Abuse and Mental Health Services Administration (SAMHSA) reports a 50 percent prevalence [3]. Data from the National Comorbidity Survey found a smoking rate of 55.3 percent in those with lifetime mental illness [4]. The association of smoking and psychiatric diagnoses remains even after controlling for other substance use, which is highly prevalent in people with mental illness [14]. Smoking prevalence increases with severity of mental illness, and rates are high in the SMI population [15]. In one sample of 991 SMI patients, the prevalence was 64 percent in schizophrenia and 44 percent in bipolar disorder patients [16]. Smoking-related mortality is also high in this population, with half of all deaths attributable to smoking [3]. In a large cohort of more than 600,000 patients, tobaccorelated conditions comprised 53 percent of total deaths in schizophrenia, 48 percent in bipolar disorder, and 50 percent in major depressive disorder patients [17]. Chronic lung disease and obstructive sleep apnea are highly prevalent in the SMI population [18,19] and contribute to the increased smoking-related mortality risk. Although people with mental illness are as motivated to quit as the general population [20], smoking quit rates are lower [6]. Data from the 2007 National Health Interview Survey on 23,393 adults showed lower success rates of quitting in mentally ill people [15]. Possible reasons for high prevalence of smoking and low quit rates in the SMI population are explored in the next section. FACTORS LEADING TO MAINTENANCE OF SMOKING IN MENTALLY ILL PATIENTS Smoking incidence in patients at the onset of illness is comparable to the general population but increases significantly as early as 1 year after treatment initiation [11]. Both biologic and social factors likely play a role in this escalation. Several studies suggest a strong predisposition to smoking and less likelihood of cessation in people with depression [21]. The worsening of symptoms seen in depressive disorders [22,23] leads to the hypothesis that tobacco is a form of self-medication. Nicotine in cigarette smoke binds to nicotinic acetylcholine receptors in the brain, and while it causes an immediate increase in receptor activity, there is a long-term decrease due to receptor desensitization. It is postulated that increased cholinergic activity causes a state of depression and smoking alleviates symptoms due to nicotinic acetylcholine receptor desensitization [24]. However, there is evidence that clinically depressed smokers can receive treatment for smoking without worsening in depressive symptoms [25]. Among patients with schizophrenia, nicotine may normalize neuronal deficits; this effect may contribute to the high rate of smoking in this population [26]. Nicotinic acetylcholine receptors have been found to be important for maintaining optimal cognitive performance [27], and so smoking may improve cognitive deficits seen in schizophrenia. Cigarette smoking is also thought to normalize several sensory deficits seen in schizophrenia such as abnormal auditory sensory gating (P50) and smooth pursuit eye movements [28,29]. The P50 system measures the response with 50ms latency to paired auditory stimuli; in people with schizophrenia, the normal decrease in response to the second stimuli does not occur. Smooth pursuit eye movements refer to the movement of the eyes when tracking an object. It is abnormal in those with schizophrenia due to deficits in attention and failure of inhibitory cortical control. In addition, the role of nicotine in the dopaminergic mesolimbic pathway is well characterized [30]. The net effect of nicotine is to increase dopamine, and this may result in use of smoking to ameliorate negative symptoms or, alternatively, represent an attempt to overcome dopamine blockade by antipsychotic medications. Also, nicotine alters pharmacokinetics of many psychotropic medications, resulting in lower drug levels [31] and leading to a hypothesis that people with SMI use smoking as a means to alleviate adverse effects from antipsychotics. Haloperidol, a potent dopamine blocking agent, is associated with an increase in smoking [32], and clozapine, a highly effective atypical agent, is associated with a decrease in smoking [33]. Clozapine impacts the P50 system [34], and this may also contribute to its role in smoking reduction. Co-existing substance use is highly prevalent in people with mental illness, and there is some suggestion that treatment for substance dependence is associated with lower odds of quitting smoking [35]. However, this may be due to providers not addressing smoking during treatment for active substance dependence. A meta-analysis of 19 studies evaluating outcomes for smoking cessation treatment for people in addictions treatment or recovery showed positive results for short-term smoking cessation and long-term abstinence from alcohol and illicit drugs [36]. Finally, social reasons contribute to smoking in the SMI population. Historically, mental health providers have been permissive toward tobacco smoking among patients [37]. A survey of mental health providers at community mental health settings showed that only a minority advised or assisted in smoking cessation [12]. Barriers cited were a lack of training of providers and a perception that patients were not interested in quitting. In recent years, however, mental health centers are moving toward becoming tobacco-free [38], and this will help foster the recognition of tobacco dependence as a serious health problem for mentally ill patients. Given the social and biologic predisposition to smoking and its maintenance, it is not surprising that people with serious mental illnesses have a very high prevalence. However, there are effective evidence-based treatments for this population. Treatment of Tobacco Dependence in the General Population Effective tobacco cessation treatments exist in the general population and in those with SMI [39]. Tobacco cessation treatment requires involvement of health care professionals at multiple levels. Combination of medications and counseling is more effective than either alone. Counseling includes practical problem solving skills and supportive engagement and can be delivered in telephone, group, and individual counseling formats. Bupropion, nicotine replacement therapy (NRT), and varenicline are all considered first line medications. Bupropion is an antidepressant, but effect on smoking cessation seems independent of antidepressant effect [40]. Varenicline is a nicotinic receptor partial agonist and simultaneously stimulates dopamine and blocks nicotine receptors. A 2013 large Cochrane review of a total of 101,804 adults showed that all three agents are superior to placebo; all forms of NRT are effective; and varenicline is superior to bupropion and to single type of NRT but not combination NRT. Adverse effects from the active treatments were not significantly different from each other or significantly higher than placebo [41]. Combination of long-acting NRT such as a patch and a short-acting NRT such as a gum, lozenge, or inhaler is more effective than a single type of NRT [42]. There is some evidence that combination of varenicline with the nicotine patch, a form of NRT, is more effective than varenicline alone [43]. In 2009 and 2011, the U.S. Food and Drug Administration (FDA) issued a boxed warning for varenicline alerting health care professionals to unusual mood or behavior changes and suicidality; recently, it also added a risk of seizures and reduced tolerance to alcohol [44]. However, many studies have demonstrated superior efficacy of varenicline without increased neuropsychiatric adverse effects [45][46][47][48]. The FDA is continuing to evaluate the risk of neuropsychiatric effects of varenicline, and further updates to the label are pending. In weighing risks, the prescribing label notes the significant efficacy of varenicline and that the benefits of quitting smoking are "immediate and substantial." Given the high relapse rate after smoking cessation treatment, multiple approaches have been studied for relapse prevention. A 2013 Cochrane review did not find evidence for any specific behavioral intervention; extended treatment with varenicline had some efficacy in preventing relapse [49]. Treatment of Tobacco Dependence in Seriously Mentally Ill People Evidence shows that smoking cessation treatments are effective in the SMI population. In large meta-analyses, response to treatment is seen to be less robust in people with SMI compared to those without mental illness [41,50]; however, individual studies have shown similar success rates in people with and without mental illness [51]. For patients with schizophrenia, a Cochrane review showed significantly high cessation rates with varenicline and bupropion compared to placebo (risk ratio RR 4.74, 95% confidence interval CI 1.34 to 16.71 and RR 3.03, 95% CI 1.69 to 5.42 respectively). Evidence was insufficient for the efficacy of NRT in this group [50], though there may be some utility of NRT for long-term maintenance [52]. Varenicline appears to be well tolerated in those with SMI [53,54]. Varenicline may even improve sensory deficits and some cognitive deficits in schizophrenia [55]. There is insufficient evidence to recommend specialized psychosocial interventions tailored to people with schizophrenia [56]. There is some evidence for utility of cognitive behavioral treatment and motivational interviewing techniques along with pharmacological treatment [57][58][59]. Financial contingency reinforcement has shown short-term improvement in smoking reduction and cessation [50]. Maintenance treatment with continued pharmacological therapies and cognitive behavioral treatment was effective in reducing high relapse rates [60,61]. A longer duration of treatment may be effective in maintenance of cessation. Smoking reduction is also a potential goal. Varenicline recently has been shown to increase abstinence rates in smokers who initially were only willing to reduce smoking [62]. This strategy of initial reduction may predict future abstinence in the schizophrenia population as well [63]. For patients with current or past depression, NRT and buproprion have shown efficacy in smoking cessation [64]. Varenicline also was found to increase smoking cessation rates in stably treated depressed smokers without symptom recurrence [65]. Behavioral mood management and staged care interventions with motivational feedback and psychological counseling may also be effective [64,66]. Patients with bipolar disorder also have high rates of smoking and increased vulnerability to treatment failure [67]. Some treatment trials of people with SMI included those with bipolar disorder, but there are very few trials specifically studying smoking cessation treatments in this group. One small trial showed varenicline was safe and showed efficacy for smoking cessation at 3 months compared to placebo [68]. The majority of data show no worsening of psychiatric symptoms with smoking cessation treatment [50]. Abstinence from smoking is not associated with an increase in depressive episodes in those with a history of depression [23], and, in fact, symptoms have been shown to improve with cessation [25,69]. However, more research is needed on long-term effects of smoking cessation on mental health, especially after the initial years of smoking cessation. Electronic cigarettes have been considered as a mechanism for harm reduction in smokers. There is limited evidence of efficacy of electronic cigarettes as a means to smoking cessation [70]. But there is concern that they may serve as a gateway to continued smoking or even serve to initiate smoking behavior. Currently, these products are unregulated, although the FDA recently announced its intention to regulate the devices [71]. At this time, given that the risks and benefits of electronic cigarettes are unknown, they are not recommended for use in treatment. Purpose The Connecticut Mental Health Center (CMHC) adopted a smoke-free policy in 2008 [72]. As part of a multifaceted approach, patients who wished to quit smoking were provided either a type of NRT or varenicline by the center pharmacy free of cost. A chart review was conducted to describe the experience of patients who were prescribed these medications for smoking cessation. Method Seventy-eight patients who received smoking cessation pharmacotherapy in 2009 were identified from pharmacy records. Each chart was reviewed retrospectively by one of two reviewers. Data was obtained from the pharmacy database and the physical chart. Information was collected on type of pharmacotherapy, side effects, number of cigarettes smoked, number of days quit, motivation to quit, selfreported cravings, and self-reported emotional state during smoking cessation. The study was approved by the Yale Human Investigation Committee. Results Among the 78 people who received smoking cessation treatment, there were 32 males and 46 females. Mean age was 45. The major psychiatric diagnoses were schizophrenia and other psychotic disorders (42.3 percent), major depressive disorder (28.2 percent), and bipolar disorder (15.4 percent). Forty-four patients were prescribed varenicline and 34 NRT as the initial pharmacotherapy. Sixteen of 78 people (20.5 percent) quit smoking, and an additional 14 (17.9 percent) reduced smoking, making a total of 30 people (38.5 percent) who either quit or reduced. The number was higher with varenicline (22/44) than with NRT (8/34) (Table 1). Also, three in the NRT group who failed treatment subsequently initiated varenicline and two reduced smoking, bringing the total quit/reduction number for varenicline to 24 (54.5 percent). Higher quit rates were seen with varenicline irrespective of diagnostic group ( Table 1). The average duration of varenicline treatment was 63.5 days for all patients. Among the 12 people who quit on varenicline, three relapsed within a month soon after varenicline was stopped due to adverse effects. Among 12 patients who reported side effects, nine were on varenicline but only four reported neuropsychiatric complaints (irritability, nightmares, anxiety, paranoia). Two people reported nausea. At least two people did not start the prescribed varenicline, citing fear of psychiatric side effects. a Three patients who initially tried NRT with no response subsequently tried varenicline, and two of them reduced smoking; including these patients, the overall quit/reduction rate for the sample is 32/78 (41%), reduction rate for varenicline is 12/44 (27.3%), and total quit/reduction rate for varenicline is 24/44 (54.5%). b Three of the 12 people relapsed within 30 days following early discontinuation of varenicline. c Includes people with anxiety disorders, unspecified mood disorders and primary substance use disorders. Among motivators to quit, personal or family history of physical illness such as lung disease, cardiac illness, and cancer was common. Less common reasons were planning pregnancy, preparation for surgery, and housing restrictions. Many people were motivated to initiate treatment in spite of previous failed attempts. People who quit reported feeling good, though some complained about weight gain. Even people who did not reduce smoking reported decrease in cravings on varenicline and wanted to continue treatment. A few patients received specific education and counseling to address anxiety or other symptoms experienced from nicotine withdrawal. People who were using other addictive substances did not report increased cravings for those substances. Many patients were on more than one psychotropic medication and did not report any medication changes during the immediate smoking cessation treatment period. Discussion In this naturalistic follow-up of patients on smoking cessation pharmacotherapy at CMHC, a clinically significant percentage of people with SMI achieved abstinence with pharmacotherapy and no other targeted interventions. An equal number of people reduced smoking, which has health benefits and may lead to future abstinence. Quit rates were lower than reported in studies of people without mental illness, consistent with lower rates seen in some studies of individuals with SMI. This SMI group showed motivation to quit for reasons similar to people without mental illness. However, the limited qualitative data on self-reported motivators to quit and cravings during treatment did not allow for assessment of these factors in predicting smoking cessation. Overall, patients had a positive experience with treatment, regardless of actual outcome on abstinence. CONCLUSION Patients with SMI have a much higher prevalence of cigarette smoking than the general population, and this contributes to the already high medical morbidity and mortality in this group. Smoking is hypothesized as a form of self-medication in people with SMI. But SMI people are motivated to quit, and treatments are effective for smoking cessation and maintenance. SMI people report an overall positive experience with treatment. Mental health providers, as well as primary care providers, should routinely offer evidence-based treatments for smoking cessation for people with SMI.	2017-09-16T15:34:52.740Z	2015-09-01T00:00:00.000	{ "year": 2015, "sha1": "db43f8bf51344b965f278af8342ce10bc3c9641a", "oa_license": "CCBYNC", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "db43f8bf51344b965f278af8342ce10bc3c9641a", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
213278085	pes2o/s2orc	v3-fos-license	Climate research in Arctic regions of Siberia The paper shows the impact of recent changes in air temperature and wind speed on the living conditions of human population in the Arctic regions of Siberia (to the north of 66°N and within 66-162°E). The combined effect of subzero temperatures and wind speed on the thermal status of the exposed surface of the human body was evaluated as a proxy indicator for the severity of the climate. As average monthly temperatures increase, this indicator decreases in the Arctic territories of Siberia. The dynamics of daily fluctuations of threshold values of the weather severity index from October to April from 1966/67 to 2017/2018 has been analysed for the Verkhoyansk station. We found a decrease in the number of days prohibiting and an increase in the number of days restricting a person’s stay outdoors. In the more recent period from 1981/82 to 2017/18, a noticeable decrease in the number of days with prohibitive weather conditions was observed. At the same time, little variation was observed in the average number of days with restrictions in outdoor activities over longer time intervals: the difference between 1936-1964, 1966-2018, and 1981-2018 periods was less than five days. Introduction The geographical position of the Arctic regions of Siberia (66-162°E longitude) has extremely unfavourable climatic conditions for humans. The combination of excessive humidity and lack of heat contribute to the formation of Arctic deserts, tundra and forest-tundra. These landscapes are retreating in the eastern part of the territory (80-162°E longitude) to the north of the Arctic circle, being replaced with Northern taiga landscapes [1,2]. Here, long (approximately six months) periods with lack of ultraviolet and daylight, low temperatures combined with frequent strong winds, and sharp weather and climatic contrasts influence human activities. Based on the combined effect of these factors, territories are ranked by the level of climate discomfort as very strong, severe and extremely severe. Previous studies identified an increase in annual temperatures due to fewer days with an average daily temperature below minus 30 ºC. The highest rates of annual temperature increase (up to 1.2ºC over ten years) were observed in the Arctic deserts. Other territories warmed by 0.4-0.6ºC over 10 years [3]. These massive temperature fluctuations in recent decades highlight the importance of assessing their impact on the living conditions in the area. Some publications [4][5][6] suggest a possible reduction in the frequency of the most severe conditions. At the same time, frequent and rapid temperature changes impose additional requirements for the infrastructure and compliance with policies and regulations for working outdoors. The purpose of this work is to evaluate the dynamics of fluctuations in the number of days prohibiting and restricting human activity outdoors and quantify these changes and extreme events in the Arctic regions of Siberia. Objects, data and methods Changes in air temperature for October-April in the Arctic regions of Siberia were analyzed based on observation records from 23 hydrometeorological stations [7]. The Arnoldi indicator [8] was used to quantify the combined effects of subzero temperatures and wind speed on the thermal status of the exposed surface of the human body. The impact of the weather conditions is most severe at the threshold values of this indicator: the range between 30 and 45 units is considered restrictive, and above 45 units − prohibitive for human activity outdoors. The number of days with such weather conditions is also considered. Restrictive and prohibitive conditions are associated with the risk of frostbite, reduced labor efficiency and increased likelihood of respiratory disorders and exacerbation of chronic illnesses. The Arnoldi indicator is calculated as follows: where t -is the temperature, °C; v -wind speed, m/s. Daily meteorological data recorded the closest to 13:00 of the local time were used to calculate TA to characterize typical workday conditions. The dynamics of daily values of the Arnoldi indicator was analyzed for the October-April period from 1966/67 to 2017/2018 (52 years) and from 1981/82 to 2017/18 years (37 years) for the Verkhoyansk station [8]. The latter time interval is the closest to the current climatic conditions [9]. The null hypothesis was to find no significant difference between the mean values for the two-time intervals. The change in air temperature was estimated using regression analysis to determine the statistical significance of the trend (p < 0.05) and the proportion of variance of the dependent variable explained by the model (R²). In the analysis of inter annual oscillations, extreme values were identified based on the standard deviation of a series of more than 2 σ. Results and discussion Air temperature regime in the polar Siberia varies significantly across the region. The average annual temperature in the west (station Marresale, 69°43'N, 66°48'E) is much higher than in the intermountain depressions in the east (station Verkhoyansk, 67°34'N, 133°24'E). Average January temperatures vary from -48.6°C to -21.8°C. To the west of 80°E longitude, average January temperature does not fall below -27°C, and to the east of 80°Eit varies from -28°C in the Yenisei River valley (station Igarka, 67°28'N, 86°34'E) to - As a result of increasing average monthly temperatures, as well as some decrease in wind activity, there was a minor decrease in the weather severity index in the polar regions of Siberia. It does not have a significant impact on the level of climate discomfort. The lowest number of days rated as restrictive for human activity outdoors was observed in subarctic tundra and subarctic forest-tundra landscapes to the west of 80°E as well as in areas with less climate discomfort (Igarka). The increasing severity of the climate in subarctic forest-tundra landscapes to the east of 80°E is related to the windtemperature regime in these territories [13]. The previous study [14] indicated that days with weather severity index (Arnoldi indicator) above 45 units were recorded for 1981-2015 only in Verkhoyansk. For that station, we analyzed the dynamics of the number of days rated as restrictive or prohibitive based on the Arnoldi indicator. Only small fluctuations not exceeding 3-5 days were identified for the three-time periods (table 1). A statistically significant decrease in the number of days rated as prohibitive for human activity outdoors Frequency of extreme weather conditions presents a considerable concern for the population health and economic activity. During the observation period, the number of days rated as prohibitive ranged from 26 days in 2005/06 to 77 days in 1971/72, and the number of days rated as restrictive -from 94 days in 1980/81 and 1990/91 to 98 days in 2011/12. Conclusion Modern climate fluctuations in the Arctic regions of Siberia have a complex effect on the living conditions of the population. While the average temperatures increase in the Arctic, the climate in the region remains severe. This is reflected in the dynamics of the number of days when weather conditions limit outdoor activity. During the observation period from 1966/67 to 2017/2018, there was a decrease in the number of days with weather conditions prohibitive for human activity outdoors, and the number of days rated as restrictive has increased. During the period from 1981/82 to 2017/18, the number of days with prohibitive weather conditions continued to decline. Extreme weather conditions and events pose a considerable danger to life and health of the human population and place additional demands on the infrastructure and compliance with policies and regulations for working outdoors.	2019-11-28T12:15:49.733Z	2019-11-22T00:00:00.000	{ "year": 2019, "sha1": "053fc4a6334ffb0bc65918d2e0eeb1439eb2a3ca", "oa_license": null, "oa_url": "https://doi.org/10.1088/1755-1315/381/1/012061", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "bb5c27732cee86c0af1fde3ab66a871ad10078b2", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Geography" ] }
257889466	pes2o/s2orc	v3-fos-license	Modeling of dynamic and spectral viscoelastic characteristics of composite materials . When designing products made of composite materials intended for use in difficult conditions of inhomogeneous deformations and temperatures, it is important to take into account viscoelastic, including spectral and dynamic, properties of the binder and fillers. The article considers dynamic characteristics (complex modulus, complex malleability, their real and imaginary parts, loss angle tangent) and spectral characteristics of relaxation and creep and their dependence on each other. The above-mentioned characteristics were found for all known types of creep kernel and relaxation kernel. To find the spectral characteristics, one of the numerical methods of reversing the Laplace transformation was used - the method of quadrature formulas. Algorithms and computer programs have been compiled to implement this method Introduction When designing products made of composite materials intended for use in difficult conditions of inhomogeneous deformations and temperatures, it is important to take into account the viscoelastic properties of the binder and fillers [1].All viscoelastic characteristics, both static and dynamic, are expressed, ultimately, through relaxation and creep nuclei [2,17]. Relaxation and creep kernel reflect the specific properties of a particular material.There are many model variants of such cores proposed at different times scattered in the literature.All of them were systematized and brought together in the work [17], and a mutual connection between them was established there [17].An alternative and generalized approach is to determine the spectral characteristics of relaxation and creep -the density of the relaxation spectrum and the creep spectrum.It is necessary to link all the defining viscoelastic functions through spectral characteristics.It is also necessary to establish the relationship between the relaxation spectral density and the creep spectral density. Thus, the density of relaxation spectra, for example, becomes the primary and main viscoelastic characteristic of the material, it is specific to each specific material and is subject to experimental determination in each specific case.In this work, a partial implementation of this program is proposed. The function G t describes the change in stress over time with constant deformation. This process is called stress relaxation.In this process, the tension decreases with time, that is, the function G t is decreasing. The function J t describes the change in deformation over time at constant stress.This process is called creep deformation.In the process of creep at constant stress, the deformation increases, that is, the function J t is increasing. In the theory of elasticity between an elastic module G and malleability J there is a simple connection [1][2][3][4] 1. GJ (2) However, in the theory of viscoelasticity, there is no such simple connection between the relaxation and creep functions. Let's write down formulas (1) in the Laplace image space using the convolution theorem , .p pG p p p pJ p p (3) From formulas (3) follows 2 1. p G p J p (4) This expression defines the relationship between the images of relaxation and creep functions. In the theory of the Laplace transform, the following limiting relations take place That is, relations of type (2) in the theory of viscoelasticity take place only in two limiting cases: when 0 t and when t Dynamic characteristics of viscoelastic materials and the relationship between them The most well-known dynamic characteristics are the complex module * G i and the complex malleability Where and g g are the images of the relaxation kernel and the creep kernel, respectively, in the Fourier space. There is the following relationship between formulas (9) [14-16]: 1. G i J i (10) Complex modulus and complex malleability, as the name implies, have real and imaginary parts: J are real and imaginary parts of complex compliance, 1 .Pa By substituting formulas (11) into (10) Let's return to expressions (12) and write the second equation in the form Where the left side of the equality, by definition, is called the tangent of the loss angle Then it follows from ( 14) and ( 15) Relaxation and creep spectra and their relation to relaxation and creep kernels In [2][3], the relaxation function G t is defined as follows: Where H is the relaxation time distribution function (relaxation spectrum), 1 . Pa s Taking into account (6), the relaxation kernel t is expressed in terms of the relaxation spectrum H From this formula by substitution 1 we can get the expression Where 1 L is the inverse Laplace transform operator; 1 is the Dirac delta function, Then (20) takes a simpler form: In [2][3], the creep function J t is defined as Where j is the lag time distribution function, or creep spectrum, Relation of relaxation and creep spectra to each otheR According to (8), there is a mutual relationship between the relaxation kernel t and the creep kernel g t in the Laplace image space.Taking into account ( 18) and ( 23), it becomes obvious that the functions of the relaxation spectrum H and the creep spectrum j are also not independent. Let's define formulas linking the spectra together.Let's start with the fact that from (18) we find the image of the relaxation kernel.Then, using (8), we find the image of the creep kernel.Applying the inverse Laplace transform operator j . By reverse substitution 1 we find the creep spectrum j , and then, taking into account ( 5) and ( 7), the final formula takes the form L and 2 L are operators of direct and inverse Laplace transformations, respectively, applied twice.Similarly, we can derive a formula expressing the relaxation spectrum through the creep spectrum: Known types of relaxation and creep functionS In [17], the known types of relaxation and creep kernels and their derivatives (Abel, Rabonov, Rzhanitsyn functions, etc.) were presented in the form of tables.Using these data, the dynamic and spectral characteristics of each of these kernels were further found. Dynamic and Spectral Characteristics of Creep and Relaxation Kernels Using the data from the tables from [17] and the formula ( 9)-( 16), ( 21) and (24) the dynamic and spectral characteristics of the known creep kernels and relaxation kernels were obtained. The results are shown in Tables 1-6. Maxwell functions Table 1 shows the dynamic and spectral characteristics of Maxwell kernels. Kohlrausch functions Table 2 shows the dynamic and spectral characteristics of Kohlrausch kernels. Table 2. Dynamic and spectral characteristics of Kohlrausch kernels.In Table 2 takes values 0 . When 0 , Kohlrausch's characteristics turn into Maxwell's characteristics.Abel functions Table 3 shows the dynamic and spectral characteristics of Abel kernels.4 shows the dynamic and spectral characteristics of the Rabotnov kernels. Table 4. Dynamic and spectral characteristics of Rabotnov kernels. Rzhanitsyn functions Table 5 shows the dynamic and spectral characteristics of the Rzhanitsyn kernels. № Relaxation Creep In tables 1-6, the functions of the kernels are indicated under number 1, the spectra of the kernels are indicated under number 2, the complex module and complex compliance are indicated under number 3, respectively, and their real and imaginary parts are indicated under numbers 4-5.Number 6 indicates the tangent of the loss angle and its limit values at 0 and at , respectively.13), it is possible to obtain the following functions already in an analytical form: the Abel accumulation modulus and the Abel loss modulus, the Rabotnov accumulation modulus and the Rabotnov loss modulus, the real and imaginary parts of the complex malleability of Rzhanitsyn. Numerical method for solving the problem Relaxation and creep spectra, according to ( 21) and (24), are found through relaxation and creep kernels by reversing the Laplace transform.We will solve this problem using one of the numerical methods of reversing the Laplace transform -the method of quadrature formulas with equal coefficients [20].This method was described in detail in the previous work [17]. Examples of numerical solution of the problem Computer programs were written that implement the method of quadrature formulas for finding spectra.Here are some calculations obtained using these programs. Figures 1-6 show graphs obtained by the method of quadrature formulas.To construct the functions of the spectra, the following parameters were set: The green graphs correspond to the analytical functions (or in the form of an approximate finite series) of the spectra, while the red graphs correspond to the functions of the spectra obtained by the numerical method mentioned above. Conclusions After analyzing the results, we draw the following conclusions: 1) the graphs of the relaxation and creep spectra were obtained fairly accurately (the maximum error of calculations does not exceed 5% on average), despite the fact that the error is very noticeable in the initial time sections.Numerical finding of the functions of the Kohlrausch and Gavriljak-Negami yield spectra caused difficulties associated with a complex recursive formula in these kernels; 2) for all kernels (except for the kernels of Kohlrausch and Gavrilyak-Negami), their dynamic characteristics were obtained in the form of analytical functions, even though the functions of some kernels are represented as an approximate infinite series; 3) formulas defining the spectra through each other have not proved to be very effective in practice (with the exception of Maxwell spectra).It is possible that for greater efficiency, the direct and inverse Laplace transform in these formulas should be replaced by an approximate one (the integrand function should be written as an approximate infinite series). , we get an expression similar to(20) from which we can get the function 2 1 -Negami 's characteristics turn into Rzhanitsyn's characteristics, and when 1 they turn into Maxwell's characteristics. Figures 1 , Figures 1, 3 and 5 show graphs of the relaxation spectra of Abel, Rabotnov and Rzhanitsyn, respectively.Figures 2, 4 and 4 show graphs of the creep spectrum of Abel, Rabotnov and Rzhanitsyn, respectively.The green graphs correspond to the analytical functions (or in the form of an approximate finite series) of the spectra, while the red graphs correspond to the functions of the spectra obtained by the numerical method mentioned above. Table 1 . Dynamic and spectral characteristics of Maxwell kernels. Table 3 . Dynamic and spectral characteristics of Abel kernels.	2023-04-02T15:09:28.571Z	2023-01-01T00:00:00.000	{ "year": 2023, "sha1": "638a70ad351fe344c413759c76f2ed6b3ebaf3ec", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1051/e3sconf/202337601042", "oa_status": "CLOSED", "pdf_src": "ScienceParsePlus", "pdf_hash": "4f4d0682194feb73303c86b6c53399dcbc4c55b2", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [] }
97962661	pes2o/s2orc	v3-fos-license	USING A BLACK BODY SOURCE TO ENHANCE LINE EMISSION IN A LASER PRODUCED PLASMA REKHA TAMBAY Well defined homogeneous metal vapors with precise information on the density and temperature needed for spectroscopic applications are usually generated with a heatpipe oven. Metal vapor-inert gas mixtures have been used extensively in non-linear optics for generation of tunable narrow band coherent light sources in the UV and VUV regions where non-linear crystals can not be used.4 Recently, heatpipe ovens have been used in a soft X-ray source5,6 which subsequently pumps surrounding metal vapors for production of short wave length lasers. Early experiments on population inversion in UV and X-ray regions used X-ray filters to eliminate unwanted spectral components from the pumping source. In order to attain efficient pumping, Harris et al suggested the use of a black body source resulting from a laser produced plasma (LPP) to excite the vapor or gas. Using a similar configuration Silfvast et al were the first to demonstrate a photo-ionization laser. The pumping rate depends on the density of the photons from an LPP source interacting with metal vapors. Since the emission from the LPP source is absorbed as it propagates through the vapor medium, it is essential to look for the coupling of the black body emission with the surrounding metal vapors. In the present paper we report results of studies of cadmium plasma in a crossed heatpipe. The studies were done with and without a background LPP. We also discuss the conditions for optimum working of a heatpipe. We report the formation of Cd clusters in the over heated heatpipe. The present paper is organized as follows: In Section 1, we discuss the details of the .experimental setup used. Section 2 describes the optimum working conditions for the crossed heatpipe and INTRODUCTION Well defined homogeneous metal vapors with precise information on the density and temperature needed for spectroscopic applications are usually generated with a heatpipe oven.Metal vapor-inert gas mixtures have been used extensively in non-linear optics for generation of tunable narrow band coherent light sources in the UV and VUV regions where non-linear crystals can not be used. 4Recently, heatpipe ovens have been used in a soft X-ray source 5,6 which subsequently pumps surrounding metal vapors for production of short wave length lasers.Early ex- periments on population inversion in UV and X-ray regions used X-ray filters to eliminate unwanted spectral components from the pumping source.In order to attain efficient pumping, Harris et al suggested the use of a black body source resulting from a laser produced plasma (LPP) to excite the vapor or gas.Using a similar configuration Silfvast et al were the first to demonstrate a photo-ioniza- tion laser.The pumping rate depends on the density of the photons from an LPP source interacting with metal vapors.Since the emission from the LPP source is absorbed as it propagates through the vapor medium, it is essential to look for the coupling of the black body emission with the surrounding metal vapors.In the present paper we report results of studies of cadmium plasma in a crossed heatpipe.The studies were done with and without a background LPP.We also discuss the conditions for optimum working of a heatpipe.We report the formation of Cd clusters in the over heated heatpipe.The present paper is organized as follows: In Section 1, we discuss the details of the .experimentalsetup used.Section 2 describes the optimum working conditions for the crossed heatpipe and the formation of Cd clusters.In Section 3 studies on cadmium metal vapors in the presence of a black body are presented. EXPERIMENTAL DETAILS The crossed heatpipe used was a 1" diameter stainless steel pipe, with a stainless steel rolled wire mesh in all the four arms.The central zone was heated using a heating tape wrapped around the pipe and a fire brick structure covered the heated zone to reduce convection losses.To avoid deposition of vapors on the windows, a water jacket was provided for cooling at the end of each of the four arms.To study the effect on efficiency of getting metal vapor plasma in the presence of background plasma, a high Z metal target, tungsten, was inserted in one of the arms, opposite to the direction of the incident laser beam.The plasma was produced by focusing radiation from an Nd" YAG laser with a 18 cm focal length quartz lens onto a tungsten target for tungsten plasma and at the center of the heatpipe for metal vapors.We have used a Nd YAG laser (DCR-4G Spectra Physics) with Gaussian limited mode structure and its harmonics 2o, 3Oo, 4Oo delivering up to 900 mJ in 2.5 ns (FWHM) at the fundamental with a repetition rate of 10 pps.In order to expose a fresh surface of the target to the laser pulses the target was continuously rotated using a small electric motor.The other two arms of the heat- pipe were used for viewing and recording of emission from plasma.The plasma was imaged on to the slit of a monochromator (HRS-2, Jobin Yvon) and detected using a photo multiplier tube (PMT) (IP-28, Hamamatsu).The PMT has a reasonable flat response in the visible region.The signal from the PMT was fed into a strip chart recorder. The density of the laser produced plasma was estimated using a Mach- Zehnder Interferometer. 9The experimental layout is shown in Figure 1.A 2Oo (0.532 tm) radiation was used as a probe beam.The collimated probe beam was split into two beams of equal intensity at the beam splitter BS and were recombined at the beam splitter BS2.The heatpipe was placed in one arm and a compensating glass plate was placed in the other arm of the inter- ferometer.The total phase difference between the beam passing through the heatpipe and the reference beam is [ Irl-t)dx[ where is the refractive L, A index of the dispersive medium, ro the radius of the plasma and is the probe wavelength.The interferogram was recorded on a pan chromatic film using a lens less camera.The interferogram were enlarged, digitized, and analyzed for fringe shift and density evaluation.With the assumption of cylindrical symmetry around the central axis of the expanding plasma, the electron density could be calculated at various distances from the target surface by using a cubic polynomial Abel's inversion.The density ne of the tungsten plasma could be estimated by measuring the shift in the fringes arising from changes in the refractive index and using, , where A is the fringe spacing and where ajk are the Abel coefficients.'2 N k is the value of the fringe shift N(x), with x kro n is the number of channels. WORKING OF THE HEAT PIPE AND CLUSTER FORMATION To start with, the system was baked by heating it to temperatures exceeding 300C for several hours with helium pressure in excess of 100 Torr.The system was evacuated to a pressure of <10 -4 Torr and then filled with helium at required pressure.The laser beam was focused at the center of the heatpipe using a quartz lens to generate cadmium plasma.The temperature was slowly raised to 450C with He gas pressure of 7-8 Torr, but no cadmium plasma was observed visually.However, when the buffer gas pressure was increased to 52 Torr and the tempera- ture kept at 400C a week cadmium plasma was observed.Increasing temperature further at the same buffer gas pressure, increased the intensity of the plasma.When the heatpipe was operated at 500C, the corresponding Cd density being 2.73 x 107/cm3, an intense green ball of cadmium plasma could be seen on viewing through one of the side arms.Figure 2 shows a Cd spectrum at oven temperature of 500C.The He pressure was 52 Torr and laser energy 900 mJ.An estimate of the temperature of the metal vapor plasma was made by taking ratio of the intensity of the spectral lines. 3The observed temperature is 0.5 eV.The intensity of the emitted cadmium lines decreased with increase in the He gas pressure.This is because at higher pressures the atomic velocity is so low Figure 2 Visible spectrum of Cd metal vapor plasma; T 500C, He: 52 Torr.that the plasma plume significantly reduces in volume and hence has less atomic density.It was also observed that increasing the wall temperature beyond 500C decreased the intensity of the green blob.This could be due to particulate formation, 4,15 the so called 'fogging' in the heatpipe.To confirm and visually observe the formation of clusters, a weak He-Ne laser beam was sent close to the center of the heatpipe.The pattern in transmitted laser beam was observed on a wall, about 4 m away from the heatpipe.The convective movement of the dust-like particles was clearly visible in the beam.We feel that this particulate formation is due to cadmium clusters formed by condensation of the metal vapors in the cold zone at the boundary with the buffer gas.The latter acts as a third body to stabilize collision complexes between atomic and small molecular species to foster growth of the cluster species.It is similar to the effect used for production and characterization of small metal clusters 16 where an ablated plasma expands into sufficient pressure of the background gas.A slight variation of the technique has been used for the deposition of C60 on various substrates. 7Though at times a useful phenomenon, the poor conditions of operation for the heatpipe result in cluster formation of the metal.The presence of clusters might severely affect the plasma emission resulting in a decrease in plasma intensity.Similar effect was also observed when the central part of the heatpipe is suddenly over heated.Generation of a high Cd vapor density required for efficient plasma production appears to be the most difficult technical problem in experiments such as ours.Our experience shows that the temperature of the heatpipe should be increased very slowly and should not exceed the value at which the vapor pressure of the metal vapor and the buffer gas pressure are equal. COUPLING OF BLACK BODY EMISSION WITH METAL VAPORS One of the aims of the present work is to report the use of laser produced soft X-rays from high Z laser produced plasma for pumping metal vapors.It is known that by focusing the laser radiation to a small area on to solid target it is possible to create a high temperature high density recombining plasma.Near the surface of the target, the plasma emission is primarily due to free-bound and to line radiation.However, for high Z target continuum radiation dominates over the line radiation.The broad band emission can be approximated to a black body with the same characteristic temperature as that of the plasma. 8,9The broad band soft X-ray emission from a laser produced plasma has been used to directly populate the upper laser level from the ground level, 5,6 the pumping rate is essentially determined by the photon density of the pumping source.The fact that at short wavelength, inner shell photo-ionization has a higher probability than ionization of a outer electron, the most probable process with the radiation peaked in the range 200-300 for high Z target is photo-ioniza- tion.Based on this excitation scheme population inversion has been observed in the wavelength range 109-748 nm.We have also reported 2 cadmium photo-ionization laser between the 4d 5s 2D5/2 and the 4d 1 5p2P3/2 levels at 441.6 nm. In order to study coupling between background plasma and Cd plasma, a tungsten target fixed to a rod was inserted into one of the arms of the heatpipe.The laser radiation was focused to a spot of 100 #m on to the tungsten target with helium gas at a pressure of 7-8 Torr.No heating of the heatpipe was done.A bluish white plasma of tungsten could be seen visually at the target surface.In order to photo- ionize the cadmium metal vapor, the heatpipe temperature was raised to 420C nd the laser radiation was focused on to the tungsten target.A bluish white plasma very close to the target surface and a green plasma due to cadmium vapor extending up to 7-8 mm away from tungsten surface were observed.It is worth reiterating that no Cd plasma was observed when the heatpipe was not heated.The Cd plasma was imaged on to the slit of the monochromator and the resulting spectrum recorded at 4-5 mm away from tungsten surface is show in Figure 3.The He pressure was about 7 Torr and laser energy 900 mJ.On comparing the two spectra (Figure 2 and figure 3) it becomes clear that the intensity of all lines increases drastically (sensitivity in figure 2 is five times more than that of Figure 3) and Cd II transitions which were absent in Figure 2 become observable in Figure 3. Thus it is seen that LPP can be a very effective pumping source for higher ionic states of Cd.Since we are interested in Cd II transitions it is worthwhile to consider the possible routes for a Cd II transition at 441.6 nm in particular.Though the process of inner shell photo-ionization is dominant, one should also look at the effect of photo-electrons which are immediately present after photo-ionization.The energy distribution of the photo-electrons has a maximum around 40 eV and is determined by the photon energy distribution and wavelength dependence of the photo-ionization cross sec- tion.If we compare the ionization cross sections 2,22 for various levels of Cd we find that the cross section for 5s level is larger than 5p states in the region above 50 eV.Thus using a black body emission peaked in the range 200-300 / one would expect that 5s D states are formed almost exclusively by removal of inner shell electron while the ordinary 5p, 5d, 6p states are formed by ionization of one electron and excitation of another electron.The emission from the LPP source is absorbed as it propagates through the vapor medium and hence in our case affects the density and temperature of the medium.Thus the coupling between the back- ground plasma and the Cd plasma affects the pumping rate for a photo-ionization process.Figure 4 shows the radial density distribution of tungsten plasma at 2 mm away and parallel to the tungsten target surface.The delay between the 1.06 tm beam and the probe beam is 19 ns.Using the configuration in Figure 1 the density of the cadmium plasma in the presence of the background target was also estimated. Figure 5 shows a radial profile of cadmium plasma density at 5 mm away and parallel to the tungsten surface.The density decreases as we move out radially at various axial positions of the laser produced plasma.Comparison of Figure 4 and 5 reveals that even at 5 mm away and parallel to the black body source the density of cadmium plasma is larger than at 2 mm for tungsten plasma alone.Thus the black body emission does help in pumping the cadmium vapor.We estimate the temperature of the Cd plasma at 4-5 mm away from the surface of the target to be 0.3 eV.At this distance the influence of plasma electrons is negligible.Radial Distance (r) IJm Figure 4 Radial density profile of tungsten plasma at 2 mm away and parallel to the target when no heating of the heatpipe was done; He: 7 Torr.In conclusion, we have studied the Cd metal vapor plasma in a heat pipe.Cluster formation was observed when the heatpipe was over heated.To study the effect of black body pumping, Cd metal plasma was studied in the presence of tungsten target.Increase in intensity of the emitted lines from the higher ionic states of metal plasma in the presence of the black body source suggests that laser produced plasma can be very effective as a pumping source. Figure 1 Mach-Zehnder Interferometer set up for density measurement. Figure 3 Figure 3 Visible spectrum of Cd metal vapor plasma in presence of tungsten plasma; T 420C, He: 7 Torr. Figure 5 Figure 5 Radial profile of cadmium plasma density at 5 mm away and parallel to the target; T 420C, He: 52 Torr.	2019-04-06T13:08:55.912Z	1994-01-01T00:00:00.000	{ "year": 1994, "sha1": "27b16507500dffdef264e89469f37b89a592dad5", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/archive/1994/083157.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "33987f17787e9bb4268bc1c7f5f137df3b0d196d", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Chemistry" ] }
221860081	pes2o/s2orc	v3-fos-license	In vitro Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Salmonella Outer Membrane Vesicles Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of Salmonella (S.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. In vitro, these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system. INTRODUCTION Bacteriophages are ubiquitous in the microbial world and major players in many bacterial ecosystems. Here, they perform a variety of life styles, with prophages or mature phage particles as prominent examples of prevalent phage states (Feiner et al., 2015). Key event in a phage's life cycle is the interaction with host surface receptors to initiate infection of the bacterial host. If this step is successful, the resulting genome transfer to the bacterial cytosol will open the route for a plethora of phage genome functions inside the host cell (Salmond and Fineran, 2015;Erez et al., 2017;Rostol and Marraffini, 2019). Diverse bacterial cell surface structures serve as bacteriophage receptors (Silva et al., 2016). Besides protein-based phage receptors, glycan structures, the major building blocks of the bacterial envelope, represent a very important and structurally diverse phage receptor class (Pires et al., 2016;Latka et al., 2017). Phages do not only exploit glycans to start cell entry (Wang et al., 2019) but also may alter these structures to prevent superinfection (Kintz et al., 2015). In turn, bacteria constantly modify their surface composition to escape from phage attack (Rostol and Marraffini, 2019). O-antigen-specific phages are specialists that use lipopolysaccharide (LPS) as an essential receptor for infection initiation in Gram-negative hosts (Broeker and Barbirz, 2017). These tailed phages link recognition and enzymatic processing of the O-polysaccharide to a so far unknown membrane interaction step that leads to conformational rearrangements in the tail and subsequent particle opening in vitro (Andres et al., 2010(Andres et al., , 2012Broeker et al., 2018Broeker et al., , 2019. In vivo, full genome transfer additionally requires other factors like outer membrane (OM) proteins or a membrane potential (Leavitt et al., 2013;Parent et al., 2014). In vitro, purified and protein-free LPS alone is sufficient to inactivate O-antigen-specific phages, because binding to membrane-mimicking LPS aggregates can trigger DNA release from the capsid (Broeker et al., 2019). In vitro studies furthermore revealed that the kinetics of this process were dependent on the architecture of the tail, but not on the chemical composition of the O-antigen receptor. Moreover, to control phage populations and to achieve superinfection exclusion, O-antigen-specific phages may modify the O-antigen structure of their host cell (Susskind et al., 1974;Cenens et al., 2015). In response to bacteriophage attack, bacteria control the number of phage particles in their environment in multiple ways, and, in turn, phages respond by own strategies to overcome bacterial defense (Doron et al., 2018;Vidakovic et al., 2018;Rostol and Marraffini, 2019). Among the large number of defense mechanisms determining the ecosystem in which bacteria and phages coexist, as a matter of fact, the host surface is a critical point for bacteriophage entry control (Leon and Bastias, 2015). For example, O-antigen phase variations may result in mixed bacterial populations and an increased resistance to phage attack (Seed et al., 2012;Schmidt et al., 2016). Moreover, like all living cells, bacteria can also shed outer membrane vesicles (OMVs; Schwechheimer and Kuehn, 2015). In OMVs, bacteria may control the content of bacteriophage surface receptors and thus exploit this extracellular material as buffer against phage attack (Manning and Kuehn, 2011;Biller et al., 2014;Reyes-Robles et al., 2018). The ubiquitous budding of spherical OM structures is highly conserved in both pathogenic and non-pathogenic Gram-negative bacteria (Bai et al., 2014;Celluzzi and Masotti, 2016;Guerrero-Mandujano et al., 2017). OMVs are composed of OMs, containing LPS, phospholipids and OM proteins, and soluble periplasmic components like DNA and proteins. OMVs function in bacterial communication, cell wall remodeling, horizontal gene transfer, defense, and pathogenicity (Mashburn and Whiteley, 2005;Bonnington and Kuehn, 2016;Crispim et al., 2019); their composition may thus differ from that of OM and periplasm (Schwechheimer et al., 2013;Kim et al., 2018;Malge et al., 2018). The size of the vesicles depends on the given strain but generally ranges from 50 to 250 nm in diameter (Beveridge, 1999). Vesiculation is induced as response to different stress factors, like the presence of antimicrobial or toxic particles or unfavorable environmental conditions, for example, nutrient deficiency or elevated temperatures (Loeb, 1974;Manning and Kuehn, 2011;Schwechheimer et al., 2013;Bai et al., 2014). Host cell OMVs can bind and rapidly remove free phage particles from a solution and effectively reduce phage activity (Manning and Kuehn, 2011;Biller et al., 2014;Parent et al., 2014;Reyes-Robles et al., 2018). However, the mechanisms of this interaction, that is time-resolved observation of the all involved steps on a molecular level, have not been studied in detail so far. In this work, we have analyzed Salmonella phage P22 in the presence of OMV prepared from its host, Salmonella enterica ssp. enterica sv. Typhimurium (S. Typhimurium). Podovirus P22 is an O-antigen-specific, transducing phage with a short, non-contractile tail. P22 is a well-established model system for studying DNA transduction, Salmonella genetics, and lysogeny (Prevelige, 2006;Casjens and Grose, 2016). P22 uses LPS as its receptor, and tailspike protein (TSP)-mediated cleavage of the O-antigen part then positions the phage particle on the cell surface, where the particle opens (Andres et al., 2010). In vivo, genome transfer is mediated by a set of P22 ejection proteins that contribute to forming a channel-like structure that provides access to the cytosol by bridging the cell wall (Wang et al., 2019). In vitro, purified protein-free LPS aggregates mimicking the OM were sufficient to elicit DNA release into the solution (Andres et al., 2010). We wanted to extend this in vitro system to a more complex receptor system and have analyzed dynamics of DNA release from phage P22 in the presence of OMVs. We used large-scale OMV preparations to obtain sufficient material for fluorescence spectroscopy studies (Thein et al., 2010). Our in vitro analyses show that OMVs efficiently rendered P22 phage particles non-infective. OMV-triggered P22 phages ejected their DNA more rapidly than LPS-triggered P22 phages. However, and in contrast to pure LPS preparations, when triggered by OMVs, only a subfraction of these phages also injected their DNA into the vesicles. Preparation of Outer Membrane Vesicles From Cell Lysates OMVs present after mechanical cell lysis were prepared according to Thein et al. (2010). Briefly, S. Typhimurium AroA::Tn10 iroBC::kan were grown in LB medium supplemented with 50 μg ml −1 kanamycin overnight at 37°C. After cell harvest, cells were incubated with 0.1 mg ml −1 lysozyme and 10 μg ml −1 DNase I on ice and lysed with French press. Membranes were collected by centrifugation at 75,400 × g for 30 min at 4°C and suspended in standard buffer. For OMV purification with sucrose density gradients ("OMV Suc"), total membranes were loaded on a three-step sucrose density gradient (3 ml 75% w/v, 5 ml 50% w/v, and 3 ml 25% w/v) and centrifuged at 250,000 × g for 12 to 16 h at 4°C to separate inner membrane (IM) and OM fractions. The OM accumulated at the 50/75% sucrose interface. The harvested OM fraction was diluted in water, collected at 30,000 × g (30 min) and stored at 4°C. Alternatively, OMVs were obtained by selective detergent solubilization ("OMV Det"). For this, the total membranes after French press lysis were incubated with 1% N-lauroylsarcosine and centrifuged at 30,000 × g for 30 min at 4°C to solubilize the IM. To wash OMV, they were suspended in water, collected at 30,000 × g (30 min) and stored at 4°C. Preparation of Outer Membrane Vesicles Naturally Budded Into Salmonella Culture Supernatants Hypervesiculating S. Typhimurium ATCC 14028 MvP2390 were grown in LB medium supplemented with 50 μg ml −1 kanamycin at 37°C to an OD 600 nm of 1.5. Cells and cell debris were removed by two subsequent centrifugation steps at 4000 × g (15 min each, 4°C). The supernatant was filtered twice on Whatman cellulose acetate 0.45 μm and 0.20 μm (GE Healthcare Europe GmbH, Freiburg, Germany), and OMVs ("OMV budded") were collected from the filtrate at 38,500 × g (3 h, 4°C) and stored in phosphate-buffered saline (PBS) at 4°C. Electron Microscopy EM images were recorded on a JEOL JEM1400-Plus transmission electron microscope (TEM). Staining methods for Gram-negative bacteria samples have been described (Grin et al., 2011). Samples were loaded on Formvar-coated copper grids, stained with 1% (w/v) uranyl acetate and embedded in 1% (w/v) uranyl acetate and 0.4% (w/v) methyl cellulose. Plaque-Forming Assay The plaque-forming assay monitors the decrease of infectious particles due to co-incubation with LPS or OMV. 5 × 10 3 pfu ml −1 P22 bacteriophages were incubated with 2.5 μg ml −1 LPS or OMV LPS equivalents in standard buffer at 37°C. After different incubation times, 100 μl were taken, mixed with warm soft agar, and plated on thin LB agar plates. After overnight incubation at 37°C, the plaques were counted and plaque-forming units (pfu ml −1 ) were determined. As control, P22 bacteriophages were incubated with buffer only. DNA Ejection Monitored by Yo-Pro Fluorescence Bacteriophage P22 DNA ejection was followed by fluorescence spectroscopy as described before (Andres et al., 2010). OMVs for these experiments were prepared with the sucrose gradient method (OMV Suc). Briefly, 4 × 10 9 pfu ml −1 P22 bacteriophages were incubated at 37°C with 10 μg ml −1 LPS or OMV LPS equivalents in the presence of 1.1 μM Yo-Pro ® -1 iodide. The fluorescence signal was detected at 509 nm after excitation at 491 nm. After 170 min, DNase I was added to a final concentration of 10 μg ml −1 . Curves were corrected for Yo-Pro phage staining in presence of O-antigen-depleted OMVs. Curves were fitted to a monoexponential function as described before (Andres et al., 2010;Chiaruttini et al., 2010). G Converted PEG weight percent (w) concentration: Preparation and Characterization of S. Typhimurium Outer Membrane Vesicles To investigate differences in preparation of OMVs and to study the effects of altered vesicle characteristics on bacteriophage interactions, different preparation protocols were employed ( Table 1). Gram-negative bacteria shed OMVs in culture supernatants; however, the low concentrations usually require large preparation scales to obtain sufficient amounts for quantitative functional analyses. We therefore generated OMV samples by accumulation of OMs after mechanical lysis of S. Typhimurium cells. Either IMs and OMs were separated on sucrose density gradients or IMs were solubilized by detergents to collect the OM fraction by centrifugation (Thein et al., 2010). Moreover, and for comparison, we isolated OMVs from culture supernatant Frontiers in Microbiology \| www.frontiersin.org 4 September 2020 \| Volume 11 \| Article 510638 filtrates of the hypervesiculating S. Typhimurium strain MvP2390 that is defective in Braun's lipoprotein (Dramsi et al., 2008). Electron microscopy analysis showed that with all preparation methods, samples were obtained with spherical vesicles of 50-100 nm diameter (Figure 1). Stokes radii measured by dynamic light scattering confirmed these OMV size distributions (Supplementary Figure S1). To obtain homogeneously round-shaped vesicles from membrane accumulation, we noticed that a pre-lysis treatment of cells with DNase I and lysozyme was mandatory, because otherwise only large and irregular membrane aggregates were formed ( Figure 1B and Supplementary Figure S2). The vesicles' protein: LPS ratio was calculated from analyses with BCA and Purpald assays, respectively ( Table 1). In contrast, IM fractions that could be isolated from sucrose gradients contained notably less vesicle-like structures (Supplementary Figure S3). OMVs from membrane accumulation contained typical OM markers, like OM proteins and LPS (Supplementary Figure S3). SDS-PAGE with glycan staining confirmed that they contained LPS with a typical O-antigen chain length distribution (Supplementary Figure S4). Hence, LPS in OMVs could be quantified with the Purpald assay. Furthermore, co-expression of a fluorescently labeled OmpA-mCherry construct as OM marker protein showed that it was located in the OMV fraction after membrane accumulation and sucrose density gradient separation (Supplementary Figure S3). The budded OMVs from culture supernatants showed an increased protein content compared to OMVs collected by membrane accumulation and also a different protein composition as estimated from SDS-PAGE analysis (Supplementary Figure S5). As shown earlier, under typical growth conditions, naturally budded Salmonella OMVs contain an increased amount of cytosolic proteins (Bai et al., 2014), which may account for the elevated protein content. Moreover, EM images of our samples revealed flagella-like structures that apparently had co-purified with the OMVs. In principle, it would be also possible that additional periplasmic proteins are present in the lumen of these naturally budded vesicles or that they display increased amounts of OM proteins (Volgers et al., 2018). However, the small yield of these OMVs isolated from culture supernatants impeded a further detailed proteomic analysis. Interaction of Vesicles With Bacteriophage P22 Transmission electron microscopy showed that P22 bacteriophages interact with OMVs. After overnight incubation at 37°C, the uranyl acetate stain reveals the OM structure as a bright ring enclosing the vesicle lumen that appeared in a darker, "negative" stain ( Figure 2). All OMV preparations showed vesicles being bound by phages. Phages either showed intense staining of the capsid shell or a lighter appearance of their capsids, which we assign to DNA-ejected and non-ejected particles, respectively. Phages were attached to the vesicle surface with their tail apparatus (arrows in Figure 2), but at the given resolution, differences in the tail conformation of ejected and non-ejected phages cannot be verified. As a rough estimate from manual image inspection, we found about 25% of the vesicles with bound phage (Supplementary Figure S6). A quantitative analysis would require cryo-EM sample preparation and variation of the phage-OMV ratio to estimate proportions of bound and ejected versus bound and non-ejected phages (Parent et al., 2014;Reyes-Robles et al., 2018). To further validate irreversible binding and inactivation of phage P22 by OMVs, we analyzed P22-OMV mixtures for their ability to form plaques on the P22 S. Typhimurium host. With prolonged incubation times, we found that the plaque-forming units in the presence of membrane preparationderived OMVs were reduced rapidly to less than 5% of the initial value (Figure 3). Interestingly, the inactivation of P22 by OMVs was well comparable to that found by a purified, protein-free LPS sample; accordingly, we assume that the proteins present in the OMVs did not influence the phage inactivation. Rather, as previously described, the isolated LPS receptor is a sufficient inhibitor to inactivate the O-antigenspecific P22 phage (Broeker and Barbirz, 2017;Broeker et al., 2019). However, whereas inactivation profiles for OMVs obtained from OM and LPS were highly similar, budded OMVs obtained from culture supernatants showed a much slower inactivation effect on phage P22, with about 25% of active phage particles remaining after 6 h of incubation. This might be related to their increased protein content, probably reducing the number and altering the distribution of surface-accessible LPS receptors, resulting in a slower decrease of infective, unbound phage particles. Flagella contaminations in this preparation might additionally have affected their capacity for phage inactivation. P22 Bacteriophage Particle Opening Analysis With Vesicles Lysis and plaque-forming assays only show irreversible binding and inactivation of phage P22, but no information is obtained whether inactivated bacteriophage P22 particles have lost their DNA upon contact with OMVs. Moreover, from negative stain EM analyses, neither quantitative amounts of ejected phage nor dynamics of the DNA ejection process were accessible. We therefore monitored DNA ejection of bacteriophage P22 with a fluorescence ejection assay that has been described previously (Andres et al., 2010(Andres et al., , 2012Broeker et al., 2018Broeker et al., , 2019. In this assay, purified host LPS is mixed with the bacteriophage. This triggers opening of the phage capsid and release of DNA in the surrounding solution that contains the fluorescent DNA-intercalating dye Yo-Pro. This leads to a rapid and intense fluorescence signal increase with increasing phage DNA concentrations in solution. In contrast, in the absence of LPS, phage particles remain intact and only show a weak background fluorescent signal, because the dye Yo-Pro has weak affinity to the highly condensed DNA inside the phage capsids. As a consequence, O-antigen-specific phages only show a DNA ejection signal when triggered with an intact LPS, whereas O-polysaccharide or lipid A alone or rough LPS do not elicit DNA release (Andres et al., 2010). For P22, as for other phages, monoexponential kinetic ejection profiles were measured with this method that describes the rate-limiting phage particle opening step (Chiaruttini et al., 2010;Andres et al., 2012). Co-incubation of P22 phage and OMVs in the presence of Yo-Pro resulted in a monoexponential increase of the fluorescence signal, reaching a plateau after about 170 min (Figure 4). Neither OMVs alone nor a control in which phage was incubated with OMVs that lack the phage O-antigen receptor showed a notable fluorescence signal increase. Hence, the fluorescence increase in the presence of OMVs monitors phage DNA liberated from the phage capsids. We monitored similar DNA release profiles from phage P22 with all OMVs irrespective of their source and purification protocol (Supplementary Figure S7). However, the naturally budded OMVs contained protein impurities and were available only at an overall poor yield (see above). In all fluorescence experiments, we therefore worked with OMVs obtained from accumulated membranes with the sucrose gradient method (cf. Materials and Methods). To check whether phages ejected their DNA into the vesicles or into the solution, we added DNase I after the ejection signal had reached a plateau. If DNA is free in solution, nucleotides produced by DNase I will no longer intercalate with Yo-Pro, resulting in a decrease of the fluorescence signal (Andres et al., 2010). However, the fluorescence signal obtained from P22 DNA ejection in the presence of OMVs did not decrease (Figure 4). Apparently, when triggered by OMVs, all DNA ejected from phage was not accessible to DNase I, because it was protected either inside the vesicles or inside the phage capsids. The plaque-forming assay showed that OMVs can rapidly inactivate phage particles in solution. To check whether all phages in the fluorescence experiment were associated with OMVs, we added free LPS to the mixtures 170 min after the reaction was started. If at this time point a significant amount of phage particles had remained free in solution, a signal increase due to DNA release upon phage interactions with the newly added free LPS receptor should be observed. However, we did not observe any further signal increase upon LPS addition (Supplementary Figure S8). This result is in agreement with the plaque-forming assay where OMV rapidly cleared off phage particles from the solution. We therefore conclude that all P22 phage particles in the fluorescent assay were rapidly and irreversibly associated with OMVs and could no longer interact with newly added LPS. In the fluorescence assay, the OMV concentration was calculated as LPS units determined with the Purpald test to be able to compare OMV-triggered phage ejection profiles with those triggered with purified LPS. Comparison of kinetic constants for DNA ejection triggered either by LPS or OMVs showed that particle opening velocities with OMVs were increased about 2-fold relative to LPS (Figure 4). However, only about 30% of the signal amplitude were reached compared to phages triggered with a pure LPS sample. Even if we increased OMV concentrations 3-fold, we did not observe an increase in the amount of ejected DNA (Supplementary Figure S8). This means that only a part of the phage particles have lost their DNA in the presence of OMVs, in contrast to purified LPS, in presence of which all particles completely exposed their DNA to the solution (Andres et al., 2010). OMVs used in the fluorescence experiments were prepared from accumulated OMs after French press cell lysis. We cannot fully exclude the idea that a part of the OMVs obtained by this method might be inverse, i.e., with the LPS at the inside and the phospholipid part at the outside. O-antigen-specific phage P22 would not bind to a vesicle with O-antigen chains on the inside and not eject its DNA and would thus remain inert against the inside-out vesicle fraction. OMVs in this study were quantified via their LPS concentration. We did not find an increase in DNA ejection when we increased the OMV concentration from 10 μg ml −1 LPS to 30 μg ml −1 LPS (Supplementary Figure S8). This means that at 10 μg ml −1 LPS OMV concentration, all phage particles in the system can interact with the OMVs. If a part of the vesicles were inside out, we should observe an increase in ejected phages because with increased OMV concentration, more phage O-antigen receptors become exposed. Moreover, we enzymatically removed the O-antigen from the vesicle surface by incubation with purified P22 tailspike protein prior to exposing them to phage P22. The resulting O-antigen-depleted vesicles could not trigger DNA ejection from phage P22 (Supplementary Figure S8). From these controls, we conclude that the fluorescence signal observed in our experiments is exclusively related to a phage interaction with vesicles that expose LPS on the outside. DNA Ejection in the Presence of Polyethylene Glycol (PEG) Protein-free, highly pure LPS preparations that are present as aggregates free in solution could trigger particle opening in all P22 phages present in a reaction. However, for P22 DNA FIGURE 4 \| DNA release from bacteriophage P22 triggered by OMV or LPS. Incubation of 4 × 10 9 pfu/ml P22 phages at 37°C in the presence of a fluorescent DNA-binding dye (1 μM Yo-Pro) with 10 μg ml −1 LPS (blue circles) or OMVs (white triangles; 10 μg ml −1 LPS equivalents, see Materials and Methods). Rate constants for phage particle opening were calculated from monoexponential fluorescence increase to k open,LPS = 5.89 ± 0.01 × 10 −4 s −1 , and k open,OMV = 10.00 ± 0.01 × 10 −4 s −1 , respectively (Chiaruttini et al., 2010). After 170 min, DNase I (10 μg ml −1 ) was added to the ejected phages, either LPS-triggered (cyan circles) or OMV-triggered (black triangles). Plot shows mean values of three technical replicates. For clarity, only every tenth point was plotted and exemplary error bars are given for the last points of the ejection curves and for data in presence of DNase I only. Plots with all error bars can be found in Supplementary Figure S9. As an O-antigen-free control, 10 μg ml −1 OMV LPS equivalents were incubated overnight with 10 μg ml −1 P22TSP at 37°C prior to the ejection experiment (white squares). ejection triggered by OMVs, only about 30% of DNA signal was obtained. Moreover, this signal was not accessible to DNase I. These findings imply two scenarios for P22 DNA ejection in the presence of OMV, either (i) 30% of the particles have completely ejected their DNA, whereas for the remaining fraction no DNA release occurs, or (ii) all particles have released their DNA, but only to about 30%. To distinguish between these two situations, we analyzed DNA release from phage P22 in the presence of increasing PEG concentrations. It has been shown previously for siphovirus λ that DNA ejection is driven by the intra-capsid osmotic pressure and that at increasing osmotic pressures, less DNA could leave the λ capsids (Evilevitch et al., 2003(Evilevitch et al., , 2005. Also for P22, it was shown that at increasing osmotic pressures and when triggered with LPS and OM proteins, part of its DNA remained in the phage capsid and was protected from DNase cleavage (Jin et al., 2015). We therefore analyzed the DNA fluorescence increase in the presence of different concentrations of PEG 8000, when P22 was mixed with OMVs ( Figure 5A). At 15% (3.58 atm) PEG, we obtained about 40% of the initial signal. This shows that an external osmotic pressure could effectively reduce DNA ejection from vesicle-associated phages. However, earlier work showed that, at this osmotic pressure (3.58 atm), still around 70% of the P22 DNA could leave the capsid when triggered with LPS or a mixture of LPS and the outer membrane protein OmpA (Jin et al., 2015). Hence, P22 triggered by OMVs ejected less DNA at increased external osmotic pressures, illustrating that OMVs represent an OM receptor system different from pure LPS or OM protein-LPS mixtures. Furthermore, the notable effect also of lower PEG concentrations on DNA release confirms that only about 30% of the P22 particles are actively ejecting their DNA in the presence of OMVs. At 5% PEG (0.46 atm), we already observed a signal decrease to 70% (Supplementary Figure S10). If all particles in the system were actively releasing only a part of their DNA, these low osmotic pressures would not elicit a pronounced effect on ejection because the initial release of only a small fraction of DNA is also possible against elevated osmotic pressures. It was shown for other systems that at higher PEG concentrations, the DNA ejection can be completely stopped (Evilevitch et al., 2003;Jin et al., 2015). However, in our fluorescence setup, we could not further increase PEG concentrations, as this resulted in high signal-to-noise ratio and large standard deviations. This was probably due to vesicle solubility hampered by PEG or slow equilibration of the mixtures in the viscous solution. At an osmotic pressure of 6.77 atm (20% PEG), we can thus only estimate that P22 was able to still release about 20% of its DNA. Moreover, and as described above, also in the presence of PEG, it was not possible to notably reduce the DNA signal by the addition of DNase. Again, we assume that the DNA was injected into the vesicle lumen, impeding quantification of DNA that had remained in the phage capsid at the given PEG concentration by DNA accessibility. As DNA protection from DNase inside the P22 capsid upon ejection against increasing osmotic pressures had only been accessed on agarose gels before (Jin et al., 2015), we also analyzed purely LPS-triggered DNA release in the presence of PEG with time-resolved Yo-Pro fluorescence ( Figure 5). Interestingly, we found that much lower osmotic pressures could completely halt DNA ejection from phage P22 than it was reported in the previous work. Already at 15% PEG (3.58 atm), nearly no free DNA was detectable in the solution and was protected from DNase cleavage, indicating that DNA release from phage P22 had come to a nearly complete stop. We assume that this different result might be due to our different LPS preparation method that ensured the complete absence of even minute contaminations of other OM components like proteins or phospholipids. In summary, our results confirmed that irrespectively of the triggering system, either OMVs or LPS, particle opening and DNA egress from P22 are driven by osmotic pressure DISCUSSION Host infection of O-antigen-specific bacteriophages is initiated by the binding of phage particles to LPS. Degradation or deacetylation of the polysaccharide anchors the phage irreversibly to the cell surface, presumably in a defined distance to ensure proper spatial positioning for building a membrane-spanning infection apparatus (Andres et al., 2010;Broeker and Barbirz, 2017;Prokhorov et al., 2017;Wang et al., 2019). It is characteristic for O-antigen-specific phages that in vitro preparations of LPS render them non-infectious because they elicit particle opening and DNA release (Andres et al., 2012;Broeker et al., 2018Broeker et al., , 2019. In contrast, neither O-polysaccharide alone nor lipid A or rough-type LPS with short O-antigen chains is sufficient to trigger particle opening. Apparently, the membrane-like character of LPS preparations that form multilamellar aggregates in solution is a prerequisite to trigger irreversible rearrangements in the phage tail apparatus, ultimately leading to DNA ejection (Richter et al., 2011). However, to also release P22's ejection proteins, the presence of OM proteins is necessary, illustrating that LPS-triggered DNA release only covers a subset of the initial phage infection steps (Parent et al., 2014). In this work, we have investigated the interactions of the O-antigen-specific bacteriophage P22 with OMVs that present LPS and other OM components in a defined way to the phage. OMV Composition and Structure Is Highly Dependent on the Formation Process OMVs are small vesicles with the same membrane organization as found in the Gram-negative OM, i.e., an inner leaflet of phospholipids and an outer LPS layer (Whitfield and Trent, 2014). OMVs thus resemble the OM to a much higher extent as compared to pure LPS aggregates. From this, it naturally follows that vesicles can be active in controlling bacteriophages in a bacterial population, and although these interactions have been unambiguously observed before with electron microscopy, they have so far only been addressed by a few studies (Manning and Kuehn, 2011;Biller et al., 2014;Parent et al., 2014;Reyes-Robles et al., 2018). One major drawback is the difficulty to obtain enough OMV material from culture supernatants (Thein et al., 2010). We therefore used OM fractions where the vesicles were formed during cell lysis. As a matter of fact, OMV composition, especially with respect to the protein content, is highly dependent on the conditions under which the vesicles are formed. During a natural budding event, bacterial cells control the OMV protein load, for example in response to nutrient availability in the medium or to distribute antibiotic resistance factors like β-lactamases (Bai et al., 2014;Kim et al., 2018). Moreover, the size of budded vesicles is dictated by the strain of origin, formation mechanism, and LPS and protein composition (Deatherage et al., 2009;Kulp and Kuehn, 2010;Schwechheimer and Kuehn, 2015;Toyofuku et al., 2019). We have used different techniques to obtain vesicles from OM preparations. Here, the formation of vesicles from the enriched OM is driven by hydrophobic effects and leaflet asymmetry (Park et al., 2015;Rozycki and Lipowsky, 2015). We found that DNaseI and lysozyme treatment were important prerequisites to obtain OMVs with size and shape similar to naturally budded OMVs. This emphasizes that inefficient separation of IMs and OMs most probably resulted in contamination with IM components. In the accumulated OM fraction this hence changed membrane thickness and resulted in large and irregular vesicle size. Moreover, the separation method, i.e., either sucrose gradient or detergent treatment, can influence the extent of residual IM contaminations in OMVs, as well as the protein content (Thein et al., 2010). Accordingly, we observed differences in the protein pattern in OMVs prepared via sucrose gradient or with detergent, although the total protein content was approximately the same. All quantitative bacteriophage-OMV interaction fluorescence analyses in this work were thus carried out with OMVs prepared by a single technique, i.e., the sucrose gradient method. We are aware that OMVs obtained from different preparation methods might show different results. Even more, to further address OMV function in bacterial control of bacteriophage populations, naturally budded vesicles should be tested, for which large-scale preparations have also been reported (Hsia et al., 2016). Nevertheless, we claim that our reductionist in vitro system can add insight into bacteriophage interactions with their O-antigen receptors when part of a more complex membrane system as present in OMVs. Bacteriophage P22 OMV Interactions Clearly Differ From Interactions With Isolated LPS Receptors OMVs produced from bacteriophage P22's S. Typhimurium host act as a decoy for phage particles. Like free LPS aggregates, incubation with OMV preparations rapidly reduced the number of infective particles in the solution. Phages that are in contact with OMVs in the majority were not primed for genome transfer. Rather, they were inactivated, mostly by simply binding and blocking the phage on the OMV by a so far unknown mechanism and, to a smaller extent, by genome loss as shown in this and other studies. Differences in the decrease of infective particle numbers were found with OMVs from different preparations. In particular, the naturally budded OMVs isolated from culture supernatants reduced infective P22 particle numbers to a lesser extent compared to OMVs prepared from OM fractions. It is important to note here that in the experiment, all OMV samples were adjusted to the same LPS content. However, lacking proteome characterization of budded OMVs, we can only speculate whether they contained a different OM protein decoration that interfered with LPS receptor accessibility or influenced the membrane curvature. Moreover, additional periplasmic proteins inside the OMVs could have slowed down P22 inactivation, for example, by increasing the osmotic pressure in the vesicle lumen. Also, if these OMVs presented less LPS on their surface, TSP cleavage of the O-antigen receptor might compete with irreversible phage attachment (Broeker et al., 2019) and prevent phage inactivation. During P22 isolation, OMVs Frontiers in Microbiology \| www.frontiersin.org were not pulled down together with the phage, in contrast to podovirus Sf6 that co-purified with OMVs through interactions with OmpA and OmpC (Parent et al., 2014). In vitro fluorescence analyses have been used regularly to monitor DNA ejection from phages triggered with their isolated receptors, either OM proteins or LPS, in bulk or on a single particle level (Mangenot et al., 2005;Andres et al., 2010Andres et al., , 2012Chiaruttini et al., 2010;Gonzalez-Garcia et al., 2015;Broeker et al., 2019). In these experimental setups, a DNA-specific dye fluorescence increase is observed due to DNA release from a capsid-confined state into a more relaxed state in the solution. For P22 it was shown that particles open and eject about 80% of their DNA content when triggered with LPS (Andres et al., 2010). The kinetic profile of this process is conserved in other podovirus systems analyzed with the same method, but different LPS receptors (Broeker et al., 2018). This reflects that the rate-limiting particle opening step most probably involves rearrangements in conserved parts of the podovirus tail assembly. In this work, we have now used OMVs to trigger in vitro particle opening of bacteriophage P22 and could monitor timeresolved DNA release with a DNA-sensitive fluorescence stain. This is in contrast to cryo-EM analyses that compared end points of ejection by quantifying phages that had lost their DNA (Reyes-Robles et al., 2018). Phage-released DNA in the presence of OMVs was fully protected from DNase degradation, in contrast to the DNA released with pure LPS in solution. This indicates that OMVs and the phage were intimately linked in a stable complex where DNA ejection was exclusively targeted to the vesicle lumen. Similarly, EM images of bacteriophagebound OMVs of the marine cyanobacterium Prochlorococcus suggested that DNA transfer into the vesicle lumen had taken place, as the tail of the myovirus was contracted and the capsid was empty (Biller et al., 2014). Our fluorescence bulk kinetic study typically monitored approximately 10 9 P22 particles, of which only about 30% ejected their DNA upon contact with OMVs. Also for other phages, cryo-EM imaging and quantification showed that a majority of particles had retained their DNA inside the capsid when bound to OMVs, for example, Vibrio cholerae phages in up to 90% of all particles imaged (Reyes-Robles et al., 2018). This fraction is similar to what was found with cryo-EM for OMV-bound Shigella podovirus Sf6 (Parent et al., 2014). So far, we have failed with attempts to physically separate the OMV-phage P22 complexes to individually analyze the vesicles' DNA content after phage contact. Similar problems had also been reported before for OMVs binding to bacteriophage T4 (Manning and Kuehn, 2011). Apparently, it is difficult to find solubilization conditions that either address the OMV or the phage without affecting the integrity of the other. Moreover, irreversible phage attachment to OMVs might already destabilize the phage even if it has not released its DNA. Our experiments furthermore showed that, compared to using pure LPS as receptor for triggering DNA release from phage P22, the fluorescence signal obtained with OMV receptors went into saturation twice as fast. In OMVs, P22 now encountered a more complex membrane system compared to LPS. LPS has a unique solution behavior, forming multilamellar aggregates that presumably mimic membrane-like structures addressed by the phage in vitro, but do not represent all characteristics of the OM heterobilayer (Andres et al., 2010;Richter et al., 2011). In OMVs in contrast, OM proteins are present that might in a so far unknown way accelerate the phage particle opening step and the subsequent DNA release. This becomes evident especially when analyzing P22 at elevated osmotic pressures. In this work, we showed that DNA release triggered by highly pure LPS lacking OM proteins already ceased at external osmotic pressures of about 3.6 atm. However, it has been shown earlier that in the presence of OM proteins at this osmotic pressure, P22 still released a fraction of its DNA (Jin et al., 2015). Moreover, OM proteins enabled the release of P22's ejection proteins, in contrast to LPS alone. Our actual DNA ejection studies are in line with these findings. P22 in the presence of OMVs at 6.8 atm osmotic pressure still showed partial DNA loss, emphasizing that progression of DNA release from the phage particle is intimately coupled to the receptor type. For P22, it was shown that this can be pure LPS aggregates, a combination of LPS and OM proteins or OMVs. We note that in comparison of the present study with the one cited above (Parent et al., 2014), the latter showed no difference in fractional DNA release between samples triggered with LPS alone or with a mixture of OmpA and LPS. As a reason for this ambiguity, we assume that different LPS preparation protocols may have resulted in incomplete OM protein removal. In summary, the new results emphasize the critical role of OM composition for phage control. Bacteria might exploit this by adjusting their OMV contents in response to phage attack. Our in vitro experiments again illustrate that all energy needed for DNA release from bacteriophages is already stored in the system (Keller et al., 2017). The phage genome is highly confined inside the capsid, packaged against the DNA intramolecular electrostatic repulsion forces by an ATP-hydrolysis-driven motor (Smith et al., 2001). Accordingly, increasing external osmotic pressures can counteract genome liberation (Evilevitch et al., 2003). For siphovirus λ with a long, non-contractile tail, an internal capsid pressure of about 20 atm was estimated that was dependent on the relationship of genome length and capsid size (Grayson et al., 2006). However, external factors were shown to significantly influence the genome ejection characteristics of λ, illustrating the complex behavior of the DNA polyelectrolyte both inside the capsid and when transferred into the solution (Evilevitch, 2018). Also, the osmotic status of OMVs interacting with phage P22 might play a role in the observed DNA release process. If OMVs had a significant turgor, for example, due to their luminal protein content, the amount of DNA release from P22 would be insensitive to low osmotic pressures in the surrounding solution. However, also with OMVs, we observed that the amount of ejected DNA decreased with increasing PEG solution concentrations. This indicates (i) that presumably no osmotic pressure existed in the OMVs and (ii) that ejecting P22 particles could transfer substantial amounts of DNA inside the vesicles. As already discussed above, we could not directly quantify how much DNA remained inside the phage capsid and how much was translocated into the vesicle lumen. Nevertheless, the notable effect of low PEG concentrations on the ejection signal shows that more than 30% of the DNA must have left the capsid on the level of an individually ejecting phage, in agreement with cryo-EM studies with other phage systems (Parent et al., 2014;Reyes-Robles et al., 2018). Hence, the reduced signal amplitude observed in fluorescence curves obtained with OMVs is due to a reduced number of particles actively ejecting when compared to particles triggered with free LPS. In the future, this bulk study-derived hypothesis needs further evaluation to understand molecular details of OMV-mediated bacteriophage inhibition. CONCLUSION Bacteriophage P22 in vitro particle opening studies with OMVs revealed that the action of the LPS receptor on the phage is modulated when LPS is part of a more complex OM system. Although OMVs could also effectively reduce the number of infective particles, inactivation was not driven by DNA release from all particles as observed with pure LPS fragments. Rather, a remarkable amount of phages did not eject their DNA. The mechanism of this OMV-driven bacteriophage inactivation remains to be elucidated and may be steered by different OMV properties. For example, vesicular curvature and protein decoration result in enhanced membrane tension and variable membrane thickness, respectively (Wu et al., 2014;Huang et al., 2017). Also, the presence of OM proteins as co-receptors influences the phage interactions (Parent et al., 2014). Moreover, multiple binding of P22 phages on small vesicles may induce mutual obstruction between the phages, prohibiting the DNA ejection. Importantly, we found that a preparation of naturally budded vesicles showed a much smaller capacity to inhibit the phage compared to the fraction obtained during mechanical cell lysis. This emphasizes that vesicle composition is a major control point for their function as phage blocking agents. From a more general point of view, if bacteria shed OMVs to block a phage population, they might employ a mechanism to inhibit substantial DNA liberation into the OMVs. As OMVs may also act in gene transfer, bacteria thus avoid another route with which phage genes could enter the bacterial cell. Further analyses will elucidate how the biochemical composition and properties of OMVs are linked to bacteriophage population regulation in a given functional context. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the article/Supplementary Material. AUTHOR CONTRIBUTIONS SB, DL, NR, and NB conceptualized the study and methodology. MS, NR, and AS performed the experiments. MS, NR, NB, and SB evaluated and visualized the data. MS, SB, DL, and AS wrote the manuscript. SB, DL, AS, NR, and NB supervised the study. SB and DL acquired funding. All authors contributed to the article and approved the submitted version. ACKNOWLEDGMENTS We thank Antje Hofgaard, Jens Wohlmann, and Martin Wolff for assistance with electron microscopy and dynamic light scattering, respectively. We thank Mandy Schietke for excellent technical support. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.510638/ full#supplementary-material OMV size distributions from dynamic light scattering (Supplementary Figure S1), additional TEM images (Supplementary Figures S2, S3, and S6) and characterization of OMVs ( Supplementary Figures S3-S5) as well as additional DNA ejection curves (Supplementary Figures S7-S9) can be found in the Supplementary Data Sheet 1.	2020-09-24T13:09:37.857Z	2020-09-24T00:00:00.000	{ "year": 2020, "sha1": "bba78c0664ecb805e3a0362861010476a9dd1081", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmicb.2020.510638/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bba78c0664ecb805e3a0362861010476a9dd1081", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
214295222	pes2o/s2orc	v3-fos-license	Research on M&A Performance of Listed Companies in Internet Industry Based on Engineering Management In recent years, the in-depth application of the Internet and other new generation of information technology in the engineering construction industry has given birth to the intelligent construction platform mode in the Internet era. Under this new model, the organization mode, operation mode and management mode of the existing project construction have undergone profound changes. Therefore, exploring the development of Internet industry is of great significance to project management. This paper selects 36 independent companies in Internet industry during the M&A announcement period in 2014 as research samples. Then it uses accounting research method and constructs the financial index system in order to compare the comprehensive performance of companies in the short term. The empirical research draws the following conclusions: First, M&A activity has not significantly promoted the performance of Internet industry companies. Second, different types of M&A produce different effects. Mixed M&A are more beneficial to the improvement of the performance of companies in the Internet industry. Finally, combined with the empirical results, this paper proposes suggestions for M&A activities of listed companies in the Internet industry from the government and enterprise levels. 1.Introduction Mergers and acquisitions(M&A) are a major strategic measure for enterprises to expand their scale and achieve long-term development. In China, with the concept of the supply-side reform and economic transformation and upgrading proposed in recent years, the development of the domestic M&A transaction market has gradually accelerated. The transaction scale has generally shown an overall growth trend, which has greatly promoted the rapid development of China's social economy. Among numerous industries, the number of enterprises in the Internet industry has also shown an obvious growth trend, and the competition is becoming increasingly fierce. Therefore, integration and M&A have become the key words for the development of this industry in recent years, so as to optimize the company's corporate structure and social resource allocation, and increase the core competitive advantage. According to the consolidation and statistics of China's 2013-2017 M&A data, there were 3,313 M&A in the Internet industry, second only to those in the industrial sector. And the amount involved is 12, 955. 873 yuan, accounting for a large proportion of the total amount of M&A. Therefore, this paper focus on the listed companies in the Internet industry as the research object and analyzes the impact of M&A events on the comprehensive performance of listed companies. The next article is divided into four parts. Section2 is to summary and comment on the research status at home and abroad. Section3 is the empirical process, including hypothesis, data collection, sample selection and research design. Section4 is mainly to analyze the empirical results. Section5 is the conclusion of the study and related recommendations. 2.Literature review The research on M&A performance at home and abroad is mainly divided into short-term performance research and long-term performance research. Most of the research shows that there is no obvious promotion of the performance of M&A entities in the short term due to the need for more time or other unstable factors after M&A integration. However, in the long term, M&A activity has a significant effect on corporate performance improvement. In 2016, Rekha and Julie studied M&A activities in ASEAN countries and collected financial indicators from 2001 to 2012 to observe changes in performance before and after mergers and acquisitions. As a result, in the three years after the merger, the adjusted operating results of M&A activities generally showed a downward trend [1]. In terms of research methods, the scholars at home and abroad mainly adopt event research method and accounting research method. But the conclusions obtained by the two methods may not be consistent. Arvanitis and Stucki (2012) used accounting research method to analyze the post-merger performance changes and the impact on various economic performance indicators. Three of the five key performance indicators used in the empirical study reflect that M&A has a significant positive impact on firm performance [2]. Zhang Benzhao and Sheng Qianwen (2017) selected 55 Internet finance companies as research samples and They used the event research method to calculate the average return rate and cumulative abnormal return rate of the sample enterprises. The results showed that the overall performance of the sample company is fully improved in the short term [3]. The event research method is based on the assumption of effective capital market. But China's capital market has not yet matured. There are many uncertain factors and the effectiveness has not yet reached the standard. Therefore, this paper chooses the accounting research method for empirical research. Primary hypothesis According to the M&A performance theory, M&A transactions can improve the market share, reduce transaction costs and agency problems between management and owners. It is beneficial to achieve partial or overall improvement in terms of operation, management and finance. But the conceited hypothesis (Roll, 1986) argues that management may have misunderstandings about M&A activity because of overconfidence, and then make wrong decisions [4]. In addition, domestic scholars' understanding of M&A performance is not consistent. Pang Qi and Zhang Jianping (2015) believe that corporate performance will be improved in the short term due to M&A activities [5], while Li Siyong and Zhang Jing (2017) believe that the merger of Chinese bosses in the short term did not reach the expected state [6]. Therefore, this paper propose a competitive hypothesis: H1a: M&A activities have contributed to the improvement of short-term performance of listed companies in the Internet industry. H1b: M&A activities have no significant effect on the short-term performance improvement of listed companies in the Internet industry. From the perspective of different industries, the role of different M&A types varies from one to another depending on the path of action. According to the development characteristics of the Internet industry, horizontal M&A and vertical M&A may cause employees to stick to the rules and poor work enthusiasm for innovation. As a result, investment in technology research and development has increased, and performance has decreased. By contrast, mixed M&A can expand the scope of business, thereby popularizing the application of Internet technology in many business areas, or gaining more flexible funds and benefits in other business areas. Therefore, mixed M&A can promote the development of enterprises to a certain extent. Therefore, this paper proposes hypothesis 2: H2: Compared with horizontal M&A and vertical M&A, hybrid M&A are more beneficial to the short-term performance improvement of Internet industry companies. Data and sample selection This paper takes listed companies in the Internet industry with M&A transactions in 2014 of Shenzhen stock exchange and Shanghai stock exchange as the original research samples, and the research window is from 2013 to 2016. The sample screening conditions are as follows: a. The company that has completed the merger and acquisition, and the amount of the merger is more than 10 million yuan. b. The control of the target company has changed. c. The financial data of the sample company during the study period is complete and available. d. Excluding ST and *ST companies. e. Excluding companies that frequently make multiple M&A transactions during the study period. f. A company that has had multiple M&As in the same year, taking the largest M&A event. Finally, a total of 36 listed companies in the Internet industry were identified as research samples. According to the type of M&A, 23 horizontal M&A, 6 vertical M&A, and 7 M&A. All data comes from the Wind database and is collected manually. Research design Table1. Comprehensive financial index system [7], this paper selects 16 financial indicators to build an evaluation system for M&A performance, as shown in Table1. In this paper, SPSS 22.0 is used to reduce the dimension of 16 original variables into a few common factors by factor analysis, so as to reduce the correlation between independent variables. First, the feasibility test of factor analysis is carried out. The test results showed that the KMO in each year is greater than 0.5, and the sig. value in Bartlett sphericity test is all 0, proving that the data come from the normally distributed population and meet the necessary conditions of factor analysis. And by testing the commonality of the variables, the results show that the commonalities of all variables are above 55.2%, and 56.25% of the variables have a commonality close to or exceed 90%. This proves that the common factor to be extracted can explain the variables to a large extent. And table2 shows that the cumulative variance contribution rate of the first five components in each period are all greater than 70%. This indicates that these five components have a higher degree of interpretation of the original data, so the first five components are extracted as common factors. Then, according to the component score coefficient matrix, Xj is represented by a linear combination of factor scores fi, thereby obtaining a score of the factor variable fi. Finally, the formula of the comprehensive performance score function is obtained by taking the ratio of the variance of the factor after rotation to the total variance as the weight. Corporate performance analysis of the sample as a whole Table4. Statistical table of the difference in company performance scores for each year(t=2014) As can be seen from the above table: First, the number of companies with improved performance in the year of mergers and acquisitions is less than the number of companies with deteriorating performance. Second, the number of companies whose performance has improved in the first year after M&A. However, compared with the year before the merger, the number of companies with improved performance is only 17, indicating that the first year of M&A has only a slight promotion effect on the company's performance. Third, compared with the year of M&A and the year before the merger, more than half of the companies in the second year after the merger have improved their performance, but the proportion has not reached the expected level. Moreover, the number of companies with reduced performance exceeds those with improved performance compared to the first year after the merger. In summary, the financial situation of the year of merger and acquisition may not be improved, and even financial performance may decline. Therefore, H1b is verified. Index It can be seen from the sign of the mean of the difference that the average performance of the listed company rises and then falls in the horizontal M&A, and then rises and in the vertical M&A it first drops and rises. Both are in an unstable change. However, the performance of listed companies under the mixed M&A method has been greatly improved after the M&A event, far exceeding the comprehensive performance before the M&A of listed companies. Moreover, the average performance of the mixed M&A is significantly higher than that of horizontal M&A and vertical M&A, and the percentage of positive value increases year by year. Therefore, according to the above analysis, different types of M&A have produced different effects. Mixed M&A is more conducive to the performance improvement of listed companies in the Internet industry. Hypothesis 2 is established. 5.Summary and conclusions Based on the current market economy environment, this paper takes the performance of listed companies in the Internet industry as the research object, and builds a performance evaluation system to study the impact of M&A activities on the short-term performance of listed companies in the Internet industry. The study found that the performance of listed companies in the Internet industry within two years after M&A has not been significantly improved. And among different types of M&A, the positive promotion effect of mixed M&A is more significant. Combined with the conclusions, this paper proposes the following suggestions. First, companies must make appropriate M&A strategic decisions based on their operational conditions and long-term development. Although mixed M&A are more conducive to the current development of the company in the short-term, enterprises should carefully select the type of merger based on their own needs and existing conditions in the long term. Second, companies should strengthen internal control. The owner should supervise the decision-making process of the manager and correct the deviation of the decision in time to ensure the smooth implementation of the M&A. It is beneficial to prevent the failure of the merger due to the wrong judgment. Third, the government should establish and improve the legal system related to M&A, reduce or even eliminate unfair trade practices, punish illegal M&A, and provide a standardized external environment for corporate mergers and acquisitions activities.	2019-12-12T10:51:14.611Z	2019-12-06T00:00:00.000	{ "year": 2019, "sha1": "528fde27eb66d4f43a2a30c01da4a6018674021d", "oa_license": null, "oa_url": "https://doi.org/10.1088/1757-899x/688/5/055050", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "73a47629c9150fe2c11e22cd0945bf640f0764c0", "s2fieldsofstudy": [ "Engineering", "Computer Science", "Business" ], "extfieldsofstudy": [ "Physics", "Business" ] }
11611455	pes2o/s2orc	v3-fos-license	Hepatic Tissue Engineering Using Scaffolds: State of the Art Severe hepatic failure accounts for many deaths and raises medical costs each year worldwide. Currently, liver transplantation is the most common therapeutic option for patients with end-stage chronic liver disease. Due to decrease in the number of organ donors, many in need of transplantation continue to remain on the waiting list. Hepatic Tissue Engineering is a step toward alleviating the need for organ donors. Regenerative medicine and tissue engineering require two complementary key ingredients as follows: 1) biologically compatible scaffolds that can be readily adopted by the body system without harm, and 2) suitable cells including various stem cells or primary cells that effectively replace the damaged tissues without adverse consequences. Yet many challenges must be overcome such as scaffold choice, cell source and immunological barriers. Today, hepatogenic differentiation of stem cells has created trust and promise for use of these cells in hepatic tissue engineering and liver replacement. However, using suitable scaffolds is an important key to achieving the necessary functions required for hepatic replacement. In recent years, different scaffolds have been used for liver tissue engineering. In this review, we have presented different concepts in using cell /scaffold constructs to guide hepatic tissue engineering. Introduction Every year, the number of patients needing a hepatic transplant increases. Many of those in need of a transplant have suffered from full hepatic failure caused by disease, genetic complications or adverse drug reactions. Currently, there are many people waiting to have a liver transplant. However, there are not enough organ donors. At the moment, there are about 700 patients waiting to have a liver transplant in Iran, but the number of liver donors is less than 200. At present, liver transplantation is the only therapeutic option for patients with end-stage chronic liver disease and severe liver failure. However, the efficacy of liver transplanta-tion is limited by the shortage of available organ donors, risk of rejection, infections, and other complications caused by the lifelong immunosuppression (1) . Tissue engineering proves to be a temporary treatment for patients suffering from hepatic failure (2) . For successful tissue regeneration, the cells constituting tissues to be regenerated are necessary. Considering the proliferation activity and differentiation potential of cells, stem cells are practically promising. Self-renewal is a unique property of stem cells that gives multi-potential differentiation ability to them. Today, there are different studies showing hepatogenic differentiation capacity of the stem cells (3)(4)(5) . However, the challenge remains to develop robust protocols, to generate functional hepatocytes from stem cells suitable for the transplantation. A complementary key ingredient in regenerative medicine and tissue engineering is to make a use of biologically compatible scaffolds that can be readily adopted by the body system without harm (6) . Advances in polymer chemistry have facilitated the engineering of synthetic matrices that can be precisely manipulated with regard to physical and mechanical characteristics. This review has presented some directions that the field of liver tissue engineering is heading. Hepatic biology The liver is a highly metabolic, complex array of vasculature, endothelial cells and parenchymal cells that performs many functions in the body. The bulk of the liver is primarily composed of parenchymal cells such as hepatocytes, hepatocyte precursor cells (oval cells or Ito cells), stellate cells, kuppfer cells, epithelial cells, sinusoidal epithelial cells, biliary epithelial cells and fibroblasts (7) . Hepatocytes constitute approximately 70% of the cellular population of the liver and perform major metabolic functions such as plasma protein synthesis and transport, xenobiotic metabolism, glucose homeostasis, urea synthesis, and ketogenesis (8) . Thus, hepatocytes used for tissue engineering purposes must be able to perform these basic functions. Cell source In the field of hepatic tissue engineering, choosing cell type and cell source is important because it is necessary to choose cells that demonstrate the particular phenotype of interest. The various cell types that have been studied include stem cells, hematopoietic cells, oval progenitor cell and mature hepatocytes (9)(10)(11) . Deciding which cell type to use is dictated by the need and desire for the cells to perform in a predicted manner, exhibiting certain characteristics. Hepatic progenitor cells found within the liver have already begun to differentiate, but still have several options before becoming destined to a specific cell line. These cells will not necessary become mature hepatocytes, but may in fact differentiate into other functional cells of the liver, such as bile duct cells (12,13) . Hepatic progenitor cells are often distinguished as primary or small hepatocytes. Mature hepatocytes can be obtained either from the perfusion of an intact or resectioned liver or from an established cell line. Currently, primary mature hepatocytes, the most common cellular component in current liver tissue engineering, do not replicate sufficiently in vitro to meet the requirements of clinical use and do not maintain their differentiated properties in vitro (14) . So the search for novel cell resources has prompted investigations into generating hepatocytes from stem cells of extra hepatic origin, based on their availability and unrestricted potential to propagate and differentiate (15)(16)(17) . The stem cells found in sources such as bone marrow, are the most flexible cells in terms of their undetermined pathway and they express a remarkable ability to differentiate into desired cell types (10,12,13) . The interest in adult stem cells has in particular been triggered by the numerous ethical dilemmas surrounding the use of embryonic stem cells in pre-clinical and clinical research (18) . Among the adult stem cells, human Bone Marrow derived Mesenchymal Stem Cells (hBMSCs) have great potential for liver tissue engineering because autologous BMSCs can be harvested, expanded extensively ex vivo, and differentiated into a hepatic phenotype for transplantation back into patient (18,19) . From stem cells to hepatocytes Until recently, it was believed that hepatocytes could only be derived from cells of endodermal origin and their progenitors. However, recent studies suggest that nonendodermal cells may also form hepatocytes in vivo and in vitro. At present, it is believed that stem cells divide asymmetrically to produce a new stem cell and a progenitor cell that subsequently undergoes differentiation and maturation to form functional tissues (25) . It seems the microenvironment or niche of the stem cell is likely to be one of the factors dictating the type of mature functional cells. The original idea of a stem cell ''niche'' evolved from the concept that stem cells inhabit tissues within an ''inductive microenvironment'' that directs their self-renewal, differentiation, and cell fate in both normal physiology and disease (26)(27)(28) . The coordinated signaling between component cells and scaffold, (in) direct cell-cell contacts, and integration of stem cell autonomous properties represent an interactive and dynamic system, organized to facilitate cell fate decisions in a proper spatiotemporal manner (26,30,31) . The microenvironment of developing hepatocytes is a continuously changing process of successively occurring biological events (34) . Each step of cell growth and differentiation is tightly regulated by intra extracellular communication as well as cell autonomous mechanisms (35) . Activin, Fibroblastic Growth Factor (FGF), Bone Morphogenesis Protein (BMP), Hepatocyte Growth Factor (HGF) and Oncostatin M (OSM) are the most essential extracellular signals. HGF is known to mediate growth, proliferation, angiogenesis and cell motility. Growth and differentiation of hepatocytes are known to be controlled by the Epidermal Growth Factor (EGF), FGF, Interleukin-6 (IL-6), Transforming Growth Factor (TGF-a), and Insulin-like Growth Factors (IGF). Corticosteroids, amino acids, OSM, nicotanimide, and Dimethyl sulfoxide (DMSO) stimulate function and differentiation (25,31,34,(35)(36)(37) . At the intracellular level, the liver-enriched transcription factors including Hepatocyte Nuclear Factor (HNF) 3α,β, HNF4α, HNF1α, β, and HNF6 act consecutively in essence in a cross-regulatory manner at specific development stages to regulate liver-specific gene expression. Interactions between these various compartments accomplish homeostatic regulation of stem/ progenitor cell functioning in vivo (25,30,31) . Consequently, identification and simulation of these in vivo signaling patterns might comprise an approach to contribute to fate reprogramming of stem/ progenitor cells in vitro. Cell-matrix constructs Generating cell/ matrix constructs to guide tissue regeneration involves isolating appropriate cell populations and transferring these to polymer scaffolds for in vivo implantation. The scaffold functions to (a) provide structural integrity and to define a potential space for the engineered tissue, (b) guide restructuring that occurs through proliferation of cells donor and ingrowths of host tissue, (c) maintain distances between cells that permit diffusion of gas and nutrients and possibly the ingrowths of vasculature from the host bed and (d) to transmit tissue-specific mechanical forces to cue the behavior of cells within it. A biodegradable polymer will degrade and gradually be replaced by regenerated tissue, minimizing the substrate for an inflammatory response (6) . Employing cell/ polymer matrices for tissue regeneration is an approach that permits experimental manipulation at three levels to achieve optimal constructs for individual tissues, i.e. the cells, the polymer scaffolds and the methods used for construct assembly. Polymer scaffolds There are multiple approaches to engineering a viable liver, with variables such as cell type, structure, and material. The scaffold is a common feature of many liver tissue engineering projects. Its benefits include providing a place for attachment, increased surface area, support for a larger cell mass, and the capability of shaping specific structures. Importantly the scaffold must be biocompatible and biodegradable allowing the organ to grow "in lieu" of the scaffold and support itself over time. Other aspects that have been studied include surface pattern and structure for optimal attachment, porosity for nutrient and gas exchange, surface factors for increased and supported growth and function, and surface coating of the scaffold (1,6,38) . Scaffolds provide a site of attachment for hepatocytes and are a delivery vehicle for transplantation. In addition to the basic structural vehicle, several other conditions must be met before a scaffold may be used in tissue engineering applications. The scaffold must be biodegradeable and biocompatible in that they do not leach harmful materials as they degrade. Pore size must be controllable to allow for prevascularization or angiogenesis occurring. Also, the scaffold should have sufficient surface area for cells to attach and be able to provide enough room for the cell colony to expand and proliferate (6,38) . Polymer scaffolds can be constructed from natural or synthetic biomaterials. The hepatogenic differentiation of stem cells in natural matrix such as collagen, fibronectin, gelatin and matrigel has been the subject of different reports (39)(40)(41)(42) . Natural polymers are suitable for cell interaction, however, scaffolds fabricated purely from these molecules exhibit poor mechanical strength and are not easy to handle. Large batch to batch variations upon isolation from biological tissues as well as restricted versatility in designing devices with specific biomechanical properties are other limitations assigned to the natural scaffolds (43) . Advances in polymer chemistry have fa-cilitated the engineering of synthetic matrices that can be precisely manipulated with regard to physical and mechanical characteristics. Variables such as polymer porosity and degradation rate can be systematically regulated by altering either the materials employed or polymer-processing methods. A variety of synthetic polymers exist, including polyesters, synthetic polypeptides and hydrogels. The most widely used polymers in tissue engineering have been aliphatic polyesters i.e. Polyglycolic Acid (PGA), Polylactic Acid (PLA), Poly Lactic-co-glycolic Acid (PLGA) and Polycaprolactone (PCL) (44) . These synthetic polymers have advantages in pro-accessibility, good mechanical properties and manipulating degradation rate, but they lack cell recognition signals and hinder successful cell seeding because of their hydrophobic trait. Therefore, to encourage cell ingrowths for better integration between cells and the scaffold, the biologically inert synthetic materials need effective hybridization with bioactive molecules (45,46) . The scaffolds should mimic the structure and biological function of native Extracellular Matrix (ECM). A well known feature of native ECM structures is the nanoscaled dimensions of their physical structure (6,47) . In recent years, with respect to nanofibers for tissue engineering purposes, a wide variety of nanofibrous scaffolds have been produced (48)(49)(50)(51)(52) . Design of nanofibers is an important concern in the effective applications of these nanostructured materials. Different techniques have been used for formation of nanofibrous materials (53) . There is increasing interest towards employing electrospinning for scaffold fabrication because the mechanical, biological, and kinetic properties of the scaffold are easily manipulated by altering the polymer solution composition and processing parameters. It has been shown that electrospun 3D nanofibrous structures share morphological similarities to ECM, and are capable of promoting favorable biological responses from seeded cells (54,55) . Kazemnejad et al (2007) designed an artificial nanofibrous matrix that can mimic ECM, to support hepatic tissue engineering. They introduced a scaffold composed of Poly (ε-caprolactone), collagen and polyether sulfone was fabricated by electrospinning technique. It has been reported that the engineered nanofibrous scaffold was a conductive matrix which supports and enhances stem cells development into functional hepatocyte-like cells (56) . Hepatic tissue engineering using scaffolds Hepatocytes are known to better maintain their differentiated functions in three dimensional (3D) multicellular aggregates or spheroids than in monolayer culture (57,58) . Extensive cell-cell contact between hepatocytes grown in aggregates promotes the formation of gap junctions, tight junctions, and bile canaliculi that are important for stabilizing the hepatocyte phenotype (59,60) . Cells in spheroids also have a morphology and ultra structure similar to those found in a native liver lobule (61)(62)(63) . It has also been demonstrated that an increased level of Ecadherin mediated cell adhesion between cultured hepatocytes, induces higher levels of liver-specific functions (64) . Many studies have also highlighted the benefit of matrigel, a basement membrane extract from the Engelbreth-Holm-Swarm mouse sarcoma that serves as a complex ECM, in prolonging the maintenance of adult hepatocyte functions and in promoting the maturation of hepatic progenitor cells. Differentiation of stem cells and hepatic progenitor cells is the most complete on 3D matrigel (40,65,66) , and liver-specific functions of adult hepatocytes are better maintained when they are plated on a combination of ECM molecules (67,68) . To date, various coatings like fibronectin, collagen, and matrigel have been used to support the differentiation of stem cells to hepatocytes (40,(69)(70)(71) . Therefore, in vitro selective growth and differentiation of MSCs in 3D biocompatible polymer scaffolds could be very efficient to develop a liver tissue having a clinically significant mass and maintain liver-specific functions. However, the use of such natural scaffolds has been associated with some limitations. The problem with the control of pore size and porosity, large batch to batch variations upon isolation from biological tissues and poor biomechanical strength are the major concerns. As such artificial microenvironments including nanofibers designed to produce differentiated cells from stem cells and progenitor cells need to support both adult progenitor cell proliferation and differentiation (72)(73)(74)(75)(76)(77)(78)(79) . Although there is a significant interest in using nanofibers in tissue engineering from stem cells, reports on the transdifferentiation of stem cells into the hepatic lineage in a nanofibrous configuration is scanty. More recently, differentiation of Human cord blood-derived unrestricted somatic stem cells into hepatocyte-like cells on poly (epsilon-Caprolactone) nanofiber scaffolds has been reported (80) . Thereafter, Kazemnejad et al (2009) differentiated hBMSCs into hepatocyte-like cells on an artificial nanofibrous matrix composed of PCL, collagen and PES. Based on the experimental evidences the expression of liver specific genes such as albumin, alpha fetoprotein, cytochrome P450 3A4, cytokeratin-18 and cytokeratin-19 detected by RT-PCR, showed progressive expression during 3 weeks of differentiation on 3D scaffold. Moreover, the hepatocyte like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, Serum Glutamic Pyruvic Transaminase (SGPT) and Serum Oxaloacetate Amino Transferase (SGOT) (81) . In an another study, Kazemnejad et al demonstrated that the PCL/ collagen/ PES nanofibers not only allow the hBMSCs to differentiate into hepatocyte, but also enhance MSCs development into functional hepatocyte-like cells when compared to the conventional culture system. They reported that the levels of the mentioned markers (except SGOT) in differentiated cells on scaffold were significantly greater than that in 2D culture system (p<0.05). So it seems that the high porous PCL/ collagen/ PES architecture provides an ECM-like nano-environment that is conducive to normal hepatic differentiation (82) . Detailed knowledge about the part played by the scaffold architecture for enhancing the stem cell differentiation especially into hepatocytes needs further studies. It is assumed that the presence of biological signals from the biomimetic nanofibers provides a nano-environment resembling a 3D natural ECM which would enhance the biological activity of growth factors and cytokines for inducing differentiation. In vivo studies of hepatic tissue engineering Hepatic tissue engineering is focused on creating a whole, implantable and functional liver. Many approaches have been used such as direct cellular injection onto present vascular beds, micro-carrier attachment and scaffold implants seeded with cells. One obstacle that must be overcome in complex, vascular organs such as the liver is the feeding of nutrients and removing waste from the cells in the interior. Two approaches to solve this problem are to allow the process of angiogenesis to occur into the cell aggregate or preestablish a vasculature bed and seed the cells around the network. The success of tissue engineering over other organs such as bone, cartilage and skin has been achieved, because they are not as highly metabolic and do not require an extensive vasculature. In addition, these vascularized organs do not need to achieve the large dimensions that many vital organs do. Direct cellular injection into a vascularized area of the body has been the focus for the cells to sustain the metabolic activity of the organ. Cells have been injected with or without a hydrogel into various vascular beds, cavities, and organs within the body (83) . Several areas that have been injected with cells are the spleen, pancreas and peritoneal cavities. In the case of transplantation into the liver through the portal vein, the number of trans-planted cells is limited because intraportal injection of too many cells might cause lethal portal embolism and liver necrosis. The spleen is considered to be a suitable site for implantation because hepatocytes can be stably viable and maintain their functions (84,85) . The only disadvantage is that the number of transplanted cells might be limited. The peritoneal cavity seems to be the most likely candidate site for implantation, because invasive treatments are not necessary and a large number of cells can be transplanted. However, the disadvantage is the difficulty in maintenance of hepatocyte viability in the peritoneal cavity. Cells have also been implanted in prevascularized polymer sponges to improve cell survival in vivo. Although success has been achieved, the cells are limited by the size and the aggregate they can achieve due to lack of vasculature in the construct (1) . Three dimensional printing of biodegradable polymers allows the ability to create complex shapes and exact replicas of existing structures from Computed Tomography (CT) scans. Isolated hepatocytes on PGA meshes were first transplanted into the mesentery and omentum of syngeneic rats. Cells in these constructs expressed liver specific functions prior to transplantation (86) and survived for extended periods of time following transplantation, organizing into liver-like structures (87) . However, a significant loss of cells was noted post-transplant. This was felt to be due to (a) a failure to meet the oxygen/nutrient requirements of the cell mass and (b) insufficient stimulation of the transplanted cells or to both of these factors. To augment the supply of these essentials, Stein et al performed prevascularization on a polymer scaffold composed of polyvinyl alcohol and transplanted into recipient animals, i.e. partial hepatectomy and a portal-caval shunt (88) . These procedures resulted in an increased delivery of hepatotrophic factors to the systemic circulation and diminished their clearance by the native liver and led to significant improvement in cell survival. Higashiyama and his colleague transplanted rat hepatocytes seeded in porous Hydroxyapatite (HA) disks into the peritoneal cavity of Nagase Analbuminemia rats (NARs) (89) . Angiogenesis was observed inside the pores in HA disks, and hepatocyte viability was shown to be maintained for at least 3 weeks, as evidenced by the increase in the serum albumin level. Moreover, these researchers have been attempting to maintain the viability and functions of hepatocytes by co-culturing them with various cells, such as Nonparenchymal liver cells (NPLCs) (90,91) . They then, co-cultured hepatocytes with BMSCs in HA disks and transplanted the disks into NARs and liver-damaged mice. The increase of serum albumin level in the liver-damaged mice was reported by the transplantation of hepatocytes and BMSCs. The serum level of IL-6 in the liver-damaged mice was also increased by the cotransplantation of BMSCs and hepatocytes (92) . Polymer implantation, surgical stimulation and cell transplantation technologies have been extended to large animal models. Dalmation dogs have a genetic deficiency in uric acid uptake in the liver which results in elevated serum and urine uric acid levels. Implantation of hepatocytes on PGA sheets resulted in partial correction of the enzyme deficiency for up to six weeks (93) . Successful hepatocyte engraftment has also been achieved in swine (94) . These evidences clearly show that more in vivo work needs to be accomplished before a viable organ will be available for transplanttation. Conclusion With the recent advances in the field of hepatic tissue engineering, there is much promise of working towards an implantable whole organ. Many new polymers are being developed that respond to thermal changes, release imbedded or attached growth factors and other mediators, and have degradation characteristics and properties that is ideal for growth, viability, and attachment. An optimal polymer is being developed based on desired characteristics. Recently, electrospun nanofibrous scaffolds showed great promise and potential for liver tissue engineering. Many other factors are being studied that contribute to cell growth and differentiation. However, further studies need to be performed for the development of a bioartificial liver system. With the growth of the tissue-engineering field, many ethical considerations must be recognized. Determining which cell source is safest for patients, which cells should be used, whether they are embryonic stem cells, oval progenitors or adult stem cells, and how the cells should be stored and cultured are important issues to take into consideration. Hepatic tissue engineering is an ever expanding field encompassing and including new areas of study. Because of its multidisciplinary nature, it is important for clinicians, basic scientists and engineers to collaborate and explore all areas of possibilities. With each new advance in the field of tissue engineering, a step towards an implantable liver is realized. Even though the goal of creating an entire implantable organ has not yet been reached, the progress towards this goal is proving to be fruitful to all those involved, mainly the patients who will benefit from the advancements being made. Development of a novel three-dimensional biocompatible nanofibrous scaffold for the expansion and hepatogenic differentiation of human bone marrow mesenchymal stem cells. Iran J Biotechnol 2007;5(4):201-211.	2017-06-18T09:43:57.350Z	2009-03-06T00:00:00.000	{ "year": 2009, "sha1": "1b4019f52cfdb1a84033e94ebfbb3e62580d2c85", "oa_license": "CCBYNC", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "1b4019f52cfdb1a84033e94ebfbb3e62580d2c85", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
52842998	pes2o/s2orc	v3-fos-license	Evaluating Economic Growth, Industrial Structure, and Water Quality of the Xiangjiang River Basin in China Based on a Spatial Econometric Approach This research utilizes the environmental Kuznets curve to demonstrate the interrelationship between economic growth, industrial structure, and water quality of the Xiangjiang river basin in China by employing spatial panel data models. First, it obtains two variables (namely, CODMn, which represents the chemical oxygen demand of using KMnO4 as chemical oxidant, and NH3-N, which represents the ammonia nitrogen content index of wastewater) by pretreating the data of 42 environmental monitoring stations in the Xiangjiang river basin from 2005 to 2015. Afterward, Moran’s I index is adopted to analyze the spatial autocorrelation of CODMn and NH3-N concentration. Then, a comparative analysis of the nonspatial panel model and spatial panel model is conducted. Finally, this research estimates the intermediate effect of the industrial structure of the Xiangjiang river basin in China. The results show that spatial autocorrelation exists in pollutant concentration and the relationship between economic growth and pollutant concentration shapes as an inverted-N trajectory. Moreover, the turn points of the environmental Kuznets curve for CODMn are RMB 83,001 and RMB 108,583 per capita GDP. In contrast, the turn points for NH3-N are RMB 50,980 and RMB 188,931 per capita GDP. Additionally, the environmental Kuznets curve for CODMn can be explained by industrial structure adjustment, while that for NH3-N cannot. As a consequence, the research suggests that the effect of various pollutants should be taken into account while making industrial policies. Introduction Water is an indispensable substance for human life, economic development, and social progress. With the rapid development of the economy and frequent human activities, the demand for water is increasing, which gives rise to pressure on ecosystems, and especially water ecosystems. At present, a water crisis that includes water shortages, pollution, and quality deterioration is increasingly prominent and has become the main bottleneck in restricting sustainable development of human economy and society [1]. Xiangjiang River is the largest river in the Hunan Province, China. With dense towns, a large population, and concentrated industry, the river basin hosts 60% of the population and 81% of the total industrial output value of Hunan Province, which makes it the main economic belt with the densest population and fastest urbanization development in Hunan [2]. However, the rapid development of the economy and constant discharge of industrial, domestic, and agricultural wastewater in recent years have resulted in the degeneration of the Xiangjiang River Basin's ecological function. Even worse, the discharge of pollutants in some river sections has exceeded the maximum capacity that a natural water environment can stand. The severe exceeding of ammonia nitrogen, total phosphorus, and chemical oxygen demand in Xiangjiang River Basin has led to horrible river pollution in the Hunan Province [3,4]. In view of this, pollution control in the Xiangjiang river basin is highly valued by the state and local governments of China. In July 2009, the Chinese government launched a special treatment project for heavy-metal pollution in the Xiangjiang basin, investing 595 billion yuan in total. In March 2011, the State Council of China approved the implementation plan for the treatment of heavy-metal pollution in the Xiangjiang basin, which has become the first pilot project of regional heavy-metal pollution control by the State Council of China. The main flow area in the Xiangjiang basin, Chang-Zhu-Tan city group, became the pilot area constructing a two-oriented society in 2007, which is a major policy measure to explore ways for conserving resources and protecting the environment without damaging economic growth in China. A series of policies to protect the Xiangjiang river were also implemented here, such as emission trading and ecological compensation institutions. Economic growth has had significant impact on changes of environmental quality. Consumption of resources and pollutant discharge caused by economic growth inevitably bring about environmental degeneration. Meanwhile, economic growth could reduce pollutant discharge and improve the environment by advancing technology, improving the economic scale, as well as increase revenue. The debate over the role that economic growth plays in determining environmental quality has been rapidly gaining importance [5,6]. Starting with the influential studies of Grossman and Krueger [7], and Selden and Song [8], a number of studies on the hypothesis of an Environmental Kuznets Curve (EKC) have investigated this relationship for various pollutants, regions, and time periods [2,9]. The EKC hypothesis depicts a long-term relationship between economic growth and environmental quality [10]. According to this view, the link between environmental quality and economic levels varies with the economic development stage. As a consequence, in the early stages of the development process, economic growth could lead to environmental degradation in the early stages of the development process, but once economic development exceeds a certain threshold, further increase in per capita income would help reduce pollution emissions and improve environmental quality. In addition, according to Panayotou [11], a high development level would lead to better regulations, greener technologies, and higher expenditures to protect the environment. At present, a large number of studies on EKC at home and abroad have selected many specific pollutant indicators such as atmosphere, water bodies, and solid waste in different regions for analysis and verification. In a meta-analysis of 121 usable observations gathered from a set of 25 studies, Cavlovic et al. [12] found that environmental indicators are divided into 11 categories, namely, toxic emissions, urban air quality, deforestation, heavy particulate, urban quality, water quality/pollution, heavy metals, SO 2 , combustion byproducts, hazardous waste, and CO 2 . It is possible that, in analyzing situations of different countries or different periods of a single country, researchers have drawn different conclusions because of the use of different regression equations, pollution indicators, or control variables. In other words, the relationship presented multiple and mixed types [13,14]. Some scholars found a traditional inverted U-shaped trend [15][16][17], yet others found that the turning point does not exist, but shows a linear relationship [18]. Moreover, some scholars found an N-shaped or inverted U-shaped relationship [19,20], while others, like Moomaw and Unruh (1997) [21], found that the relationship varied greatly in different countries [22][23][24] or even that there was no significant relationship between economic growth and environmental quality [25]. In addition, there was also evidence that testing results depended on specific econometric models [26,27]. In summary, the EKC has shown various forms, such as U-shaped, N-shaped, and linear. In other words, various environmental indicators of different regions do not follow a specific evolutionary trajectory. In existing empirical studies on EKC, model analysis is usually performed based on cross-sectional data from different geographic regions or administrative divisions. A general assumption is that pollution emissions between regions are uncorrelated. That is to say, the economic growth of a region only affects the environmental quality of itself, rather than the surrounding environment, which is obviously inconsistent with reality. It should be taken into consideration that environmental pollution may exist in spatial autocorrelation or spatial dependence [28]. On the one hand, almost all spatial data might have spatial or spatial autocorrelation characteristics [29], and pollution-emission data may be no exception. On the other hand, there are strong regional clusters of pollution-source distribution, energy-consumption structure, and pollution-control investment capacity in different regions, which also makes pollution discharge have a certain spatial correlation [30]. If the effects of this spatial correlation are ignored, the estimation of the EKC model might have a bias or a parametric test that produces errors. Therefore, spatial metric analysis methods should be used when dealing with EKC-related regional data [31][32][33]. However, there are not many literatures using spatial econometric models to study EKC, or especially dealing with the watershed environmental problem. Su et al. [34] employed spatial regression to analyze the determinants of dissolved oxygen (DO) and nutrients in the Qiantang River, China. Chang [35] examined spatial patterns of water-quality trends for 118 sites in the Han River basin of South Korea. Wang et al. [36] employed statistical tools, the GIS technique, and the EKC method to analyze the spatiotemporal relationship of riverwater quality with the economic growth in the Jiulong River Basin of the Western Taiwan Strait Economic Zone. Yang et al. [37] utilized spatial regression to evaluate the impact of watershed characteristics on stream NO 3 NO 2 -N concentration in the Cedar River Watershed, Iowa. In a meta-analysis of 255 EKC studies published between 1995 and 2010 [38], only 35 studies used water-quality indicators as the dependent variable in comparison to 214 studies that used air, climate, and energy indicators. Of the few studies that focused on water pollution, only 10 have performed at a lake and watershed level. Paolo et al. [39] examined the nexus between economic growth and water usage using a country's water footprint as an indicator of water impact and found an N-shaped curve for the relationship between water footprint per capita and GNI per capita, and grey water footprint per capita and GNI per capita. In view of this, this research utilizes the environmental Kuznets curve to demonstrate the interrelationship between economic growth, industrial structure, and water quality of the Xiangjiang river basin in China by establishing spatial panel data models. First, it obtains two variables (COD Mn and NH 3 -N) by pretreating the data of 42 environmental monitoring stations in the Xiangjiang river basin from 2005 to 2015. Afterward, Moran's I index is adopted to analyze the spatial autocorrelation of COD Mn and NH 3 -N concentration. Then, a comparison between the nonspatial panel model and spatial panel model is conducted and the mediating effect of the industrial structure is evaluated. The remainder of this paper is organized as follows: the second part introduces the EKC model and the spatial econometric model with an EKC hypothesis; the third part describes the research site, data processing, and the correlation and multicollinearity of the variables; the fourth part is the empirical results and discussion; the fifth part draws conclusions and policy recommendations. We contribute to the emerging literature in several ways: first, for the research method, the inverted N-shaped space panel EKC model is adopted; second, regarding the research content, the EKC formation mechanism of a watershed is analyzed from the perspective of industrial-structure adjustment; and third, for the research data, nonpublic pollutant-concentration data collected by the government, rather than commonly used emission data, are used. Study Site The Xiangjiang River, which traverses from its source in Xin'an of the Guangxi Province to its confluence with the Yangtze River in Dongting Lake, is one of the major and important rivers in Hunan Province, southern China. The location of each environmental monitoring station in Xiangjiang River Basin is shown in Figure 1. The river has a length of approximately 856 km (with 660 km in the Hunan Province), a catchment area of approximately 94,660 km 2 [40], and it covers eight cities, namely, Yongzhou, Chenzhou, Hengyang, Loudi, Zhuzhou, Xiangtan, Changsha, and Yueyang. The water of the Xiangjiang River is used for irrigation, and domestic and industrial purposes. In the Hunan Province, there are abundant reserves of nonferrous metals. Most of the ores used for mining, mineral processing, and the smelting of nonferrous and rare metals are found in the middle and lower reaches of the Xiangjiang River, while effluents from these intensive mining and industrial activities are heavily loaded into the river [41]. Moreover, many large cities, such as Changsha, Xiangtan, and Zhuzhou, are located in the middle and lower reaches of the Xiangjiang River. With the development of industrial production and the enlargement of cities, the middle and lower reaches of the Xiangjiang River have been seriously polluted in recent years [42]. Various heavy industries operate in the Xiangjiang river basin, including chemical, dyeing, electroplating, and food plants. In the last eight years, this area has nearly doubled its GDP, increasing from about 190 billion dollars in 1996 to 360 billion dollars in 2003. This basin exemplifies some direct water-pollution problems, and many typical water-pollution problems that occur during the process of urbanization in developing countries. The case study of the Xiangjiang River, therefore, provides a good opportunity to examine the spatial determinants of water pollution. Study Site The Xiangjiang River, which traverses from its source in Xin'an of the Guangxi Province to its confluence with the Yangtze River in Dongting Lake, is one of the major and important rivers in Hunan Province, southern China. The location of each environmental monitoring station in Xiangjiang River Basin is shown in Figure 1. The river has a length of approximately 856 km (with 660 km in the Hunan Province), a catchment area of approximately 94,660 km 2 [40], and it covers eight cities, namely, Yongzhou, Chenzhou, Hengyang, Loudi, Zhuzhou, Xiangtan, Changsha, and Yueyang. The water of the Xiangjiang River is used for irrigation, and domestic and industrial purposes. In the Hunan Province, there are abundant reserves of nonferrous metals. Most of the ores used for mining, mineral processing, and the smelting of nonferrous and rare metals are found in the middle and lower reaches of the Xiangjiang River, while effluents from these intensive mining and industrial activities are heavily loaded into the river [41]. Moreover, many large cities, such as Changsha, Xiangtan, and Zhuzhou, are located in the middle and lower reaches of the Xiangjiang River. With the development of industrial production and the enlargement of cities, the middle and lower reaches of the Xiangjiang River have been seriously polluted in recent years [42]. Various heavy industries operate in the Xiangjiang river basin, including chemical, dyeing, electroplating, and food plants. In the last eight years, this area has nearly doubled its GDP, increasing from about 190 billion dollars in 1996 to 360 billion dollars in 2003. This basin exemplifies some direct water-pollution problems, and many typical water-pollution problems that occur during the process of urbanization in developing countries. The case study of the Xiangjiang River, therefore, provides a good opportunity to examine the spatial determinants of water pollution. Data and Processing Existing research indicates that water quality is mainly affected by point-source pollution that is caused by industrial wastewater and urban/domestic sewage and nonpoint-source pollution that is caused by agricultural activities, soil erosion, and solid waste [43][44][45]. As GDP represents the level of social and economic development of a place, in general, the higher the GDP is, the more frequent corresponding economic activities are, and the more obvious the environmental impact is. With the development of economy and society and the constant improvement of urbanization, the contradiction between population increase and a serious shortage of water resources is becoming Data and Processing Existing research indicates that water quality is mainly affected by point-source pollution that is caused by industrial wastewater and urban/domestic sewage and nonpoint-source pollution that is caused by agricultural activities, soil erosion, and solid waste [43][44][45]. As GDP represents the level of social and economic development of a place, in general, the higher the GDP is, the more frequent corresponding economic activities are, and the more obvious the environmental impact is. With the development of economy and society and the constant improvement of urbanization, the contradiction between population increase and a serious shortage of water resources is becoming increasingly conspicuous. Population density can directly reflect the social production activities of a region. Higher population density leads to a higher demand for water and greater pollutant emissions. Natural precipitation also brings the pollutants of the surrounding areas into the river and further pollutes the water. With a dense population, developed economy, and high-level urbanization, the Xiangjiang river basin area enjoys the rapid development of agriculture and forestry, a large number of enterprises, and abundant rainfall. The main pollution of the area is derived from industrial pollution, domestic wastewater pollution, and agricultural pollution, among which heavy-metal pollution is particularly serious [46,47]. Pollutants mainly include COD Mn , NH 3 -N, mercury, arsenic, and hexavalent chromium. In this paper, we focus on the eutrophication of the Xiangjiang river basin that is caused by organic pollution and nutrients. The quality of the measured data of total phosphorus is extremely poor, many of which are lower than the minimum detection index of 0.01. Consequently, we selected COD Mn and NH 3 -N as the water-quality indicators of Xiangjiang river basin; and took per capita GDP (pergdp), primary industry (primary), industry (indus), population (pop), urbanization level (ur), and precipitation as control variables. In this paper, we extracted water-quality data from the 42 environmental monitoring stations in the Xiangjiang river basin. Then, we matched the 42 monitoring stations with corresponding counties according to map coordinates. It should be noted that (1) there are more than one monitoring sites in some areas, for example, Bailang, Toushan, and Xiao Dongjiang are nestled in Zixing City, so we regarded the average data in the above three places as the environmental quality data in Zixing City; (2) several monitoring stations measure more than one district. For instance, the Lilang monitoring station supervises the Yuhua District and Changsha County. Thus, we merged the above two places into one area in our article and took the sum data as the research object. Based on this, 26 observations were selected. Additionally, the data related to the economic income and social development of the Xiangjiang river basin were obtained from the "2005-2015 Statistical Yearbook of Hunan Province", among which GDP (pergdp) was treated at a constant price based on the 2005 price index. The unit of population (pop) density was "Ten thousand people per square kilometer", primary industry (primary) refers to the proportion of the added value of the primary industry to the GDP, and industry (indus) refers to the proportion of industrial added value to the GDP. Correlations and Multicollinearity of Variables The monitoring results revealed that the average concentration of COD Mn ranks the highest from 2005 to 2015 of all the pollutants in the Xiangjiang River Basin. Table 1 shows that the maximum value was 8.27 mg/L, while the minimum was 0.25 mg/L. In addition, average concentration of NH 3 -N was nearly 0.64 mg/L, which is also at a relatively high level. Specifically, the concentration of COD Mn and NH 3 -N in the Liuyang and Zhenshui rivers (the tributaries of Xiangjiang and the corresponding monitoring stations are SanJiaoZhou, HeShiDu, and ZhenShuiRuXiangJiangKou) were higher on the whole, while reaching an average of 4.65 mg/L and 1.89 mg/L, respectively. The main reason is that a large number of industrial and papermaking enterprises are distributed on both banks of the Liuyang and Zhenshui rivers, but sewage discharge has not been effectively controlled. However, the concentrations of the two pollutants showed a trend of rising first and then decreasing because of the comprehensive remediation of environmental pollution in the basin. The main measures include the regulation of livestock and poultry-farming pollution, elimination and renovation of industrial pollution sources, and strengthening centralized and harmless disposal of domestic sewage and domestic waste in towns and townships. Data-analysis results demonstrate that the concentration of major pollutants in each section of the Xiangjiang river basin is gradually increasing from upstream to downstream. Figure 2 reveals the pollution of major cities along the Xiangjiang river basin. The darker color indicates the higher concentration of contaminants. It can be seen from the figure that the concentrations of COD Mn and NH 3 -N of Changsha, Loudi, and Xiangtan in the lower reaches of the basin are significantly higher than those of Chenzhou and Yongzhou in the upstream part. Table 2 shows the variable correlation coefficient matrix. It can be seen that the correlation coefficients of most variables are low or moderate. Most variables have strong and significant correlation, while the correlation between COD Mn (NH 3 -N) concentration and primary (precipitation) is rather weak and insignificant. In addition, there is a certain correlation between every two control variables. To further test multicollinearity, a Variance Inflation Factor (VIF) test was used. The VIF test showed that the biggest value is 9.32, which indicates that multicollinearity might exist. We also found that the variable (ur) had no significant effect on the COD Mn (NH 3 -N) coefficient. When the (ur) was deleted, the biggest value of VIF became 4.93, indicating that there was no multicollinearity between the variables [2,48]. Table 2 shows the variable correlation coefficient matrix. It can be seen that the correlation coefficients of most variables are low or moderate. Most variables have strong and significant correlation, while the correlation between CODMn (NH3-N) concentration and primary (precipitation) is rather weak and insignificant. In addition, there is a certain correlation between every two control variables. To further test multicollinearity, a Variance Inflation Factor (VIF) test was used. The VIF test showed that the biggest value is 9.32, which indicates that multicollinearity might exist. We also found that the variable (ur) had no significant effect on the CODMn (NH3-N) coefficient. When the (ur) was deleted, the biggest value of VIF became 4.93, indicating that there was no multicollinearity between the variables [2,48]. Methodology Spatial regression was employed to analyze the relationship between water-pollution indicators and economic growth considering the spatial correlation of water pollution. Spatial regression models typically include three categories, which are spatial lag (SAR, Equation (1)), spatial error (SEM, Equation (2)) and spatial Durbin mode (SDM, Equation (3)). Spatial lag regression is expressed as follows: where y it is the water-pollution indicator of monitoring station i in year t. x it is the GDP of area i in year t indicating the level of economic growth. Z it is the set of other explanatory variables, including population density, rate of urbanization, and industrial structure. α i are unobserved country-specific effects; γ t is the time-specific effect; ε it is the normally distributed error term; λ is the spatial autoregressive coefficient; and (w ij ) is the spatial weight matrix that describes the relationship between every two sites. Spatial error regression is given as: where σ it denotes the spatially autocorrelated error term. Term λ reflects the spatial autocorrelation coefficient of the error term. The other parameters are the same as the above mentioned. Some spatially autocorrelated variables in the spatial-error model were omitted in the regression, while the model consequently generated a spatially autocorrelated error. The spatial lag model assumes that, besides explanatory variables, the water-quality variable in one site is affected by the spatially weighted concentrations in its neighborhood. The SAR and SEM models both consider county interactions. However, when studying the spatial difference of river pollutants, water flow direction from upstream to downstream directly leads to unidirectional (from upstream to downstream) spatial influence. At the same time, a spatial relationship occurs when the dependent variable can be predicted as a function of spatially lagged values of the independent variables. Therefore, the two spatial-interaction situations need to be considered simultaneously. The spatial Durbin model specification provides a function containing spatially lagged values, both of the dependent variable and the independent variables, which is specified as: w 1ij is a unidirectional contiguity matrix, namely w 1ij = 1 if j is the nearest upstream region of i, otherwise w 1ij = 0. w 2ij is a threshold distance and the spatial matrix in which the element w 2ij is calculated using the square of the reciprocal of the geographic distance between counties i and j. Distance-based weights are defined as follows: where d ij is the distance in kilometers between counties i and j. The 77 km distance was the cut-off parameter above which interactions were assumed to be negligible. This distance was chosen so that each county interacted with at least one other county. This cut-off parameter is important since there must be a limit to the range of spatial dependence allowed by the spatial-weight matrix [49][50][51]. This is due to the asymptotical features required to obtain consistent estimates for the model parameters. We use the inverse-squared distance in order to reflect gravity relation. The two matrices were both row-standardized so that each row summed to one and the coefficients that emerged from the subsequent regression could be readily interpreted by virtue. Under the definitions of w 1ij and w 2ij , the spatial Durbin model took account of the two interaction cases simultaneously. Due to the introduction of a spatial-weight matrix, spatial econometric models arouse an endogenous variable problem. If spatial econometric models were still estimated by ordinary least squares (OLS), estimated coefficients would be biased. Therefore, spatial panel models are generally estimated with the maximum likelihood method [52]. As each county has characteristics that do not change or change very little over time, such as unobservable geographic characteristics and resources endowment, this can lead to parameter homogeneity in each spatial unit. In this paper, we primarily focused on the fixed effects of the estimation procedure. The time period affected control for time-specific shocks that affect economic factors such as economic crises, oil shocks, and national environmental policy, which leads the coefficients to vary in each time unit. For completeness, we also estimated the random-effect model and used the Hausman diagnostic test to determine which model provided a better fit for the data. Two models were analyzed, while the Robust Lagrange Multiplier (LM) tests and their robustness (Robust-LM) were used to determine the form of spatial relationships. To verify if the spatial panel data models offered a more appropriate specification, we tested which spatial panel data model was the best-fitting for the data with the Wald and LR tests. We used the null hypotheses (H0: γ = 0) of the Wald test to examine whether the SDM model could be simplified to the SAR model. We also explored the null hypothesis (H0: γ + βλ = 0) of the LR test to determine whether the SDM model could be simplified to the SAR model or SEM model. If both of the null hypotheses were rejected, the spatial Durbin model provided the best fitting. Spatial Autocorrelation for COD Mn and NH 3 -N This part uses Moran index analysis to comprehensively evaluate the spatial autocorrelation of COD Mn and NH 3 -N. The Moran Index was introduced by Australian statistician Patrick Alfred Pierce Moran in 1950. The range of Moran's I values concentrates in the interval (−1, 1), where 1 and −1, respectively, denote the strongest positive spatial autocorrelation and strongest negative spatial autocorrelation [53]. Table 3 suggests that Moran's I index values of the COD Mn and NH 3 -N in the Xiangjiang River from 2005 to 2015 were all positive and in the range of (0, 1), which indicates that there exists spatial autocorrelation between the two types of pollutants. Especially for NH 3 -N, its Moran index reached the highest-0.871 in 2015. Therefore, the river COD Mn and NH 3 -N patterns depended both on neighboring patterns and on a set of local independent parameters. All these results demonstrate the need to incorporate spatial analysis when interpreting the spatial determinants of COD Mn and NH 3 -N. Empirical Estimation and Results A suitable model is a prerequisite for reliable empirical results. In order to detect which model best fits the data, this paper first conducts a nonspatial panel model analysis, then examines whether there exists spatial correlation among spatial units by the classic LM tests and the Robust LM tests. As shown in Tables 4 and 5 referring to the results of classical LM tests on COD Mn , the null hypothesis of no spatially lagged dependent variable and the null hypothesis of no spatially autocorrelated error term were respectively rejected at 10% and 5% significance level. In the robust LM tests, both of the hypotheses were not rejected when the OLS is included. While referring to the results of classical LM tests on the NH 3 -N, both of the hypotheses were strongly rejected at a 1% significance level, while both of the hypotheses were rejected for the OLS in the robust LM tests. These results show that spatial panel models are better than nonspatial interaction effects of traditional mixed panel data models. In terms of a model-fitting effect, the SDM model had the most significant regression coefficient compared with the SAR or SEM model. To further judge the fitting effect of the SDM model, we estimated the spatial Durbin model and then performed the Wald and LR tests. According to the results of the Wald and LR tests on COD Mn and NH 3 -N, both of the null hypotheses were rejected at the 1% significance level. Furthermore, we conducted the Hausman test, and the result of COD Mn and NH 3 -N were respectively rejected at 5% and 1% significance level. These results imply that the SDM model is more appropriate than the SAR or SEM model in reflecting the impact of spatial autocorrelation on regression results. Therefore, we adopt the SDM model with fixed effects to study the spatial effect of economic development on water quality in the river basin. This shows that, for COD Mn and NH 3 -N, estimated coefficients of the cubic polynomial of per capita GDP (Pergdp3) are highly significant, which presents that the relationship between economic growth and the concentration of these two pollutants cannot validate the traditional environmental Kuznets curve hypothesis. However, it shows a back-N style, as displayed in Figure 3. The first turning point of the environmental Kuznets curve for COD Mn is at approximately RMB 83,001; the second turning point is at approximately RMB 108,583. By contrast, for NH 3 -N, the first turning point is at approximately RMB 50,980; the second turning point is at approximately RMB 188,931. The EKC of other watersheds are different, for example, Jiang (2014) [46] studied the EKC of the Qiantang river basin districts in Hangzhou, Jinhua, and Quzhou from 2005 to 2012; the results showed that the EKC was U-shaped, inverted U-shaped, and N-shaped. Moreover, the EKC of the Changjiang river basin and Taihu river basin was U-shaped; of the Haihe river basin, and Huanghe river basin was S-shaped; and of the Huaihe river basin and Liaohe river basin was tilted-S-shaped. Compared with these basins, the EKC of the Xiangjiang river basin was inverted N-shaped. The reason may be due to a series of targeted environmental protection measures being implemented in the Xiangjiang River Taking the 2016 data as an example, for the environmental Kuznets curve of the pollutant NH 3 -N, the per capita real GDP of 11 districts or counties of the Xiangjiang river basin are in the upward phase of the inverted "N" curve, namely, Lusong District, Shifeng District, Yuelu District, Kaifu District, Yuhua District/Changsha County, Furong District, Tianxin District, Wangcheng District, Xiangtan City, Louxing District, and Zixing City. As for the environmental Kuznets curve of the pollutant COD Mn , 10 districts or counties are in the upward phase, namely, Lusong District, Shifeng District, Yuelu District, Kaifu District, Yuhua District, Changsha County, Tianxin District, Wangcheng District, Xiangtan City, and Zixing City. Since the diagnostic results above have suggested that the SDM with fixed effects is the best fit, we limited the interpretation of coefficient estimates to it. As shown in Table 4, focusing on the estimated coefficient of primary industry (primary), industry (indus), and population (pop), the elastic coefficients were −6.726415, −2.326853 and −2.405921, respectively. By contrast, the results described in Table 5 are 1.028606, 0.6328895, and −4.974618, which indicates that primary industry (primary) and industry (indus) have a negative effect on increasing COD Mn concentration and have no obvious effect on increasing NH 3 -N concentration. Similarly, results also show that population (pop) has a negative effect both on COD Mn and NH 3 -N, while precipitation (pre) has a negative effect on COD Mn while having no obvious effect on NH 3 -N. Estimated coefficient λ is significantly positive in both Tables 4 and 5 indicating that the spatial factor has a significant effect on the concentration of these two pollutants. In summary, the per capita GDP of the Xiangjiang river basin, especially the primary industry output value and industrial output value, population density, and precipitation have different effects on the concentration of pollutants in the basin, especially the impact on COD Mn . The reason might be that the Xiangjiang river basin is an economic region in which the first and second industries are the mainstay and advantageous. It is known as the "hometown of nonferrous metals" in which the heavy chemical industry, steel, nonferrous metals, chemicals, and building materials are at the top of the industrial structure. Economic and industrial development have caused serious heavy-metal pollution in Xiangjiang, especially COD Mn . Ammonia-nitrogen pollution is mainly caused by domestic wastewater discharged from the cross-strait population and agricultural nonpoint source pollution. With the development of the first industry on the coast, the use of chemical fertilizers and pesticides has been increasing year by year, while the discharge of domestic sewage has also increased. Research results of Lesage and Pace [54], and Elhorst [55] show that the estimation coefficient of models cannot directly reflect the marginal effect of the independent variable on the dependent variables. The direct and indirect effects of each explanatory variable are listed in Table 6. As is described in the table, the direct effect on COD Mn , the per capita GDP (pergdp, pergdp2, pergdp3), primary industry (primary), industry (indus), and population (pop) are significant. The primary industry (primary) in particular has the maximum effect (−6.7732). Additionally, the population (pop) of NH 3 -N has the maximum effect (−4.841386), while precipitation (pre) has the minimum effect (−0.000111). For the direct effect on COD Mn and NH 3 -N, the per capita GDP was rejected at a 5% significance level and 10% significance level, respectively, which indicates that the per capita GDP has a large spillover effect on the water quality of adjacent areas. The main reason is that water pollution in the Xiangjiang river basin is serious, and water pollution presents a cross-regional feature. Water pollution in different regions of the basin has spatial correlation. Specifically, when per capita GDP increases in a certain region, water pollution in the adjacent areas is aggravated. The impact of the primary industry on COD Mn and NH 3 -N is not significant, which indicates that the increase in the output value of the primary industry in one region has no effect on the water quality of the adjacent regions. In addition, the impact of industrial and population density on COD Mn is not significant, but that on NH 3 -N is significant, which implies that industrial output value and population density has a positive impact on NH 3 -N pollution in the adjacent areas but has no effect on COD Mn . In summary, in the process of spatial interaction, the space effect is basically reflected in the form of direct effect, while the spatial feedback effect is relatively weak. However, we cannot ignore the indirect influence of the spatial spillover effect of economic development on water pollution. www.mdpi.com/journal/ijerph region has no effect on the water quality of the adjacent regions. In addition, the impact of industrial and population density on CODMn is not significant, but that on NH3-N is significant, which implies that industrial output value and population density has a positive impact on NH3-N pollution in the adjacent areas but has no effect on CODMn. In summary, in the process of spatial interaction, the space effect is basically reflected in the form of direct effect, while the spatial feedback effect is relatively weak. However, we cannot ignore the indirect influence of the spatial spillover effect of economic development on water pollution. Intermediate Effect of Industrial Structure The existing literature shows that there are three factors that determine the shape of the environmental Kuznets curve: scale efficiency, structural effect, and technical effect. In the case where other effects are fixed, the scale effect refers to the concentration of pollutants increasing according to the scale of economic activities; the structural effect refers to the decrease of pollutant concentration when economic activities become clean and green; and the technical effect is the reduced concentration of pollutants when clean technology is used in economic activities. The key points in this section is the structural effect, that is, whether the industrial structure adjustment can change the shape of the EKC curve. According to Judd and Kenny [56], Baron and Kenny [57], MacKinnon et al. [58], and James Kroes et al. [59], this part carries out a series of tests to verify whether industrial structure has a mediating effect in adjusting the relationship between economic growth and environmental changes. The results show that: (1) when the impact of the economy alone on the environment was considered, the per capita GDP was significantly correlated with COD Mn and NH 3 -N, which indicates that economic growth had a certain influence on the environment (as shown in Table 7); (2) per capita GDP is significantly correlated with the primary industry (Primary) and industry (Indus), implying that economic growth had a certain influence on the development of primary and industries (see Table 8); (3) when considering economic development and the impact of an industrial structure on the environment, the industrial structure has significant impact on COD Mn (see Table 4). Therefore, it can be concluded that the industrial structure has a mediating effect on adjusting the relationship between economic growth and COD Mn concentration change. The impact of an industrial structure on NH 3 -N concentration, however, is not significant (see Table 5), which shows that there is at least one nonsignificant situation. Therefore, the Sobel test was continued, in which test statistic z was −0.4228 and 0.3312, respectively, which meant it failed to pass the tests. We can then conclude that the industrial structure is not significant to adjusting the relationship between economic growth and the NH 3 -N concentration change. In our models, per capita GDP was nonlinearly related to industrial structure and environmental quality; so it was no longer possible to talk about the indirect effect of per capita GDP on environmental quality through the industrial structure as a single quantity. As Hayes et al. [60] describe, we used the rate at which a change in pergdp changes COD Mn concentration indirectly through changes in the industrial structure, denoted as θ, to measure this instantaneous indirect effect. Based on previous estimates, the instantaneous indirect effect of pergdp, corresponding to primary and indus, respectively, is as follows: Conclusions and Policy Implications This paper examined the CODMn and NH3-N EKC hypothesis using a spatial Durbin model to avoid coefficient-estimation deviation covering the period of 2005-2015 of the Xiangjiang river basin. The results showed that: (1) there exists spatial correlation between CODMn/NH3-N and the per capita GDP has a significant influence on CODMn / NH3-N; (2) the turning points of the CODMn and NH3-N environmental Kuznets curves were (83,001, 108,583) and (50,980, 188,931), respectively; (3) the industrial structure can explain the change of the EKC of CODMn, but cannot explain the EKC of NH3-N, which indicates that the industrial structure has a mediating effect on CODMn but not on Conclusions and Policy Implications This paper examined the CODMn and NH3-N EKC hypothesis using a spatial Durbin model to avoid coefficient-estimation deviation covering the period of 2005-2015 of the Xiangjiang river basin. The results showed that: (1) there exists spatial correlation between CODMn/NH3-N and the per capita GDP has a significant influence on CODMn / NH3-N; (2) the turning points of the CODMn and NH3-N environmental Kuznets curves were (83,001, 108,583) and (50,980, 188,931), respectively; (3) the industrial structure can explain the change of the EKC of CODMn, but cannot explain the EKC of NH3-N, which indicates that the industrial structure has a mediating effect on CODMn but not on NH3-N. Compared with conventional estimation techniques, the spatial econometric approach is Conclusions and Policy Implications This paper examined the COD Mn and NH 3 -N EKC hypothesis using a spatial Durbin model to avoid coefficient-estimation deviation covering the period of 2005-2015 of the Xiangjiang river basin. The results showed that: (1) there exists spatial correlation between COD Mn /NH 3 -N and the per capita GDP has a significant influence on COD Mn / NH 3 -N; (2) the turning points of the COD Mn and NH 3 -N environmental Kuznets curves were (83,001, 108,583) and (50,980,188,931), respectively; (3) the industrial structure can explain the change of the EKC of COD Mn , but cannot explain the EKC of NH 3 -N, which indicates that the industrial structure has a mediating effect on CODMn but not on NH 3 -N. Compared with conventional estimation techniques, the spatial econometric approach is seldom used in the validation of a COD Mn / NH 3 -N environmental Kuznets curve. This paper proves that taking spatial autocorrelation into consideration, the spatial panel model is a more proper method, which proposes a more reliable result. In view of the conclusions above, we make the following policy recommendations to solve environmental problems brought about by economic growth: (1) Spatial measurement results demonstrate that there is an inverted N-shaped relationship between environmental quality and economic development, which indicates a positive interaction between them. Therefore, local government should attach great importance to the inevitability that economic development brings pressure to the environment, and should not simply pursue GDP growth at the expense of environmental pollution. The relationship between economic development and environmental protection should be better dealt with. (2) The reality that upstream and downstream pollution emissions have a strong positive spatial correlation indicates that local government may choose a way to achieve economic development by sacrificing the environmental quality of the surrounding areas. Therefore, it is necessary to stop shortsighted behavior and promote overall environmental quality and consider spatial correlation in the environmental planning of each region. For local government, inter-regional environmental cooperation is of great necessity in dealing with the contradiction between economic development and environmental pollution. Additionally, the national government should pay more attention to the spatial correlation of environmental pollution in the basin so as to implement policies coordinating regional economic development and ecological protection. (3) As the impact of an industrial structure on different pollutants is different, the control of pollutant discharges should be strengthened in a targeted manner during the economic development. Moreover, the reduction effects of different pollutant emissions should be taken into consideration when formulating industrial policies.	2018-10-05T01:42:49.274Z	2018-09-25T00:00:00.000	{ "year": 2018, "sha1": "09839789e6bbda54dfb073f4c335347b571245d7", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1660-4601/15/10/2095/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "09839789e6bbda54dfb073f4c335347b571245d7", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [ "Environmental Science", "Medicine" ] }
14875598	pes2o/s2orc	v3-fos-license	The N17 domain mitigates nuclear toxicity in a novel zebrafish Huntington’s disease model Background Although the genetic cause for Huntington’s disease (HD) has been known for over 20 years, the mechanisms that cause the neurotoxicity and behavioral symptoms of this disease are not well understood. One hypothesis is that N-terminal fragments of the HTT protein are the causative agents in HD and that peptide sequences adjacent to the poly-glutamine (Q) repeats modify its toxicity. Here we test the function of the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT in a novel, inducible zebrafish model of HD. Results Deletion of N17 coupled with 97Q expansion (mHTT-ΔN17-exon1) resulted in a robust, rapidly progressing movement deficit, while fish with intact N17 and 97Q expansion (mHTT-exon1) have more delayed-onset movement deficits with slower progression. The level of mHTT-ΔN17-exon1 protein was significantly higher than mHTT-exon1, although the mRNA level of each transgene was marginally different, suggesting that N17 may regulate HTT protein stability in vivo. In addition, cell lineage specific induction of the mHTT-ΔN17-exon1 transgene in neurons was sufficient to recapitulate the consequences of ubiquitous transgene expression. Within neurons, accelerated nuclear accumulation of the toxic HTT fragment was observed in mHTT-ΔN17-exon1 fish, demonstrating that N17 also plays an important role in sub-cellular localization in vivo. Conclusions We have developed a novel, inducible zebrafish model of HD. These animals exhibit a progressive movement deficit reminiscent of that seen in other animal models and human patients. Deletion of the N17 terminal amino acids of the huntingtin fragment results in an accelerated HD-like phenotype that may be due to enhanced protein stability and nuclear accumulation of HTT. These transgenic lines will provide a valuable new tool to study mechanisms of HD at the behavioral, cellular, and molecular levels. Future experiments will be focused on identifying genetic modifiers, mechanisms and therapeutics that alleviate polyQ aggregation in the nucleus of neurons. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0063-2) contains supplementary material, which is available to authorized users. Background Huntington's disease (HD) is an incurable, autosomal dominant neurodegenerative disease. Patients with HD display progressive symptoms including psychiatric, cognitive, and motor dysfunction, and the disease onset is often defined by the onset of motor symptoms [1][2][3]. Initially, patients present with excessive movements of the extremities and face, progressing to larger movements described as chorea. Further disease progression results in the opposite effect with patients suffering from rigidity, akinesia, bradykinesia, and gait abnormalities. The disease is invariably lethal 10-20 years after onset. There is no cure for HD and treatments for the symptoms are of limited effectiveness. HD is part of a broader class of neurological disorders linked to polyglutamine (polyQ) repeat expansions within several disease proteins [4]. All of these disorders are progressive, degenerative diseases with age of onset inversely correlated with the number of polyQ repeats within the protein. Although the genes involved in polyQ diseases are, for the most part, broadly expressed, each disease has a well-defined pattern of neurodegeneration affecting selective populations of neurons. In HD, the medium spiny neurons (MSNs) of the striatum are thought to be most susceptible to degeneration and cortical pyramidal neurons are also affected [5]. The precise mechanisms underlying such selective neuronal vulnerability in HD remain incompletely understood. One hypothesis is that amino acid sequences flanking the polyQ repeat dictate the cell-type specific toxicity. HD is caused by an expanded polyQ within the Nterminus of the huntingtin protein (HTT) [6]. HTT is a large, 3144 amino acids, protein that is ubiquitously expressed, evolutionarily conserved, and has been suggested to function as a scaffolding protein for many cellular processes. Knockout of this gene is early embryonic lethal in mice suggesting an essential developmental function [7][8][9], however the specific mechanisms involved are not well understood. Histologically, a hallmark of HD is large intracellular polyQ protein aggregates [10]. These aggregates can be localized to several sub-cellular regions including cytoplasmic, perinuclear, and intra-nuclear [11,12]. Although originally thought to be disease causative, polyQ aggregates have been suggested to be neuroprotective [13] and their role in the disease is still unresolved. One hypothesis is that localization of the aggregate defines its toxicity. Experimental targeting of HTT-polyQ to different subcellular compartments suggests that the nucleus is especially sensitive to HTT-polyQ expression [14,15]. The normal subcellular distribution of HTT is mediated by proteinprotein interactions to regions outside the polyQ [16]. Most notably, the N-terminal 17 amino acids of HTT (N17) have been suggested to regulate protein localization and stability [17]. This region of the protein is evolutionarily conserved [18]. It functions as both a nuclear export signal and cytoplasmic membrane association domain [19,20]. Additionally, its amphipathic alpha-helical structure [21,22] promotes oligomer formation and accelerates HTT-polyQ fragment aggregation in vitro [23,24]. Post-translational modifications of N17 are prevalent and diverse [25]. Phosphorylation of serines 13 and 16 has been demonstrated to reduce aggregation and toxicity in vitro [26] and in vivo [27]. In vivo, BAC transgenic mice expressing human mHTT-97Q lacking the N17 domain, a deletion of 2-16 amino acid residues in otherwise fulllength mHTT context (BACHD-ΔN17), results in greatly accelerated motor deficits including adult-onset movement disorder, striatal neurodegeneration, and exclusive nuclear mHTT aggregation formation [28]. The phenotypes of BACHD-ΔN17 mice is dramatically accelerated and more closely resemble clinically-manifest HD than the BACHD mouse model expressing intact full-length mHTT of the same polyQ length [28,29]. Therefore N17 acts as a modulator of mHTT aggregation, subcellular localization, and neurotoxicity. Since the treatments available for HD are quite limited, it is important to have preclinical models of the disease that can be used to study mechanism and test candidate therapeutics. Mouse models are the most popular and have been developed over the last 18 years using conventional transgenic, YAC and BAC transgenic, and gene knock-in technologies to introduce polyQ into the genome [30]. Some models express the entire protein while others only use exon 1 which encodes the pathological polyQ expansion. These mice develop behavioral abnormalities and brain pathologies that recapitulate HD to varying degrees. Recent data suggests that proteolytic fragments containing exon 1 [31] or aberrantly spliced transcripts of exon 1 [32] are toxic and exist in patients. mHTT exon 1 based transgenic mice [33] have already been demonstrated to exhibit behavioral and pathological similarities to human disease, mHTT exon 1 based models are likely to be relevant to aspects of the disease. However, mammalian models are expensive to maintain and have limited scalability. Several non-mammalian models of polyQ toxicity have been developed in organisms such as S.cerevisiae [34], c.elegans [35,36], and Drosophila [37]. These models are scalable for screening compounds and genetic interactions, but lack high genetic similarity to humans and have significantly different, or in the case of S. cerevisiae no, nervous system. Zebrafish are an advantageous vertebrate model organism that is genetically more closely related to humans than non-vertebrate models but is still scalable and reasonably affordable as compared to mammalian models [38]. HTT-polyQ toxicity has been reported in zebrafish by using mRNA or plasmid DNA injection to acutely over-express the protein [39,40]. However, this model might not recapitulate specific mechanisms of the disease due to its early developmental effects and the extreme levels of protein expression that are necessary to cause toxicity. A second zebrafish model of polyQ toxicity has been reported in which the rhodopsin promoter drives mHTT-exon1 fragment expression in photoreceptors of the retina [41]. These zebrafish exhibit specific cellular degeneration and protein aggregation in the rod photoreceptor layer of the retina. However, retinal degeneration is not a known pathology in HD. Therefore, a zebrafish model that more closely recapitulates aspects of the human disease would be a valuable new tool for the field. We have generated a series of conditional transgenic zebrafish models of HD. Using Cre-loxP technology, we have generated inducible transgenic fish that express HTT-exon1(25Q)-EGFP or mHTT-exon1(97Q)-EGFP upon Cre recombination. We have also generated complementary HTT-ΔN17-exon1(25Q)-EGFP and mHTT-ΔN17-exon1(97Q)-EGFP lines. These latter models were created to test if the accelerated nuclear pathogenesis and disease-like phenotypes observed originally in BACHD-ΔN17 mice [28] could also be seen in our zebrafish model, and to test if N17 plays a crucial role in modifying the toxicities of mHTT-exon1, a disease-relevant toxic fragment in HD [32]. Upon ubiquitous recombination, EGFP + protein aggregates are visible within both mHTT-exon1 and mHTT-ΔN17-exon1 lines. Surprisingly, these fish develop normally up to five weeks of age at which point mHTT-ΔN17-exon1 lines begin to exhibit abnormal movement and swimming behavior that progressively worsen until the fish are unable to swim by about 12 weeks of age. The mHTT-exon1 lines present much milder swimming impairment that does not appear until 4 months of age and progresses much more slowly. Additionally, we crossed the mHTT-ΔN17-exon1 line into transgenic Cre driver lines for neurons, glia, muscle, or vasculature. Only fish expressing mHTT-ΔN17-exon1 specifically in neurons developed a progressive movement disorder. Finally, we examined the subcellular localization of mHTT-exon1 fragments in the transgenic fish and found that mHTT-ΔN17-exon1 is enriched in the nucleus of neurons, providing direct evidence that N17 is crucial for cytoplasmic targeting/nuclear export of mHTT-exon1 in vivo and further suggests the nuclear toxicity is key to the manifestation of disease-like phenotypes in an HD vertebrate model. This new zebrafish model of HD will be an invaluable tool to further dissect the mechanisms of mutant polyQ toxicity and to screen for potential therapeutics or genetic modifiers to ameliorate disease-like phenotypes in vivo. Cre-loxP inducible expression of human HTT exon 1 in transgenic zebrafish Since our earlier experiments had demonstrated significant toxicity leading to premature death of transgenic zebrafish upon over-expression of polyQ expanded HTT, we decided to create Cre-loxP conditionally inducible transgenic fish. This system allows us to maintain the transgenic lines by bypassing any toxicity by keeping the transgene switched off until expression of Cre recombinase is introduced, which also allows us spatial and temporal control of expression. We chose to use the strong, ubiquitous bactin2 promoter provided with the tol2kit to drive transgene expression [42]. Each transgene was assembled with the bactin2 promoter, floxed mCherry, followed by one of four HTT-exon1 cassettes (25Q, 97Q(m), ΔN17-25Q, or ΔN17-97Q(m)) fused to GFP flanked by tol2 transposon sites (Fig. 1a). The polyQ coding DNA sequences were mutated at wobble sites as reported previously [29] to stabilize the transgene and prevent the genomic instability associated with CAG repeats. Transgenic germlines were established for each transgene, one line was maintained for HTT-exon1 and HTT-ΔN17-exon1 control fish while two separate lines were maintained for mHTT-exon1 and mHTT-ΔN17-exon1. Transgenic lines have been maintained to at least F 6 generation with Mendelian inheritance patterns suggesting a single insertion site is present in each line. Additionally, the use of the Tol2 system should result in single copy transgene insertions. To confirm that these transgenes were inducible by Cre, we injected embryos with Cre mRNA and observed loss of mCherry expression and induction of GFP expression in all lines ( Fig. 1b and data not shown). HTT-exon1 L1 and HTT-ΔN17-exon1 L1 lines exhibited ubiquitous, diffuse GFP expression at all time points examined while mHTT-exon1 and mHTT-ΔN17-exon1 lines initially had ubiquitous expression but exhibited GFP + protein aggregates by 5 days post fertilization (dpf), most prominently in trunk muscles (Fig. 1c). To measure the expression level of each transgene in the different lines, western blots and qRT-PCR were performed. Each line was crossed to heat shock inducible Cre (HS:cre) transgenic fish and then heat shocked at shield stage resulting in robust, ubiquitous recombination. To evaluate protein levels, Western blots were performed on pools of embryos from three separate clutches harvested at 5 dpf for the HTT-GFP fusion protein and α-tubulin as a loading control (Fig. 1d, upper panel). Densitometry was used to measure relative protein abundance (Fig. 1d, lower panel). Embryos lacking HS:cre never show GFP expression (data not shown). HTT-exon1 L1 and HTT-ΔN17-exon1 L1 both exhibit strong GFP expression upon recombination at a size slightly larger than the predicted~37 kDa and~35 kDa, suggesting post-translational modification was present. Similarly, both mHTT-exon1 and mHTT-ΔN17-exon1 appear at sizes larger than their predicted~46 kDa and~44 kDa molecular masses, which may result from aberrant conformation due to the expanded polyQ and/or post-translational modification (Fig. 1d, upper panel). mHTT-exon1 L1 and L2 exhibit similar levels of expression that are significantly lower than line HTT-exon1 L1, HTT-ΔN17-exon1 L1, and both mHTT-ΔN17-exon1 lines (p < 0.01, ANOVA, Bonferroni posthoc test) (Fig. 1d). mHTT-ΔN17-exon1 L1 and L2 also exhibit similar levels of expression that are lower than HTT-ΔN17-exon1 L1 but with only L2 reaching statistical significance (p < 0.01, ANOVA, Bonferroni posthoc test) (Fig. 1d). We were surprised to observe the lower protein levels for the mHTT-exon1 lines as compared to the mHTT-ΔN17-exon1 lines since the transgene design and promoter were identical. To test whether transgene mRNA expression was responsible for the difference, we performed qRT-PCR analysis on mRNA extracted from each line. For this analysis, pools of ten GFP + embryos from six independent clutches were assayed for each line. mRNA levels were less varied between lines than protein, with mHTT-exon1 L1 the only one to reach statistical significance in comparison to HTT-exon1 L1 (p < 0.05, ANOVA, Bonferroni posthoc test) (Fig. 1e). It is important to note that mRNA levels between mHTT-exon1 lines and mHTT-ΔN17-exon1 lines are similar. This is in contrast to the protein levels, suggesting that post-translational mechanisms may be affecting protein abundance. In fact, the N17 region of HTT has been described as a modifier of protein stability, harboring both phosphorylation [26,27] and ubiquitination [17] sites that regulate protein degradation. Therefore, it appears that the intact N17 domain leads to greater protein instability in this zebrafish model and its deletion accelerates protein accumulation. The transgenic zebrafish expressing mHTT-exon1 lacking the N17 domain exhibits accelerated, progressive movement disorder Each HTT transgenic line was crossed with HS:cre and the embryos heat shocked at shield stage to induce recombination or left as non-heat shocked controls. HTT-exon1 L1 (n = 21) and HTT-ΔN17-exon1 L1 (n = 26) both exhibited robust GFP expression and developed normally with no behavioral changes up to 26 weeks of age ( Fig. 2a and c and Additional file 1: Movie S1 and Additional file 2: Movie S2). mHTT-exon1 L1 (n = 16) and L2 (n = 16) both survived early development even though mHTT-exon1 protein aggregates were present by 5 dpf. These fish behaved normally until 16 weeks of age, at which point mild behavioral abnormalities, such as abnormal swimming and immobility alternating with jerky movement, were noted in a small number of fish in mHTT-exon1 L1 with more fish displaying these behaviors over time (Fig. 2c). mHTT L2 has not displayed any behavioral abnormalities up to 32 weeks of age. mHTT-ΔN17-exon1 L1 (n = 10) and L2 (n = 24), in contrast, developed a robust, progressive movement deficit ( Fig. 2b and c, and Additional file 3: Movie S3 and Additional file 4: Movie S4). mHTT-exon1, mHTT-ΔN17-exon1 L1, and mHTT-ΔN17-exon1 L2 all exhibit statistically significant differences in survival curve analysis as compared to each other as well as HTT-exon1 L1, HTT-ΔN17-exon1 L1, and mHTT-exon1 L2 (Fig. 2c, Kaplan Meier with Log Rank, p < 0.001). We grouped the observed behaviors into three stages according to their severity ( Fig. 2d): Stage 1abnormal swimming, immobility alternating with jerky movement; Stage 2loss of lateral stability, corkscrew swimming (see Fig. 2b); Stage 3loss of vertical stability, inability to coordinate swimming, death. mHTT-exon1 L1 fish begin exhibiting symptoms at 16 weeks and slowly progress so that by 44 weeks 50 % of the fish have reached Stage 3 (Fig. 2e). However, 40 % of the fish are still unaffected. mHTT-ΔN17-exon1 L1 begins exhibiting Stage 1 symptoms by 5 weeks of age and progresses to Stage 3 by 10-12 weeks (Fig. 2f). mHTT-ΔN17-exon1 97Q L2 does not exhibit Stage 1 symptoms until 8 weeks and progresses more slowly with Stage 3 not reached for all fish until after 20 weeks (Additional file 5). These experiments have been repeated in multiple generations with similar results. Non-heat shocked control transgenic fish for all lines, including both mHTT-ΔN17-exon1 lines, develop normally and do not show abnormal movement before 1 year of age (the last observation made), although some leaking recombination was noted. Overall, we have found that mHTT-ΔN17-exon1 transgenic fish consistently exhibit a progressive movement deficit, while mHTT-exon1 fish show a later-onset and more slowly progressing behavioral impairment. Importantly, the control HTT-exon1 and HTT-ΔN17-exon1 fish are indistinguishable from wild-type fish. Since the phenotypes of the transgenic fish were analyzed up to 11 months of age, it is possible that the mHTT-exon1 zebrafish could develop more severe disease-like phenotypes as they age. (See figure on previous page.) Fig. 1 Design and demonstration of Cre-loxP inducible HTT-exon 1-GFP expression in transgenic zebrafish. a Schematic of the transgene design using the bactin2 promoter to drive expression of floxed mCherry followed by the HTT-exon 1 cassette being tested. Following Cre expression by mRNA injection or HS:cre transgene induction, mCherry is recombined out and the desired HTT-exon 1 cassette fused to GFP is expressed ubiquitously. b Photo micrographs depicting 5 day old, HTT-exon1(25Q) embryos displaying mCherry expression when Cre is absent (upper panel) or mCherry negative, HTT-exon1-GFP positive expression following cre mRNA injection (bottom two panels). c Close-up view of the trunk of HTT(25Q)-exon1-GFP and mHTT(97Q)-exon1-GFP embryos demonstrating ubiquitous, diffuse expression of HTT-exon1-GFP (left panel) and ubiquitous, aggregated expression of mHTT-exon1-GFP (right panel). d A representative western blot and densitometric measurments for each transgenic line (n = 3). Anti-GFP antibody (top panel) and anti-tubulin loading controls (bottom panel) on protein isolated from pools of 5 day old embryos for each HTT transgenic line crossed to HS:cre. 97Q and 25Q mark the location of mHTT-GFP and HTT-GFP protein bands respectively. Protein expression was highest in HTT-exon1 L1 and HTT-ΔN17-exon1 L1 lines. mHTT-exon1 line 1(L1) and line 2(L2) both express at similar levels as do mHTT-ΔN17-exon1 line 1(L1) and line 2(L2). Densitometric measurements (lower bar graph) demonstrate significantly lower levels of protein in mHTT-exon1 L1 and L2 compared to all other lines while mHTT-ΔN17-exon1 L2 is significantly lower than HTT-exon1 L1 and HTT-ΔN17-exon1 L1. Comparisons were done by one-way ANOVA with Bonferroni posthoc test, *p < 0.01, error bars are SEM. e Quantification of transgene expression by quantitative RT-PCR. Six pools of ten embryos from separate clutches of each HTT transgenic line crossed to HS:cre were analyzed. Comparisons of expression level were done by one-way ANOVA with Bonferonni posthoc test, p < 0.05, error bars are SEM The transgenic zebrafish expressing mHTT-ΔN17-exon1 exhibit reduced brain weight To examine if brain atrophy, a hallmark of neuropathology in HD, was present in the mHTT-ΔN17-exon1 fish, brain weight from Stage 3 fish was compared to nonheat shocked fish and control HTT-ΔN17-exon1 with and without heat shock. Fish from each group were age matched and raised under equal population density and tank size. This is an important consideration for zebrafish since they exhibit density dependent growth [43]. The average body length and weight of each group of fish was not significantly different from each other (n = 8 for each group) (Fig. 3, a and b). The zebrafish brains from each line (excluding the olfactory bulbs and eyes) were dissected and weighed. Interestingly, heat shocked, Stage 3 mHTT-ΔN17-exon1 fish exhibited a significantly smaller brain weight than the brains from non-heat shocked mHTT-ΔN17-exon1 fish or HTT-ΔN17-exon1 fish with or without heat shock (Fig. 3c) (ANOVA with Bonferroni post hoc, p < 0.05). This result suggests that mHTT-ΔN17-exon1 fish exhibit brain atrophy similar to that seen in HD patients. N17 domain determines the cytoplasmic versus nuclear accumulation of mutant HTT exon1 fragments in vivo The 17N-terminal amino acids of HTT have been reported to regulate protein stability, toxicity, and subcellular localization. In a mouse BACHD model with N17 deleted in the context of the full length HTT gene, dramatically accelerated and exclusive nuclear accumulation of mHTT fragments were demonstrated [28]. In the context of mHTT-exon1, an HD-relevant pathogenic fragment [32], N17 is necessary for nuclear export/cytoplasmic targeting in vitro [44,45]: its relevance in mediating nuclear versus cytoplasmic pathology in vivo remains unclear. Therefore, we assessed if the localization of aggregates was different in transgenic HD fish expressing mHTT-exon1 with or without intact N17. First, we immunostained brain sections of Stage 3, HS:cre/ mHTT-ΔN17-exon1 fish, HS:cre/HTT-ΔN17-exon1 fish, HS:cre/mHTT-exon1 fish, and HS:cre/HTT-exon1 fish with the anti-human HTT antibody S830 [46] ( Fig. 4a-d). HTT-ΔN17-exon1 and HTT fish exhibit diffuse staining throughout the brain with no visible aggregates ( Fig. 4a and c). mHTT-ΔN17-exon1 and mHTT-exon1 fish both exhibit robust aggregate staining throughout the brain ( Fig. 4b and d). The aggregates in mHTT-exon1 appear as small puncta with occasional larger accumulations (Fig. 4b) while the aggregates in mHTT-ΔN17-exon1 appear mainly as large oval shaped staining reminiscent of nuclei (Fig. 4d). To examine the cellular and subcellular localization of these aggregates in more detail, we combined immunofluorescent staining for neurons (anti-HuC) with GFP staining of the HTT-exon1 fusion protein and DAPI for nuclei. We focused our examination on the ventrolateral nucleus of torus semicircularis, a brain region with strong HuC staining and a mixture of cell bodies and neuropil. In HTT-exon1 and HTT-ΔN17-exon1 fish, diffuse GFP staining was present throughout the brain including neurons (Additional file 6). Close examination by confocal microscopy demonstrated that HTT-exon1 is largely excluded from the nucleus while HTT-ΔN17-exon1 is detectible in the nucleus suggesting the nuclear export function of N17 is intact in vivo (Additional file 6). mHTT-exon1 fish exhibited robust aggregate formation, with the majority localized away from HuC positive neuronal cell bodies (Fig. 4e-h). These aggregates are likely present in the neuropil (e.g. axons and dendrites), but it is also possible that a subset of them are present in nonneuronal cells. Occasional GFP + aggregates are present in the peri-nuclear regions stained by HuC or weakly and diffusely in DAPI positive nuclei (Fig. 4e-h, arrowheads highlight GFP + nuclei). mHTT-ΔN17-exon1 fish, on the other hand, exhibited strong and predominant nuclear accumulation of mHTT-ΔN17-exon1 that was found almost exclusively in the HuC positive neurons (Fig. 4i-l). Neuropil aggregates similar to those seen in mHTT-exon1 fish were also present, but were much less abundant. 3D confocal projections clearly demonstrated the neuropil localization of HTT aggregates in mHTT-exon1 fish (Fig. 4m) and nuclear accumulation in the mHTT-ΔN17-exon1 fish (Fig. 4n). Quantification of HTT-GFP location demonstrates significant enrichment of nuclear mHTT-ΔN17-exon1 in HuC positive neurons of mHTT-ΔN17-exon1 fish (Fig. 4o, p < 0.05, ANOVA with Bonferonni (See figure on previous page.) Fig. 2 mHTT-ΔN17-exon1 transgenic fish develop a progressive motor behavior phenotype. a Panels taken from Additional file 1: Movie S1 showing normal swimming behavior in a HTT-ΔN17-exon1 transgenic fish. b Panels taken from Additional file 3: Movie S3 showing abnormal swimming behavior, corkscrew swimming, in a mHTT-ΔN17-exon1 transgenic fish. c Disease free survival curve for each HTT-exon1 transgenic line corresponding to disease onset. Note that mHTT-ΔN17-exon1 L1 (n = 10) and L2 (n = 24) both develop symptoms earlier than mHTT-exon1 L1 (n = 16) and that mHTT-exon1 L2 (n = 16), HTT-exon1 (n = 21), and HTT-ΔN17-exon1 (n = 26) do not exhibit symptoms in the observed time frame. Kaplan Meier analysis with Log Rank test, p < 0.001 for mHTT-ΔN17-exon1 L1, mHTT-ΔN17-exon1 L2, and mHTT-exon1 L1. d Behavioral characterization within different transgenic lines. Behavior was grouped into four categories: Healthy, Stage 1, Stage 2, or Stage 3 as described. Observations were made weekly. e Disease progression of mHTT-exon1 L1 fish. Note the discontinuous x-axis to account for the extended time frame of behavioral changes (n = 18). f Disease progression of mHTT-ΔN17-exon1 L1. mHTT-ΔN17-exon1 L1 fish developed a robust, progressive motor behavioral deterioration beginning at 5-8 weeks of age and progressing to immobility and death by 12 weeks (n = 10) post hoc). No difference was detected in cell density, percent HuC positive cells, or aggregate density (nuclear accumulation was scored as a single aggregate) in mHTT-exon1 versus mHTT-ΔN17-exon1 fish (Additional file 7). Taken together, our study is consistent with the in vitro studies in cells and mouse study with BACHD-ΔN17 mice that N17 is a crucial element in preventing nuclear pathogenesis for mHTT-exon1, and our study provides the first in vivo evidence that N17 can prevent nuclear accumulation and aggregation of a known pathogenic mHTT fragment, mHTT-exon1, in a vertebrate model of HD. Mutant HTT exon1 lacking the N17 domain exerts toxicity selectively in neurons to induce movement deficits in HD transgenic zebrafish HD is generally thought to be caused by neuron specific dysfunction and atrophy. However, since HTT expression is ubiquitous it is possible that disease specific pathologies are occurring in other cell types. HTT-polyQ expression in glia [47], immune cells [48], skeletal muscle [49] and vasculature [50,51] have all been suggested to contribute to HD. A recent study using conditional BAC transgenic mouse models of HD (BACHD) revealed distinct and synergistic roles of full-length mHTT expressed in cortical pyramidal neurons and striatal MSNs in eliciting multiple behavioral deficits and selective neurodegeneration [52]. However, these prior models lack the overt and progressive movement disorder seen in our mHTT-ΔN17-exon1 fish model, and it is unclear if mHTT-exon1 lacking N17 is ubiquitously toxic or may retain pathogenic specificity similar to the intact mHTT-exon1. To begin answering such a question, we crossed mHTT-ΔN17-exon1 fish with Cre driver lines with selective Cre expression in lineages for neurons (elavl3-Cre) [53], glia (gfap-Cre) [54], skeletal muscle (mylpfa-Cre) [55], and endothelial (etv2-Cre) [56] cells. Each cross gave expected tissue and cell-type specific mHTT-exon1 transgene expression (Fig. 5a-d). GFP + fish from each cross were selected and raised for behavioral observation (n = >20 for each genotype). Only the neuron specific, elavl3:cre/mHTT-ΔN17-exon1 fish developed movement abnormalities reminiscent of the ubiquitously activated mHTT-ΔN17-exon1 (Fig. 5e, p < 0.001, Kaplan Meier analysis). Using our previously described behavioral categories, we found that elavl3:cre/mHTT-ΔN17-exon1 fish exhibit all of the progressively deteriorating movement behaviors described in the ubiquitously expressing fish (Fig. 5f). It should be noted that the movement abnormalities observed in elavl3:cre/mHTT-ΔN17-exon1 fish appeared and progressed more slowly than ubiquitously expressing fish, suggesting the possibility that nonneuronal cells modify the effect of mHTT on neurons in this model. To examine the behavior of these fish in a more quantitative manner, we performed field potential recordings [57] of freely behaving elavl3:cre/mHTT-ΔN17-exon1 (n = 6) and elavl3:cre/HTTΔN17 fish (n = 6) fish (Fig. 6). elavl3:cre/mHTT-ΔN17-exon1 fish were categorized as Stage 1 at the time of this experiment while elavl3:cre/HTT-ΔN17-exon1 fish were categorized as healthy. Field potential recordings are performed by placing individual fish into a small chamber with recording Fig. 3 mHTT-ΔN17-exon1 transgenic fish exhibit reduced brain weight. a Body length is not statistically different between HTT-ΔN17-exon1 and mHTT-ΔN17-exon1 without (−HS) or with heat shock (+HS) (n = 8 for each condition). b Body weight is not statistically different between HTT-ΔN17-exon1 and mHTT-ΔN17-exon1 with or without heat shock (n = 8 for each condition). c The brain weight of mHTT-ΔN17-exon1 after heat shock induction of the HTT transgene is significantly less than either non-heat shocked mHTT-ΔN17-exon1 fish or heat shocked HTT-ΔN17-exon1 fish (n = 8 for each condition). () p < 0.05 by Student's t-test. Error bars are SEM Fig. 4 mHTT-ΔN17-exon1 accumulates mainly in the nucleus of neurons while mHTT-exon1 aggregates are mostly outside the neuronal cell body. Brain sections from 26 week old HTT-exon1 and mHTT-exon1 fish or 12 week old HTT-ΔN17-exon1 and mHTT-ΔN17-exon1, Stage 3 fish were immunostained to observe the transgenic HTT-exon1 localization. All images are from the hindbrain region and similar staining was present throughout all brain regions in each fish. a-d S830 anti-human HTT Exon 1 antibody was used to detect transgene expression in each transgenic line. HTT-exon1 (a) and HTT-ΔN17-exon1 (c) tissue exhibits uniform, ubiquitous expression. mHTT-exon1 (b) tissue displays many small but distinct aggregates while mHTT-ΔN17-exon1 (d) tissue has many large aggregates. Scale bar equals 25 μm. e-l Immunofluorescent staining of mHTT-exon1 (e-h) and mHTT-ΔN17-exon1 (i-l) transgenic fish for neurons (HuC), transgenic HTT-exon1-GFP fusion protein, and nuclei (DAPI). mHTT-exon1 tissue exhibits many small GFP + aggregates that are not generally associated with HuC positive neuronal cell bodies suggesting they are either in the axons or dendrites or are non-neuronal (e-h). Occasional, weak nuclear accumulation is present in a few neurons (white arrowheads). mHTT-ΔN17-exon1 tissue has many neuronal, HuC positive, nuclei co-localized with HTT-exon1-GFP (i-l). Scale bar equals 10 μm. m and n 3D confocal projections of mHTT-exon1 (m) and mHTT-ΔN17-exon1 (n) tissue demonstrating weak nuclear HTT-exon1-GFP staining in mHTT-exon1 and strong nuclear staining in mHTT-ΔN17-exon1. Scale bar equals 10 μm. o Quantification of protein accumulation location. Nuclear HTT-exon1 aggregates were rare in HuC negative cells of both lines and mHTT-exon1 transgenic HuC positive neurons. mHTT-ΔN17-exon1 transgenic fish exhibited robust nuclear accumulation of HTT-exon1-GFP in~40 % of HuC positive cells. ANOVA with Bonferroni posthoc test, () p < 0.01 electrodes at each end. When a fish moves the electrical activity of the muscle creates a potential across the electrodes that can be recorded over time. This gives an indirect measurement of each fish's motor activity. elavl3:cre/HTT-ΔN17-exon1 fish exhibit short bursts of activity corresponding to swimming and turning movements within the chamber (Fig. 6a, top panel). elavl3:cre/ mHTT-ΔN17-exon1 fish exhibited bouts of prolonged activity (Fig. 6a bottom panel and c). However, elavl3:cre/ mHTT-ΔN17-exon1 fish initiated movements less frequently than elavl3:cre/HTT-ΔN17-exon1 fish (Fig. 6b). Overall, elavl3:cre/HTT-ΔN17-exon1 and elavl3:cre/ mHTT-ΔN17-exon1 fish did not differ in the total amount of time they were active during these trials (Fig. 6d). These results support the behavioral observations that Stage 1 fish exhibit bouts of jerky movement followed by inactivity. In summary, our cell-type-specific expression of mHTT-ΔN17-exon1 revealed a remarkable specificity of toxic mHTT fragments to neurons while its toxicity to several non-neuronal cell types including glia, muscle and blood vessels are unremarkable. This result suggests the neurons are particularly sensitive to the nuclear polyQ toxicity in the context of mHTT-exon1, and the presence of N17 domain can delay such toxicity in vivo. Discussion The molecular mechanisms causing HD have been difficult to identify even though the specific gene mutation has been known for 20 years. Many research models have been created in diverse organisms such as rats [58], mice [29,33,59] zebrafish [39,40], fly [37], worm [35,36], and yeast [34]. Mammalian models have been extremely valuable in validating disease mechanisms and testing candidate therapeutics [30]; they also have limitations such as lack of overt disease phenotypes and limited scalability due to the cost and effort to maintain a large rodent colony. Invertebrate models are highly scalable but lack close genetic homology to humans. The zebrafish model is an ideal compromise between the scalability of invertebrate models and genetic homology to vertebrates. Previously developed zebrafish models of HD are not ideal models of the disease due to lethality caused by high level overexpression of toxic mHTT f Disease progression of elavl3:cre/ mHTT-ΔN17-exon1 fish is similar to ubiquitious mHTT-ΔN17-exon1 fish although temporally delayed, note the discontinuous x-axis (n = 23). Behavioral Stages are described in Fig. 2 fragments in embryos or limited disease relevance due to restricted tissue expression. Here we present a novel Cre-loxP inducible zebrafish model of HD. This model allows for precise spatial and temporal control of expression of the disease linked protein, which was previously only available in the mouse [52,60]. These transgenic lines can be maintained in non-recombined form and experimental clutches of embryos generated as needed by Cre mRNA injection or intercross with Cre driver transgenic lines. By incrossing tissue specific Cre driver lines to the mHTT-ΔN17-exon1 line we found that neurons appear to be the main cell involved in the abnormal movement behaviors originally observed with ubiquitous expression of this particular toxic polyQ protein. This is in agreement with findings in conditional mouse HD models [52,60,61]. Future work will be necessary to establish the specific neuronal population responsible for the behavioral phenotype in this zebrafish model using neuronal sub-type-specific Cre driver lines. Additionally, careful cell-type specific analysis for histological and molecular markers of HD-like pathologies will be necessary to further define the similarities to human disease. These transgenic fish are based upon expression of mHTT-exon1. In recent years it has become apparent that the exon 1 fragment of the much larger HTT Fig. 6 Behavioral characterization of elavl3:cre/mHTT-ΔN17-exon1. Field potential recordings were made from freely behaving elavl3:cre/HTT-ΔN17 fish (n = 6) and elavl3:cre/mHTT-ΔN17-exon1 (n = 6) fish exhibiting Stage 1 behaviors. Field potentials represent the electrical muscle activity of a given freely behaving animal as measured by recording electrodes placed in the chamber during movements. Each fish was allowed to acclimate to the recording chamber for 5 min and then recorded for 5 min. a Representative field potential recordings from an elavl3:cre/HTT-ΔN17-exon1 fish (top) and an elavl3:cre/mHTT-ΔN17-exon1 fish (bottom). Brackets define of swimming activity. Note the short, distinct swimming activity of the elavl3:cre/HTT-ΔN17-exon1 fish versus the sustained activity of the elavl3:cre/mHTT-ΔN17-exon1 fish. b Average movement frequency as calculated by counting each distinct, non-overlapping movement waveform over time. elavl3:cre/mHTT-ΔN17-exon1 fish move significantly less often than elavl3:cre/HTT-ΔN17-exon1 fish. c elavl3:cre/mHTT-ΔN17-exon1 fish display extended periods of activity as compared to elavl3:cre/HTT-ΔN17-exon1 fish. d Total time moving is not significantly different between elavl3:cre/HTT-ΔN17-exon1 and elavl3:cre/mHTT-ΔN17-exon1 fish. () p < 0.05 by Student's t-test. Error bars are SEM protein not only contains the expanded polyQ region that is mutated in the disease but is also a known pathogenic species in the diseased as a result of aberrant mHTT splicing event [32] and proteolysis [31]. A surprising finding in our study is that zebrafish is somewhat refractory to the toxicity of ubiquitously expressed mHTT-exon1, since only a few transgenic mHTT-exon1 fish developed abnormal movements by 6 months of age. Histological examination of these fish demonstrated robust mHTT-exon1-GFP positive protein aggregates formed in the brain, and most of these aggregates are located in the neuropil (e.g. axons, dendrites). One leading hypothesis is that the nucleus is the site of pathological action of expanded polyQ in all polyQ disorders [4]. It has been reported that the N17 terminal amino acids of HTT contains a nuclear export signal [45,62]. Additionally, these amino acids have been reported to function in modifying aggregate formation and protein stability [17,23,24,26,27]. Recently, Gu et. al. [28] showed that deletion of these 17 amino acids in the context of BAC transgenic mice harboring the full length human HTT gene with 97Q causes an adult-onset progressive movement disorder and robust striatal neurodegeneration. Interestingly, these BACHD-ΔN17 mice also presents with dramatic acceleration of selective nuclear mHTT aggregates due to accumulation of small mHTT polyQ fragments. We therefore, tested whether deletion of these 17 amino acids in the context of an HTT exon 1 based transgene would cause accelerated disease in our zebrafish model. Mutant HTT-ΔN17-exon1 expression caused a rapidly developing progressive movement disorder that ultimately led to early death. The development of this behavioral phenotype was correlated with massive accumulation of mHTT-ΔN17-exon1-GFP in the nucleus of neurons. Interestingly, although expression was ubiquitous, large nuclear aggregates were only observed in neurons, suggesting mechanisms of nuclear accumulation and aggregation of the mutant polyQ protein is neuronal selective. We did not observe robust nuclear accumulation of the non-toxic HTT-ΔN17-exon1 transgene suggesting that lack of N17 function alone without polyQ expansion is not sufficient to elicit high levels of nuclear protein accumulation. Our study confirmed and extended the results found in BACHD-ΔN17 mice by demonstrating not only that N17 is crucial in preventing nuclear mHTT toxicity and onset of disease-like phenotypes in an vertebrate, but also provide strong evidence that N17 is crucial in mitigating the ability of mHTT-exon1 fragment, a known pathogenic species in HD, to mediate its nuclear toxicity and disease pathogenesis in an intact vertebrate model. A truly surprising finding in our study is that the mHTT-ΔN17-exon1 fragment elicits robust and progressive movement disorder through its toxicity in neurons, but not in astrocytes, skeletal muscles, or vascular cells. This result showed that neurons, unlike the non-neuornal cells, may have selective impairment in managing the proteostasis of the expanded polyQ proteins, especially when such protein is translocated and accumulated in the nucleus. Our result is consistent with the rich evidence that expanded polyQ protein is neurotoxic in an artificial context of an endogenous protein [63], or other polyQ diseases (e.g. SBMA or SCA1) in which nuclear translocation is essential to disease pathogenesis [4]. Our study has important implications in the context of understand HD pathogenesis and therapy. First, it suggests that despite the fact that mHTT-exon1 is naturally occurring and is more toxic than longer mHTT-exon1 fragments or full-legnth mHTT, the N17 domain can still provide substantial protection against its ability to translocate into the nucleus and elicit severe disease. Second, since in HD patients and HD mice mHTT fragments (including exon1 fragment) eventually accumulate in the nucleus and cause nuclear pathoglogy [10,31], the mHTT-ΔN17-exon1 zebrafish represent a feasible model of HD. Thus, better understanding how N17 function is compromised in the context of HD neurons or how to improve the ability of the nucleus to clear such aberrant polyQ proteins may be important steps in reducing disease burden in HD. Finally, our study may have important implications in the use of zebrafish models to study pathogenesis or testing candidate therapeutics for HD. An important advance we have made, through the use of conditional genetics in zebrafish, is the creation of a stable and robust model of HD with progressive movement deficits and brain pathology. The inducibility of the model allows us to study the toxicity of mutant polyQ protein in distinct cell populations. Since the model recapitulates aspects of the disease related to nuclear accumulation of mHTT-polyQ fragments, our model may be particularly useful to study mechanism and therapeutics aiming to improve nuclear proteostasis or reduce the consequence of nuclear mHTT toxcitiy in vivo. Moreover, due to the robust brain atrophy associated with the overt and progressive movement disorder, our model could also be used to test novel neuroprotective therapies. Given the obvious advantage of the zebrafish system in terms of its scalability and relative low cost in its maintenance, we envision that the mHTT-ΔN17-exon1 fish model will facilitate the screening of potential genetic disease modifiers or candidate therapeutics at a scale that is not likely to be achievable in the other vertebrate models of HD. Animal husbandry All zebrafish use was approved by the University of California, Los Angeles Animal Care and Use Committee. This work was performed under IACUC protocol ARC # 2001-074-41 issued by OFFICE OF ANIMAL RESEARCH OVERSIGHT, University of California Los Angeles. Adults, juveniles, and zebrafish embryos were maintained according to standard zebrafish methods [64]. Heat shock was performed on shield stage embryos for 30 min at 38.5°C after which embryos were transferred to a standard zebrafish incubator at 28.5°C to be raised. Transgene constructs and generation of transgenic zebrafish All transgenes were generated using the Multisite Gateway System (Life Technologies) and the Tol2kit developed for zebrafish [42]. p3E-HTT Exon 1 25Q-EGFP, p3E-HTT Exon 1 97Q-EGFP, p3E-ΔN17 HTT Exon 1 25Q-EGFP, and p3E − ΔN17 HTT Exon 1 97Q-EGFP were generated by BP reaction of each respective PCR product flanked with B2F and B3R sequences to pDONR™ P2R-P3. Transgene plasmids were created by LR multisite reaction of pDESTTol2pA, p5E-bactin2, pME-loxPmCherryloxP, and each respective HTT p3E vector. Resulting plasmids were injected into single cell zebrafish embryos along with Tol2 mRNA using standard methods to efficiently generate germline integrations. Resulting F 0 fish were raised and their offspring screened for ubiquitous mCherry expression. Positive F 1 embryos were raised to establish each line. Each line was derived from separate F 0 fish to control for integration effects. Cre driver lines were generated by LR multisite reaction with pDESTTol2pA, promoter specific p5E vector, pME-Cre, and p3E-pA. p5E-elav3l was generated by cloning the 3.1 kilobase zebrafish elav3l (also known as HuC) promoter, into pDONR™ P4R-P1R using the BP reaction. Similarly p5E-gfap was generated by subcloning the gfap promoter from the Tg(gfap:GFP) plasmid [54] into pDONR™ P4R-P1R. p5E-mylpfa [65] and p5E-etv2 [56] were previously reported. Each transgenic line was generated as described above. However since these lines do not have a fluorescent marker gene, founders were identified by outcross to ubi:Switch fish [66] which exhibit a GFP to mCherry fluorescent switch in tissues expressing Cre recombinase. Independent lines with strong tissue specific recombination and minimal leakiness were maintained for each promoter. qRT-PCR Quantitative RT-PCR was performed on cDNA generated using Superscript III Reverse Transcriptase (Life Technologies, 18080044) from total RNA extracted from 5 day post fertilization larvae using Trizol reagent (Life Technologies, 15596026) on a Stratagene Mx3005P qPCR system. Three pools of ten embryos from separate clutches of each transgenic line were analyzed. Oligo(dT)12-18 (Life Technologies, 18418-012) was used to prime the reverse transcriptase reaction. Western blotting Western blots were performed on protein extracted from 5 day post fertilization embryos using standard methods. Five embryos were pooled for each sample into 25 μl of 2X SDS Loading Buffer. Protein samples were run on a 12 % Tris-glycine acrylamide gel (Thermo Scientific, 25247). Protein was transferred onto a PDVF membrane (Thermo Scientific, 88520), and then probed with rabbit anti-GFP primary antibody 1:2000 dilution (Life Technologies, A11122), stripped and then reprobed with mouse anti-tubulin antibody 1:10,000 dilution (Sigma, T5168). Secondary antibodies were anti-rabbitperoxidase (Sigma, A0545) and anti-mouse-peroxidase (Sigma, A9044) both at 1:5000 dilutions. Chemiluminescent detection was performed using Lumi-Light western blotting reagent (Roche, 12015200001) and HyBlot CL autoradiography film (Denville Scientific, E3018). Film was digitized on an HP Scanjet G3110 and the image adjusted in Adobe Photoshop CS4. ImageJ was used to perform densitometric measurements. Behavioral analysis Fish were raised at similar densities and monitored weekly for abnormal movement. Fish were grouped into four categories: 1) Healthynormal swimming; 2) Stage 1abnormal swimming, immobility alternating with jerky movements; 3) Stage 2loss of lateral stability, corkscrew swimming; 4) Stage 3loss of vertical stability, inability to coordinate movement, death. When fish were no longer able to swim for feeding they were killed for further analysis. For more detailed behavioral analysis we performed electric field potential recordings on freely behaving adult elav3l:cre/HTT-ΔN17 or, Stage 1 categorized, elav3l:cre/mHTT-ΔN17 fish (n = 6 each) according to the methods of Issa et.al. [57]. Fish were placed into a recording chamber and allowed to acclimate to the chamber for 5 min. Then field potentials generated from muscles during movement were recorded for 5 min for each fish. The recording chamber was 11 cm long × 4 cm wide × 3 cm deep containing double distilled water with a resistance of~15 MΩ. Water depth was 2.75 cm. Room temperature was set to 25°C. Electric field potentials were recorded using one pair of electrodes (0.5 cm exposure, 0.1 cm thick of bare copper medal) placed on either end of each testing chamber. Electric signals were amplified 1000-fold using an AC differential amplifier (AM-Systems model 1700, Carlsborg, WA USA), lowpass filtered at 300 Hz and high-pass filtered at 500 Hz. All signals were digitized and stored with a Digidata-1322A and acquired using Axoscope software (Molecular Devices, Inc., Sunnyvale, CA, USA). Data was annotated manually and movement frequency, duration, and total time were calculated. Body length, weight, and brain weight measurements Body length and weight were measured on fish deeply anesthetized with Tricaine solution (Sigma, A5040). Length was measured from the tip of the jaw to the base of the tail since the HS:cre line was in a long-fin genetic background resulting in mixed normal and long-fin offspring. After measuring the body, fish were killed by exsanguination, the top of the skull removed, and the head fixed in 4 % paraformaldehyde overnight at 4°C to improve tissue stability for dissection. Fish heads were washed three times in PBS and gently dried. Brains were carefully dissected using watchmakers forceps from the olfactory bulb-telencephalon junction to the hindbrain-spinal cord junction. All cranial nerves were carefully removed except for the optic nerve and optic tract which remained attached to the brain. The eyes were removed. Excess liquid was removed and brain weight was measured. Histology and immunostaining To harvest tissue for cryo-sectioning, adult fish were deeply anesthetized in Tricaine solution (Sigma, A5040) and then killed by exsanguination. The head was removed as was the top of the skull. Fish heads were fixed overnight in 4 % paraformaldahyde/PBS solution at 4°C with constant movement on a nutator mixer. The following day the brain was dissected out intact from the tip of the forebrain to the hindbrain spinal cord junction. The brain was then fixed overnight in 4 % paraformaldahyde/PBS solution plus 5 % sucrose at 4°C with constant movement on a nutator mixer. The following day fixed tissue was put through an increasing sucrose gradient: 5, 10, 12.5, 15, and 20 % in PB buffer, 1 h each and then placed at 4°C with constant movement on a nutator mixer overnight. The following day tissue was infiltrated with a mix of 2:1 20 % sucrose solution to OCT Compound (Tissue-Tek, 4583) for 1 h and then snap frozen in 100 % OCT in aluminum foil molds placed into a 2-methylbutane (Sigma, M32631), dry ice bath. Blocks were stored at −80°C until they were cut at 16 μm for immunostaining on a Leica model CM3050S cryostat. Imaging Images were captured on an Axioskop 2 plus microscope (Zeiss) using 5×, 10×, or 40× objectives with a Hamamatsu ORCA-ER camera and Volocity 5 software (Improvision). Confocal images were collected on a Zeiss LSM 510 with a 63× oil immersion objective (NA 1.4). Adobe Photoshop CS4 was used to adjust brightness and contrast and assemble composite images. To quantify GFP + protein aggregates in HuC stained sections, ImageJ was used to count cell number (DAPI positive nuclei), HuC + cells, and aggregate number. Maximum intensity projections of 133.6 μm × 133.6 μm × 4.7 μm zstacks were analyzed from three non-consecutive sections of each fish. Images were acquired such that bright GFP+ aggregates did not over saturate the image. Therefore, the background level of GFP expression appears lower than it actually was. Three fish were examined for each genotype. Statistical analsysis Student's t-test was used for single comparisons, ANOVA with Bonferonni posthoc test was used for multiple comparisons, and Kaplan Meier survival analysis with Log Rank posthoc test was used for survival curve comparisons with p < 0.05 set as significant for each. All statistical analysis was performed using SPSS 14.0 software. Conclusions We have developed a Cre-inducible HD model in zebrafish base upon expression of human exon 1 of HTT. Using this model we have demonstrated that the N17 domain is protective in the context of 97Q since its deletion results in more severe phenotypes and earlier lethality. Deletion of N17 results in neuron-specific accumulation of 97Q in the nucleus and neuron specific induction of the transgene is sufficient to generate HD-like phenotypes. Together these results support a protective role of the N17 domain in HD possibly through nuclear export of the pathological 97Q fragment. This new zebrafish model will facilitate pharmacological, genetic, and mechanistic studies of HD.	2016-05-04T20:20:58.661Z	2015-12-01T00:00:00.000	{ "year": 2015, "sha1": "2d0d2eb526cae9c5deebfeff0cc58e57b9ed7ac9", "oa_license": "CCBY", "oa_url": "https://molecularneurodegeneration.biomedcentral.com/track/pdf/10.1186/s13024-015-0063-2", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bf8e60c0affb5e196f68e471cfe7b7bebf0dd169", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
244177528	pes2o/s2orc	v3-fos-license	Direct brain involvement of Takayasu arteritis treated with rituximab and infliximab: a case report Takayasu arteritis (TAK) is a systemic vasculitis involving large arteries. Reports of direct central nervous system (CNS) involvement in TAK are extremely rare in the literature. In addition, treatment for direct involvement has not been reported. Herein, we describe a case of encephalitis in a TAK patient who presented with fever and headache at the first attack, then cognitive impairment at the second attack. The patient improved with rituximab and especially infliximab. These findings indicate the usefulness of rituximab and infliximab to treat the direct CNS manifestations in TAK. gia, claudication, dizziness, dysarthria, or sided weakness). His blood pressure was mildly elevated (systolic blood pressure 140-150 mmHg) without a significant difference ( > 10 mmHg) between both arms, and his body temperature was above 38.0°C. Upon physical examination, no oral/genital ulcers or skin rashes were observed. In addition, radial and dorsalis pedis artery pulses were present. Furthermore, bruit was not observed in either the carotid or ophthalmic arteries. The detailed neurological examination revealed no focal neurological deficits, including spasticity or dyskinesia. Computer tomography (CT) of the abdomen and pelvis was unremarkable; however, chest CT revealed wall thickening of the ascending aorta, right proximal brachiocephalic artery, and left proximal common carotid artery ( Figure 1A). Brain magnetic resonance imaging (MRI) showed T2 high signal intensities and T1 contrast-enhanced lesions involving the right basal ganglia, corpus callosum genu, and both hypothalamic areas without stenotic or occlusive lesions in the intracranial and extracranial arteries ( Figure 1B-E). CNS vasculitis, demyelinating disease, brain tumor, autoimmune encephalitis, and infectious encephalitis were included as differential diagnoses. Torso 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) showed no evi-dence of tumorous conditions. In particular, brain FDG-PET showed hypometabolism of the affected lesions ( Figure 1F). Based on a cerebrospinal fluid analysis showing a normal profile, other infectious etiologies were also excluded. The autoantibodies and the vasculitis-associated tests were unremarkable. Unspecified inflammatory CNS diseases such as CNS vasculitis or demyelinating disease were suspected, and an intravenous (IV) corticosteroid pulse was administered. After pulse therapy, radiologic findings and the symptoms (fever and headache) improved. The oral steroid was maintained and then tapered off over an approximately 1-month period. Approximately 4 months after the first steroid pulse therapy, the patient developed sudden memory impairment, recurrent headache, urinary incontinence, and sleeping tendency. Approximately 2 months after the recurrence, IV steroid pulse was readministered, followed by oral steroid and cyclosporine combination therapy. Despite immunotherapy, impaired cognitive function (subjectively, 80% of baseline status) and headache remained. Although the patient was treated with oral steroid and cyclosporine combination therapy, uveitis occurred approximately 6 weeks after the second steroid pulse therapy. Follow-up brain MRI (3 months after second pulse therapy) revealed a few residual patchy T2 high signal intensities in both the basal ganglia, hypothalamic areas, and optic tracts without enhancement and diffusion restriction. Based on clinical symptoms, including remaining cognitive dysfunction, rituximab was administered as an alternative treatment. After five cycles of rituximab (375 mg/m 2 /cycle), the patient showed complete recovery of neurological symptoms. Although clinical response was remarkable, subsequent brain images showed no interval changes. Around the 12th cycle of rituximab, fever recurred with a painful erythematous patchy rash on the abdomen and right tibia. Erythrocyte sedimentation rate (ESR) was highly elevated. After infection was excluded, infliximab (5 mg/kg/cycle every 2 months) was administered and ESR levels started to normalize (Figure 2A). Furthermore, there were no definite residual patchy T2 high signal intensities in the basal ganglia, hypothalamic area, or optic tracts, which was a complete radiological response after the five cycles of infliximab ( Figure 2B). Around the eighth cycle of infliximab, wall thickening at the ascending aorta and its main branches were not observed on follow-up chest CT ( Figure 2C). Discussion TAK is a large vessel vasculitis affecting the aorta and its major branches. However, TAK can exhibit extravascular involvement, including arthritis, erythema nodosum, and uveitis [8]. In the present case, uveitis occurred after the second CNS attack, and infliximab ameliorated erythema nodosum and CNS lesions. TAK and encephalitis could have occurred independently (i.e., coincidentally). However, the temporal association and complete responses of both systemic inflammation and CNS lesions to treatment indicated that encephalitis was an extravascular manifestation of TAK. Notably, the persistent CNS lesions were reversed with infliximab. The persistent lesions indicated the remaining vasogenic edema was due to the ongoing inflammatory process. Although B cell immunity was suppressed by rituximab therapy and endothelial injury was restored, it was insufficient to reconstruct the disruption of the blood-brain barrier caused by other immune mechanisms. In contrast, infliximab therapy completely suppressed inflammation and induced a complete radiologic response, eliminating vasogenic edema. Regarding immunological aspects, Mutoh et al. [9] recently reported antiendothelial cell antibodies promote vascular inflammation by activating endothelial cells in TAK. Our patient's clinical response of CNS symptoms to rituximab therapy supports this hypothesis. Despite the clinical response, the effect of rituximab on the patient's disease activity and radiological findings was insufficient. This result is compatible with the previous studies in which rituximab was suggested to have a role as second-or third-line but not first-line therapy in TAK [6]. Hypothetically, the B cell immunity does not play a critical role but an accessory role. In several previous studies, natural killer (NK) cells and CD8 T cells played essential roles [1]. T cells, NK cells, and macrophages produce TNF-α which is important in the formation of granulomatosis in TAK. Furthermore, TNF-α induces the production of interleukin-12 from macrophages, the key cytokine involved in CD4 T cell differentiation into T helper type 1 cells and NK cell activation. Infliximab is a TNF-α inhibitor and has been proven efficacious in TAK [7]. Although we anticipated infliximab would suppress the patient's disease activity and systemic manifestations, the complete resolution of CNS lesions was unexpected. Notably, encephalitis associated with TAK was partially treatable with rituximab and completely with infliximab. To the best of our knowledge, this is the first report of the treatment options of TAK with direct CNS involvement. In conclusion, rituximab and infliximab may be useful for managing the direct CNS involvement in TAK. Future investigations regarding the efficacy of this management are needed. However, this report is limited because CNS lesions could not be confirmed using brain biopsy. Conflicts of Interest Kon Chu has been on the editorial board of encephalitis since October 2020 and was not involved in the review process of the original article. The authors declare no other potential conflicts of interest relevant to this article. Author Contributions Conceptualization: Moon J, Park JK, Chu K; Data curation: Kim SD, Ahn SJ, Lee HS, Lee WJ; Investigation: Kim SD, Ahn SJ; Writing-original draft: Kim SD, Ahn SJ; Writing-review and editing: all authors.	2021-10-18T17:05:06.832Z	2021-10-05T00:00:00.000	{ "year": 2021, "sha1": "7e30982faa158d1a5ce1f63f3f0f39a233d42983", "oa_license": "CCBYNC", "oa_url": "https://www.encephalitisjournal.org/upload/pdf/encephalitis-2021-00108.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "ebe2a90a3a68d7cda8a95133069abb9bea8e68e4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
266412702	pes2o/s2orc	v3-fos-license	Enzymatic Acylation of Black Rice Anthocyanins and Evaluation of Antioxidant Capacity and Stability of Their Derivatives In this study, the structure of the anthocyanin fractions isolated from black rice (Oryza sativa L.) was modified by the enzyme catalysis method using caffeic acid as an acyl donor. At the same time, the effects of the acylation on the lipophilicity, antioxidant activity, and stability of black rice anthocyanins were comprehensively evaluated. The structural analyses of acylated derivatives based on ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy, ultra-high-performance liquid chromatography–high-resolution mass spectrometry, and thermogravimetric analysis revealed that caffeic acid was efficiently grafted onto the anthocyanins of black rice through an acylated reaction, while the acylation binding site was on glucoside. When the mass ratios of anthocyanins to caffeic acid were 1:1, the A319/AVis-max value of acylated anthocyanins reached 6.37. Meanwhile, the lipophilicity of acylated derivatives was enhanced. The antioxidant capacity (DPPH and FRAP) and stability (thermal, pH, and light stability) were significantly increased. Overall, the study results provide deeper insights into controlling anthocyanin homeostasis in food processing, broadening the application of colored grain products. Introduction Anthocyanins are flavonoid derivatives that are powerful antioxidants against aging and oxidative stress.So far, more than 600 anthocyanins have been detected in nature, mostly derived from cyanidin, petunidin, peonidin, pelargonidin, delphinidin, and malvidin [1][2][3].The natural state of anthocyanins is in the form of glycosides, which are extensively utilized as colored pigments in various industries such as food, cosmetics, biology, pharmaceuticals, and others due to their diverse biological activities, bright and attractive colors, and high level of edible safety [4][5][6]. Grain is a general term for cereal plants or grain crops, covering a wide range of foods, including black rice, rice, corn, and other miscellaneous grains, which are rich in a variety of nutrients needed by the human body and form an important part of the human diet [7].Among them, black rice is rich in phenolic compounds, including anthocyanin, phenolic acid, flavonoid, and dietary fiber, which is a great source of anthocyanins [8].The primary anthocyanins of black rice included cyanidin and paeoniflorin, with a typical flavonoid C6-C3-C6 structure, which can be modified to prepare grain food with specific functionality to expand their applications [9,10]. Regardless of the excellent biological activity of anthocyanins, these pigments are unstable.Anthocyanins can be easily destroyed by different factors, such as pH, light, thermal treatment, enzymes, oxygen, etc., limiting their application to a great extent [11].Therefore, improving the stability of anthocyanins has been the focus of many researchers.Accumulating studies have indicated that glycosyl in anthocyanins can combine with the acylation donor to form acylated anthocyanins, exhibiting excellent stability [12,13].The Foods 2023, 12, 4505 2 of 13 primary sites for the acylation reaction of anthocyanin are the units of C3 and C5 in the glycosyl group, or C3, C6 [14].Higher steric hindrance is related to the increased stability of acylated anthocyanins, which likely protects the anthocyanins from hydration.This occurs as the aglycone cannot be attacked by water, thus preventing the chalcone from forming.Furthermore, acylated anthocyanins, especially in acidic and neutral environments, exhibit excellent stability over a wide range of pH values [15]. Recently, the acylation of anthocyanins has attracted much attention.Lv et al. [16] acylated the black rice anthocyanins using octenyl succinate anhydride in pure ethanol and evaluated their stability and color change.Dini et al. [17] found that acylation reactions with aromatic carboxylic acids improved the thermal stability and light-resistivity of anthocyanins.However, the mechanism of enzymatic acylation with carboxylic acid as a donor for the structural stabilization of anthocyanins has not yet been fully elucidated.Therefore, in this study, the anthocyanins were extracted from black rice and acylated by caffeic acid in the presence of Novozyme 435 to understand the relationship between the molecular structure of acylated derivatives and their stability and antioxidant capacity.This research will provide insights to broaden the application of black rice anthocyanins in the food coloring and healthcare industries, as well as the development of new cereal foods. Anthocyanin Extraction The method described by Hao et al. [18] was used to prepare anthocyanin extracts from black rice with some modifications.The black rice flour was dissolved in 1% hydrochloric acid/pure ethanol/water (0.5:50:50, v/v/v) (1:10 w/v) and extracted twice at 30 • C for 2 h.The supernatants were collected after centrifuging at 4000 r/min for 10 min.A vacuum evaporator was used to remove residual ethanol from the supernatant.Then, the crudely extracted black rice anthocyanins and AB-8 macroporous adsorption resin were mixed at a mass ratio of 5:1 for 24 h at room temperature.It was subsequently recovered using 70% ethanol (containing 1% HCl).The anthocyanin extracts were obtained by evaporation of the ethanol eluent under a vacuum and, finally, freeze-drying to produce anthocyanin powders. Anthocyanin Acylation The acylation of anthocyanins was performed by the method described by Zheng et al. [19] with slight modifications.A mixture (300 mg) of anthocyanins (AN) and caffeic acid (CA) with mass ratios of 1:0, 2:1, 1:1, and 1:2, respectively, was placed in a 250 mL conical flask, and 5 mL of distilled water was added to mix completely.The black rice anthocyanins and their derivatives were labeled AN/CA 1:0, AN/CA 2:1, AN/CA 1:1, and AN/CA 1:2, respectively.A total of 15 g of dry-activated 4A zeolite was mainly used in the absorption of water in the reaction system.Later, the mixture was mixed with tert-butyl alcohol (60 mL) and lipase Novozyme 435 (30 mg) and stirred at 25 • C for 24 h.Filtration was used to stop the reaction and remove the zeolite and enzyme.Organic reagents were removed by evaporation under vacuum and were freeze-dried for use. Ultraviolet-Visible (UV-Vis) Analysis The UV-vis analysis of black rice anthocyanins and their derivatives was performed by a Puruisi UV-18 ultraviolet and visible spectrophotometer (Tianjin Puruisi Instrument Co., Ltd., Tianjin, China).Briefly, the sample of anthocyanins was dissolved in methanol to obtain a 1.0 mg/mL solution, and stirred to sufficiently dissolve.Then, the UV-vis absorption measurements were recorded at a 1 nm sampling interval. FTIR Analysis Spectrum 100 FTIR spectrometer (PerkinElmer Co., Ltd., Shelton, CT, USA) was used to measure the chemical structure of black rice anthocyanins and their derivatives.The black rice anthocyanins and their derivatives were blended with potassium bromide (KBr) at a 1:100 (w/w) ratio and compressed into pellets.The wavenumber range was 4000 cm −1 to 800 cm −1 , with a resolution of 4 cm −1 and 32 scans per sample. Thermogravimetric Analysis (TGA) The thermal weight loss value of black rice anthocyanins and their derivatives during heating were studied using a Q50 TGA analyzer (TA Instruments Co., Ltd., New Castle, DE, USA).Heating was carried out to 400 • C under a nitrogen atmosphere at a ramp rate of 20 • C/min.The first derivatives of the weight loss curve thermograms were calculated (DTG curves). Determination of the Lipophilicity According to Xie et al. [20], the octanol-water partition coefficient (logP) was used to evaluate the lipophilicity of the anthocyanins and acylated derivatives.Briefly, the samples were dissolved in acidified water (2% HCl) saturated with n-ocanol, and the absorbance at 520 nm was measured (A 0 ).Then, double the amount of octanol-saturated acidified water was added, and the mixture was centrifuged for 10 min at 3000 rpm after being vigorously shaken for 1 h.At 520 nm, the absorbance (A X ) of the upper layer of octanol was determined.The following equation was used to calculate the octanol/water partition coefficient: The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay and ferric reducing antioxidant power (FRAP) assay were used to determine the antioxidant capacity.The method described by Sahreen et al. [21] was used to determine the DPPH and ABTS scavenging assays.The absorbance of DPPH and ABTS radical scavenging activity was measured at 517 nm and 734 nm, respectively, using a spectrophotometer.The following equation was used to calculate the scavenging rate: Scavenging rate (%) = (1 − A/A 0 ) × 100 where A represents the sample absorbance, and A 0 represents the absorbance of the standard solution without an added sample. The FRAP assay was performed according to the method reported by Thaipong et al. [22] with some modifications.The FRAP stock solution was made with a volume ratio of 10:1:1 of sodium acetate buffer (0.3 mol/L, pH 3.6), TPTZ solution (0.01 mol/L with 0.04 mol/L HCl), and FeCl 3 solution (0.02 mol/L), respectively.Later, 3.98 mL of FRAP stock solution was mixed with 0.02 mL of the sample and stirred at room temperature for 30 min.Sample absorbance was measured at 593 nm.Within the range of 0 and 25 mM Trolox, the standard curve was linear.The results were represented as mM Trolox equivalents (TE)/mg anthocyanin mass. Stability Assays The effect of temperature on the thermal stability of black rice anthocyanins and their derivatives was evaluated according to the method reported by Swer and Chauhan [11] with some modifications.Briefly, 1 mg/mL of anthocyanins was made.The solutions were added to test tubes with stoppers and heated at 50 • C, 60 • C, 70 • C, 80 • C and 90 • C, respectively.After cooling to room temperature, the absorbance was immediately measured at 520 nm in a spectrophotometer.Meanwhile, the same solvent was used as blank.The absorbance of anthocyanins was determined each hour for 5 h continuously.The first-order reaction rate constant (k) and half-lifetime (t 1/2 ) were calculated by the following equations: where C and C 0 are the anthocyanin content of the sample at a particular time and the initial content, respectively, k is the rate constant (per unit time), and t is the time (in hours). The dependence of the degradation rate constants on temperature was described by the Arrhenius model: where k 0 is the initial value of the rate constant for degradation (1/min), R is the universal gas constant, T is the absolute temperature (K), and E 0 is the activation energy (kJ/mol). The slope of the ln(k) vs. 1/T plot can be employed for calculating the E 0 . The pH stability (pH 3.0, 4.0, 5.0, 6.0, and 7.0) of black rice anthocyanins and their derivatives was investigated.Anthocyanin (1 mg/mL) was made at 25 • C and added to test tubes.The absorbance was measured using a spectrometer at 520 nm.The absorbance of anthocyanin was determined each hour for 5 h continuously. The light stability of black rice anthocyanins and their derivatives was assayed.A total of 1 mg/mL of anthocyanin was made and divided into two parts.One part is sheltered from light, and one part is exposed to ultraviolet light (UV intensity ≥ 90 uw/cm 2 ).After 0, 1, 2, 3, 4, and 5 h, a spectrophotometer was used to determine the change in color intensity by measuring the absorbance at 520 nm, respectively. Statistical Analysis Each experiment was repeated three times.The mean ± standard deviation (SD) was used to express the results.SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used for one-way analysis of variance (ANOVA).The graphs were created in Origin 8.5 (Origin Institute Inc., Victoria, TX, USA).p < 0.05 was considered statistically significant. UV-Vis Analysis The effects of caffeic acid grafting on the molecular structure of black rice anthocyanins were investigated using UV-vis spectroscopy, and the results are shown in Figure 1.Two absorption bands at 280 nm and in the range of 500-550 nm were ascribed to the char-Foods 2023, 12, 4505 5 of 13 acteristic structure of anthocyanins.The acylation caused slight shifts, which caused the absorption maximum of anthocyanins to move from 280 nm to 289 nm.In addition, the absorption band in the range of 300-350 nm was attributed to the acylated structure of anthocyanin.The increased peak intensity of the acylated derivatives at 319 nm indicates that the acylation degree increased.Notably, the absorption peak (A319) ratio at this wavelength to the maximum wave intensity in the visible light region (A Vis-max ) could characterize the degree of acylation of the anthocyanins [23].The A 319 /A Vis-max value of black rice anthocyanin (0.1397) increased to 1.63, 6.37, and 2.67, respectively, after grafting with caffeic acid.These results confirmed that caffeic acid was successfully grafted onto the molecules of black rice anthocyanin through the acylation reaction. UV-Vis Analysis The effects of caffeic acid grafting on the molecular structure of black rice anthocyanins were investigated using UV-vis spectroscopy, and the results are shown in Figure 1.Two absorption bands at 280 nm and in the range of 500-550 nm were ascribed to the characteristic structure of anthocyanins.The acylation caused slight shifts, which caused the absorption maximum of anthocyanins to move from 280 nm to 289 nm.In addition, the absorption band in the range of 300-350 nm was attributed to the acylated structure of anthocyanin.The increased peak intensity of the acylated derivatives at 319 nm indicates that the acylation degree increased.Notably, the absorption peak (A319) ratio at this wavelength to the maximum wave intensity in the visible light region (AVis-max) could characterize the degree of acylation of the anthocyanins [23].The A319/AVis-max value of black rice anthocyanin (0.1397) increased to 1.63, 6.37, and 2.67, respectively, after grafting with caffeic acid.These results confirmed that caffeic acid was successfully grafted onto the molecules of black rice anthocyanin through the acylation reaction. FTIR Analysis The FTIR spectra of black rice anthocyanins and their derivatives are illustrated in Figure 2. The glycosides in the anthocyanins and numerous -OH stretching vibrations on the aromatic ring were the causes of the high absorption of black rice anthocyanins at 3226.81 cm −1 .The caffeic acid was grafted on the black rice anthocyanins, and the hydroxyl groups in the structure of acylated derivatives increased, resulting in a wider and deeper waveform [24].The absorption peak of the acylated derivatives shifted from 3226.81 cm −1 to 3219 cm −1 , the wave seal became wider, and the wave peak increased compared with black rice anthocyanins.The peak located at 2929.82 cm −1 was attributed to -CH3, -CH2, and -CH [13].The C=C vibration of the benzene skeleton in anthocyanins and caffeic acid was responsible for the characteristic peak at 1522 cm −1 .The absorption band at 1300-1000 cm −1 was due to the stretching vibration of C-O, and slight blue shifts were observed at the absorption peaks of 1278.10 cm −1 , 1188.90 cm −1 , and 1119.00 cm −1 after acylation with caffeic acid, which might be attributed to the C-O-C stretching vibration in the sugar ring. FTIR Analysis The FTIR spectra of black rice anthocyanins and their derivatives are illustrated in Figure 2. The glycosides in the anthocyanins and numerous -OH stretching vibrations on the aromatic ring were the causes of the high absorption of black rice anthocyanins at 3226.81 cm −1 .The caffeic acid was grafted on the black rice anthocyanins, and the hydroxyl groups in the structure of acylated derivatives increased, resulting in a wider and deeper waveform [24].The absorption peak of the acylated derivatives shifted from 3226.81 cm −1 to 3219 cm −1 , the wave seal became wider, and the wave peak increased compared with black rice anthocyanins.The peak located at 2929.82 cm −1 was attributed to -CH 3 , -CH 2 , and -CH [13].The C=C vibration of the benzene skeleton in anthocyanins and caffeic acid was responsible for the characteristic peak at 1522 cm −1 .The absorption band at 1300-1000 cm −1 was due to the stretching vibration of C-O, and slight blue shifts were observed at the absorption peaks of 1278.10 cm −1 , 1188.90 cm −1 , and 1119.00 cm −1 after acylation with caffeic acid, which might be attributed to the C-O-C stretching vibration in the sugar ring.The above analysis results suggested that anthocyanins could maintain their structural stability during the acylation reaction.The intensity of the characteristic peaks at 1641 cm −1 and 1616 cm −1 gradually increased with acylation.Evidently, the caffeic acids grafted onto black rice anthocyanins through the acylation reaction introduced abundant C=O groups in anthocyanins, therefore increasing the peak intensity [25].Meanwhile, the intensity of the absorption peak at 892.40 cm −1 continued to increase, which could be the bending vibration of H-C=C in caffeic acid.This might have happened because the caffeic acid reacted with -OH of glycosyl in anthocyanin, generating C=C-COOAr (Ar refers to the aromatic nucleus).After the acylation reaction, the many hydroxyl groups and phenyl groups were introduced onto black rice anthocyanins, and the acyl aromatic ring underwent sandwich-type stacking on the planar pyrylium ring, which enhanced the Foods 2023, 12, 4505 6 of 13 stability of anthocyanins [26].These results confirmed that caffeic acid was authentically grafted onto the black rice anthocyanins. in anthocyanins, therefore increasing the peak intensity [25].Meanwhile, the intensity of the absorption peak at 892.40 cm −1 continued to increase, which could be the bending vibration of H-C=C in caffeic acid.This might have happened because the caffeic acid reacted with -OH of glycosyl in anthocyanin, generating C=C-COOAr (Ar refers to the aromatic nucleus).After the acylation reaction, the many hydroxyl groups and phenyl groups were introduced onto black rice anthocyanins, and the acyl aromatic ring underwent sandwich-type stacking on the planar pyrylium ring, which enhanced the stability of anthocyanins [26].These results confirmed that caffeic acid was authentically grafted onto the black rice anthocyanins. TGA Thermal Stability Thermodynamic analysis can provide a deeper insight into anthocyanin thermal stability.Figure 3 demonstrates the TGA (A) and DTG (B) curves of black rice anthocyanins and their derivatives.The anthocyanin degradation could be divided into three phases according to the TGA and DTG curves.The first decomposition stage for black rice anthocyanins and their derivatives appeared at 100 °C and 125-200 °C, respectively, due to the presence of a small amount of absorbed and bound water [27].The loss of black rice anthocyanins and their derivatives samples in the second stage was significant (p ≤ 0.05).The maximum weight loss occurred at approximately 205 °C for anthocyanins and 230 °C or 280 °C for the acylated derivatives, showing compound decomposition of anthocyanins [28].In the third stage, the weight loss for anthocyanins occurred at approximately 300 °C, which was insignificant for the acylated derivatives.The acylated derivatives showed stronger thermal stability than black rice anthocyanins at higher temperatures, indicating that the acylation reaction had a protective effect on the structure of anthocyanins.Notably, compared to black rice anthocyanins, the degradation of the acylated derivatives for AN/CA 2:1, AN/CA 1:1, and AN/CA 1:2 migrated into higher regions, but the thermal weight loss value in the first stage reached the maximum for AN/CA 1:2.It was reported that the thermal decomposition temperature of caffeic acid was between 153 and 241.2 °C.This may be due to the thermal degradation of excess caffeic acid in the AN/CA1:2. TGA Thermal Stability Thermodynamic analysis can provide a deeper insight into anthocyanin thermal stability.Figure 3 demonstrates the TGA (A) and DTG (B) curves of black rice anthocyanins and their derivatives.The anthocyanin degradation could be divided into three phases according to the TGA and DTG curves.The first decomposition stage for black rice anthocyanins and their derivatives appeared at 100 • C and 125-200 • C, respectively, due to the presence of a small amount of absorbed and bound water [27].The loss of black rice anthocyanins and their derivatives samples in the second stage was significant (p ≤ 0.05).The maximum weight loss occurred at approximately 205 • C for anthocyanins and 230 • C or 280 • C for the acylated derivatives, showing compound decomposition of anthocyanins [28].In the third stage, the weight loss for anthocyanins occurred at approximately 300 • C, which was insignificant for the acylated derivatives.The acylated derivatives showed stronger thermal stability than black rice anthocyanins at higher temperatures, indicating that the acylation reaction had a protective effect on the structure of anthocyanins.Notably, compared to black rice anthocyanins, the degradation of the acylated derivatives for AN/CA 2:1, AN/CA 1:1, and AN/CA 1:2 migrated into higher regions, but the thermal weight loss value in the first stage reached the maximum for AN/CA 1:2.It was reported that the thermal decomposition temperature of caffeic acid was between 153 and 241.2 • C.This may be due to the thermal degradation of excess caffeic acid in the AN/CA1:2. UHPLC-HR-MS/MS Analysis Figure 4 shows the UHPLC-MS spectra of anthocyanins before and after the acylation reaction.The structure of the acylation reaction products was studied in the positive ion mode.This is combined with previous research in the laboratory, which found that anthocyanins were detected at a retention time of 5.27 min, where the daughter ion at m/z UHPLC-HR-MS/MS Analysis Figure 4 shows the UHPLC-MS spectra of anthocyanins before and after the acylation reaction.The structure of the acylation reaction products was studied in the positive ion mode.This is combined with previous research in the laboratory, which found that anthocyanins were detected at a retention time of 5.27 min, where the daughter ion at m/z 287 in the MS 2+ spectra originated from the parent ion at m/z 449, which was a typical ion peak for anthocyanins.The base peak at m/z 449 represented the cyanidin-3glucoside [29].In MS + mass spectrometry, a new molecular ion signal peak of m/z 611 at retention time 21.82 min, which happened to be the total molecular weights of caffeic acid and anthocyanins, combining with the UV and FTIR data indicated that the reaction with caffeic acid resulted in the formation of an acylation product.Notably, according to the MS 2+ of AN-CA, the m/z of the other two ion signal peaks were 287 and 611, respectively.The observation of fragment ions in the same position as the AN parent ion suggested that the acylation was regioselective for the glycosidic portion and occurred preferentially on the hydroxyl group in the glycoside [23]. UHPLC-HR-MS/MS Analysis Figure 4 shows the UHPLC-MS spectra of anthocyanins before and after the acylation reaction.The structure of the acylation reaction products was studied in the positive ion mode.This is combined with previous research in the laboratory, which found that anthocyanins were detected at a retention time of 5.27 min, where the daughter ion at m/z 287 in the MS 2+ spectra originated from the parent ion at m/z 449, which was a typical ion peak for anthocyanins.The base peak at m/z 449 represented the cyanidin-3-glucoside [29].In MS + mass spectrometry, a new molecular ion signal peak of m/z 611 at retention time 21.82 min, which happened to be the total molecular weights of caffeic acid and anthocyanins, combining with the UV and FTIR data indicated that the reaction with caffeic acid resulted in the formation of an acylation product.Notably, according to the MS 2+ of AN-CA, the m/z of the other two ion signal peaks were 287 and 611, respectively.The observation of fragment ions in the same position as the AN parent ion suggested that the acylation was regioselective for the glycosidic portion and occurred preferentially on the hydroxyl group in the glycoside [23]. Determination of the Lipophilicity The results of the octanol/water partition coefficient (logP) of black rice anthocyanins and their derivatives are shown in Figure 5.The logP of black rice anthocyanins significantly increased from negative to positive after acylation with caffeic acid, indicating a characteristic shift from hydrophilicity to lipophilicity.After acylation, the logP of anthocyanins increased, presumably due to the acylation reaction resulting in acylation products, which enhanced the lipophilicity of anthocyanins.Paulina et al. [30] found that acylation of anthocyanins reduced the fluidity of the membrane hydrophobic and significantly increased the affinity of the membrane, so the above phenomenon also broadened the ideas for the study of improving the bioavailability of black rice anthocyanins.cantly increased from negative to positive after acylation with caffeic acid, indicating a characteristic shift from hydrophilicity to lipophilicity.After acylation, the logP of anthocyanins increased, presumably due to the acylation reaction resulting in acylation products, which enhanced the lipophilicity of anthocyanins.Paulina et al. [30] found that acylation of anthocyanins reduced the fluidity of the membrane hydrophobic and significantly increased the affinity of the membrane, so the above phenomenon also broadened the ideas for the study of improving the bioavailability of black rice anthocyanins. Antioxidant Capacity The antioxidant capacity of black rice anthocyanins and their derivatives is shown in Figure 6.The acylated derivatives (AN/CA 2:1, AN/CA 1:1, and AN/CA 1:2) exhibited good antioxidation activity in DPPH assays, the DPPH clearances of 28.20%, 28.10%, and 30.35%, respectively.Nevertheless, the acylated derivatives were slightly inferior to black rice anthocyanins in the ABTS radical scavenging assay.This indicated that the DPPH radical had good lipid solubility and stability in the organic solvent ethanol, the poorer water solubility of the ABTS radical, and the acylation reaction enhanced the liposolubility of anthocyanin, therefore increasing its antioxidant capacity in organic solvents [31].Additionally, the presence of many hydroxyl groups in caffeic acid gave it good antioxidant activity, and the DPPH clearance was slightly better after grafting the caffeic acid onto anthocyanins [13].The FRAP approach yielded a total antioxidant capacity for the acylated derivatives that was twice that of black rice anthocyanins, indicating that acylation enhanced the antioxidant capacity of black rice anthocyanins. Antioxidant Capacity The antioxidant capacity of black rice anthocyanins and their derivatives is shown in Figure 6.The acylated derivatives (AN/CA 2:1, AN/CA 1:1, and AN/CA 1:2) exhibited good antioxidation activity in DPPH assays, the DPPH clearances of 28.20%, 28.10%, and 30.35%, respectively.Nevertheless, the acylated derivatives were slightly inferior to black rice anthocyanins in the ABTS radical scavenging assay.This indicated that the DPPH radical had good lipid solubility and stability in the organic solvent ethanol, the poorer water solubility of the ABTS radical, and the acylation reaction enhanced the liposolubility of anthocyanin, therefore increasing its antioxidant capacity in organic solvents [31].Additionally, the presence of many hydroxyl groups in caffeic acid gave it good antioxidant activity, and the DPPH clearance was slightly better after grafting the caffeic acid onto anthocyanins [13].The FRAP approach yielded a total antioxidant capacity for the acylated derivatives that was twice that of black rice anthocyanins, indicating that acylation enhanced the antioxidant capacity of black rice anthocyanins.The influence of temperature from 50 °C to 90 °C on the stability of black rice anthocyanins and their derivatives, respectively, was investigated.Figure 7 displayed the Stability Assays 3.7.1. Thermal Stability The influence of temperature from 50 • C to 90 • C on the stability of black rice anthocyanins and their derivatives, respectively, was investigated.Figure 7 displayed the change in ln(C/C 0 ) with respect to heating time.The results were consistent with the previous studies reporting that the degradation of black rice anthocyanins and their derivatives followed a first-order reaction model.Table 1.Thermal degradation kinetics data of black rice anthocyanin and their derivatives.Values with different letters in the same graph differ significantly (p< 0.05).The stability of black rice anthocyanins and their derivatives was assessed at different pH ranging from 3.0 to 7.0.Figure 8 describes the degradation of black rice anthocyanins and their derivatives under various pH with kinetic parameters shown in Table 2.As shown in Figure 8 and Table 2, the black rice anthocyanins and their derivatives had higher k values and lower t1/2 values at high-pH treatments, implying lower stability of the sample Table 1 summarizes the kinetic parameters.As expected, the k value increased, and t 1/2 decreased gradually with increased heating temperature, implying that heat treatment promoted the degradation of anthocyanins.The anthocyanin stability could be enhanced after being acylated through self-association reactions and intramolecular and intermolecular co-pigmentation [32].This study showed that the acyl donors were arranged on one side of the pyran ring after the acylation reaction occurred, preventing nucleophilic attack by water and allowing only weak intermolecular interactions, protecting the molecular structure of the acylated anthocyanins [23,33].Notably, the k values decreased gradually, and the E 0 values improved as the caffeic acid content increased.This suggested that higher caffeic acid content led to a greater energy barrier required during degradation.The improvement of anthocyanin stability in samples (AN/CA 2:1 and AN/CA 1:1) by acylation was mainly attributed to the reduction of acyl group polarity and site resistance, which protected the chromophore from nucleophilic attack by water [34].Combined with the previous acylation degree determination results, it was speculated that in addition to the above reasons for the increased stability of sample AN/CA 1:2, co-pigmentation of acylated anthocyanins with excess caffeic acid in the reaction system effect also prevented the thermal degradation of the acylated anthocyanins [35].The stability of black rice anthocyanins and their derivatives was assessed at different pH ranging from 3.0 to 7.0.Figure 8 describes the degradation of black rice anthocyanins and their derivatives under various pH with kinetic parameters shown in Table 2.As shown in Figure 8 and Table 2, the black rice anthocyanins and their derivatives had higher k values and lower t 1/2 values at high-pH treatments, implying lower stability of the sample at higher pH values.This was attributed to the fact that the electron-deficient form of anthocyanins was affected by the charge when the environmental pH changed, and the chemical structure changed accordingly.These results were consistent with the previous study results [16].All acylated anthocyanins had greater pH stability than black rice anthocyanins.This indicated that the pyran ring of anthocyanins reduced the susceptibility to hydrophilic group attack because of acyl stacking, preventing the formation of achromatic pseudobases and chalcone structures from black rice anthocyanin [36].Meanwhile, k of the acylated derivatives slightly increased with increased content of caffeic acid and remained lower than that of black rice anthocyanins.This result confirmed that acylation improved the pH stability of anthocyanin.study results [16].All acylated anthocyanins had greater pH stability than black rice anthocyanins.This indicated that the pyran ring of anthocyanins reduced the susceptibility to hydrophilic group attack because of acyl stacking, preventing the formation of achromatic pseudobases and chalcone structures from black rice anthocyanin [36].Meanwhile, k of the acylated derivatives slightly increased with increased content of caffeic acid and remained lower than that of black rice anthocyanins.This result confirmed that acylation improved the pH stability of anthocyanin.As shown in Figure 9 and Table 3, the rate of degradation of the anthocyanins in black rice was higher than that of the acylated derivatives when exposed to UV radiation.Acylated derivatives may be attributed to the changes in the structural conformation and the presence of intramolecular and/or intermolecular stacking (such as acylation, selfassociation, and co-pigmentation) of anthocyanins, as well as the stable acryl ring in caffeic acid structure, which protected anthocyanins from degradation factors [19,37].Additionally, the increasing caffeic acid content also increased the light stability of the acylated derivatives.This may be the result of the additional caffeic acid providing intermolecular co-pigmentation, therefore increasing the stability of black rice anthocyanins. Conclusions The stability of black rice anthocyanins has an important implication for their application as a natural pigment or functional ingredient in food.Therefore, in this study, the Conclusions The stability of black rice anthocyanins has an important implication for their application as a natural pigment or functional ingredient in food.Therefore, in this study, the black rice anthocyanins were acylated with caffeic acid to address their poor stability.The analysis of UV, FTIR, and UHPLC-HR-MS/MS showed that the acylation reaction took place at the hydroxyl site of glucoside, forming an acylated derivative of anthocyanins.Acylation with caffeic acid enhanced the lipophilicity of black rice anthocyanins.The antioxidant activity of the acylated derivatives in the hydrophilic systems (ABTS assays) decreased, but the clearance ratio in DPPH and the total antioxidant capacity assay increased.Compared with black rice anthocyanins, the degradation kinetic parameters of the acylated derivatives showed a trend of lower degradation rate k and longer degradation half-life t 1/2 .The stability (temperature, pH, and light) of the acylated derivatives increased.This study establishes an acylation route for the synthesis of highly stable anthocyanin-based pigments, further broadening the application of black rice anthocyanins in functional foods or as novel functional colorants. Figure 2 . Figure 2. FTIR spectra of black rice anthocyanins and their derivatives. Figure 2 . Figure 2. FTIR spectra of black rice anthocyanins and their derivatives. Figure 3 . Figure 3. TGA (A) and DTG (B) curves of black rice anthocyanin and their derivatives. Figure 3 . Figure 3. TGA (A) and DTG (B) curves of black rice anthocyanin and their derivatives. Figure 3 . Figure 3. TGA (A) and DTG (B) curves of black rice anthocyanin and their derivatives. Figure 4 . Figure 4.The UHPLC-MS spectra of black rice anthocyanins and their derivatives.Figure 4. The UHPLC-MS spectra of black rice anthocyanins and their derivatives. Figure 4 . Figure 4.The UHPLC-MS spectra of black rice anthocyanins and their derivatives.Figure 4. The UHPLC-MS spectra of black rice anthocyanins and their derivatives. Figure 5 . Figure 5. Determination results of the lipophilicity for black rice anthocyanins and their derivatives.Values with different letters in the same graph differ significantly (p< 0.05). Figure 5 . Figure 5. Determination results of the lipophilicity for black rice anthocyanins and their derivatives.Values with different letters in the same graph differ significantly (p < 0.05). Figure 6 . Figure 6.The DPPH, ABTS, and FRAP for black rice anthocyanins and their derivatives.Values with different letters in the same graph differ significantly (p < 0.05). Figure 8 .Table 2 .Figure 8 . Figure 8. Degradation of black rice anthocyanins and their derivatives treated under conditions of different pH (■ pH 3, • pH 4, ▲ pH 5, ▼ pH 6, ◆ pH 7).Table 2. Degradation kinetics data of black rice anthocyanins and their derivatives under conditions of different pH.Values with different letters in the same graph differ significantly (p< 0.05). Figure 9 . Figure 9. Degradation of black rice anthocyanins and their derivatives under UV light. Table 1 . Thermal degradation kinetics data of black rice anthocyanin and their derivatives.Values with different letters in the same graph differ significantly (p < 0.05). Table 2 . Degradation kinetics data of black rice anthocyanins and their derivatives under conditions of different pH.Values with different letters in the same graph differ significantly (p < 0.05).	2023-12-21T16:33:44.516Z	2023-12-01T00:00:00.000	{ "year": 2023, "sha1": "7787d0b2cd6b0d11d84c1c6df10b48844874b1fe", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2304-8158/12/24/4505/pdf?version=1702803535", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "d470d6c8b38e005ff8d31ac6182e15da7d49704e", "s2fieldsofstudy": [ "Chemistry", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
231934766	pes2o/s2orc	v3-fos-license	Should treatment of low-level rifampicin mono-resistant tuberculosis be different? Background Rifampicin resistant tuberculosis (RR-TB) was frequently detected in Suriname after the introduction of Xpert MTB/RIF in 2012. Subsequent phenotypic drug-susceptibility testing (DST) was not conclusive at that moment, while RR-TB patients treated with first-line tuberculostatics had good treatment outcome. In our study, we analysed this interesting observation. Methods We collected demographic and clinical characteristics and treatment outcome of TB patients from May 2012-December 2018 and performed a univariate and multivariate analysis to assess possible associations with resistance to rifampicin. Secondly, we conducted whole genome sequencing on all available Mycobacterium tuberculosis isolates that had a rifampicin resistance in the Xpert MTB/RIF test and performed phenotypic DST on selected isolates. Findings RR-TB was detected in 59 (9.6%) patients confirmed by Xpert. These patients were treated with rifampicin-containing regimens in most (88%) of the cases. In all 32 samples examined, a D435Y mutation in the rpoB gene was identified; only one isolate revealed an additional isoniazid mutation. Phenotypic DST indicated low-level rifampicin resistance. In multivariate analysis, the Creole ethnicity was a factor associated with rifampicin resistance (aOR 3.5; 95%CI 1.9–6.4). The treatment success rate for patients with RR-TB (78.0%) was comparable to the treatment outcome in non-RR-TB patients 77.8%. Interpretation This study confirms a low-level rifampicin mono-resistance in TB patients of Suriname. These patients could benefit from a first-line regimen with high dose rifampicin (or rifabutin), rather than from the lengthy treatment regimens for rifampicin-resistant and multi-drug resistant TB, a concept of stratified medicine also advocated for the treatment of TB. Funding None. Introduction Globally, an estimated 10 million people developed tuberculosis (TB) in 2018; half a million of them had rifampicin-resistant (RR) TB [1], of which 78% had also confirmed resistance to isoniazid (INH), and were hence classified as multidrug-resistant (MDR) TB. The last decade brought large improvements in the diagnosis and treatment of RR/MDR-TB. [2] For instance, rifampicin resistance can now be diagnosed with rapid molecular tests and new effective secondline drugs as bedaquiline and linezolid, are now included in injection-free treatment regimens recommended by the World Health Organization (WHO). [3] The new diagnostic tests and drugs have become available to low and middle-income countries at special pricing schemes e.g. through support of the Global Fund to fight AIDS, TB and Malaria. Despite these advances, globally only 51% of patients with confirmed TB were tested for rifampicin resistance in 2018. Moreover, only 33% of all estimated RR/MDR-TB patients were enrolled in treatment, with a treatment success rate of 56%. [1] Suriname is a country with a multi-ethnic population of 575,991 in 2018 (https://data.worldbank.org/country/Suriname) consisting of Hindustani (27%), Maroon (22%), Creole (16%), Javanese (14%), mixed (13%) or people with other ethnic background (8%) (https://statistics-suriname.org/nl/censusstatistieken-2012-2/). The TB notification rate was 30 per 100,000 population in 2018; WHO estimated the TB incidence at 38 per 100,000. [1] Before 2012, drug susceptibility testing (DST) was not routinely available in Suriname. Samples were sent abroad, and only two MDR-TB patients were diagnosed from 2005 till 2011. In May 2012, the Xpert® MTB/RIF test (Cepheid, Sunnyvale, CA, USA) was introduced in Suriname. The unexpected high proportion of rifampicin-resistant Xpert results has puzzled national and international TB experts for several years. Phenotypic drug susceptibility testing (DST) performed by one international laboratory in 2012 showed susceptibility to rifampicin, while re-analysis by the supranational reference laboratory several years later showed rifampicin resistance in these strains. [4] In 2018, 12.4% of confirmed cases in Suriname had RR-TB, which is the highest proportion in the WHO Region of the Americas. [1] This study aims to identify characteristics of the RR-TB patients in Suriname, understand the resistance pattern found, search for similar strains in the Netherlands, a country with strong historical ties to Suriname, and identify possible implications for treatment recommendations. Patients In Suriname, TB patients are mandatory reported to the National Tuberculosis Program (NTP), which maintains an electronic national TB register. In our study, we included TB patients who were notified between May 2012 and December 2018. Patients were treated according to National Treatment Guidelines, which had been based on the 2010 Caribbean TB guidelines [5], with a standard regimen of isoniazid, rifampicin, ethambutol and pyrazinamide. Streptomycin and/or ciprofloxacin were added to the regimen in some patients who had previous TB treatment or RR-TB. Patients treated with a regimen without rifampicin were treated for 12-18 months. Patients, including those with RR-TB, were not actively followed up after treatment completion. The following data were obtained from the register: sex, age, ethnicity, site of disease, smear results, Xpert results, drug-resistance, case type (new or previously treated), current TB treatment regimen, co-morbidities (HIV, diabetes mellitus), illicit drug use (marihuana or hard drugs) and treatment outcome. Treatment outcomes (including RR-TB) were based on the definitions and reporting framework for drugsensitive TB [6], e.g. failure was defined if the sputum was microscopically or culture positive after 5 months treatment and cure if the smear was microscopically negative after 6 months treatment. A positive smear was graded as previously described. [7] We compared demographic and clinical characteristics of Xpertconfirmed rifampicin-sensitive and rifampicin-resistant TB patients. Data were analysed with SPSS version 24 (SPSS, Inc., Chicago, IL, USA), including a univariate analysis to estimate the Odds Ratio (OR) and 95% Confidence Intervals (95%CI) and a multivariate regression analysis including those variables that attained a P value < 0.25 in the univariate analysis. Mycobacterium tuberculosis isolates Suriname In Suriname, samples of all TB patients are sent to the Central Laboratory, which accommodates the national TB reference laboratory, and performs culture (Löwenstein Jensen) and strain identification (SD BIOLINE TB Ag MPT64). Cultured M. tuberculosis isolates are stored at minus 80 • Celsius. In 2018, 32 stored Xpert rifampicin-resistant Mycobacterium tuberculosis isolates were re-cultured and sent to the National Tuberculosis Reference Laboratory of the National Institute for Public Health and the Environment (RIVM) in the Netherlands for further analysis. These isolates were from patients diagnosed in 2012 (n = 4), 2013 (n = 4), 2014 (n = 10), 2015 (n = 8), 2017 (n = 3) and 2018 (the first three RR-TB patients in that year). Rifampicin-resistant M. tuberculosis isolates from 2016 were not available for analysis. At the RIVM, whole genome sequencing (WGS) was applied to detect mutations associated with resistance and to determine the genetic distance between the isolates, as a proxy for epidemiological links between cases. Additionally, reverse line blot assays (GenoType MTBDRplus® Hain Lifesciences, Nehren, Germany) and phenotypic DST were performed on selected isolates. Similar isolates in the Netherlands We searched the Dutch TB laboratory database at the RIVM for isolates with the same mutation found in Suriname, and obtained data from the Dutch National TB Register of patients infected with M. tuberculosis isolates with this mutation. Isolates that were possibly identical with the strain found in Suriname were subjected to WGS. Ethical consideration Ethical approval for this study was obtained from the Human Scientific Research Ethic Committee of the Ministry of Health of Suriname. The Dutch National TB Registration Committee approved the use of the data from the Netherlands for this study. Patients Between May 2012 and December 2018, a total of 978 TB patients were notified to the NTP in Suriname. The male-female ratio was 2.5 (696 male patients; 71%) with a median age of 42 years (interquartile range [IQR] 29-53). Patients' ethnicity was mostly Creole (32%), followed by Maroon (16%), Hindustani (15%) or mixed ethnicity (16%). Eighty-six percent (n = 838) of the patients had pulmonary TB; 599 had a smear positive sputum with microscopy (71% of pulmonary TB patients). Xpert was positive in 64% (n = 623) of the patients; whilst negative or not done in the remaining 36% (n = 355) of patients. Rifampicin resistance was determined in 59 (9.5%) of Xpert-positive patients ( Table 1). Nine percent of patients (n = 91) were previously treated for TB. HIV status was known in 939 patients (96%) and HIV coinfection was present in 23% (n = 218) of tested patients. Diabetes mellitus was prevalent in 13% of the patients (n = 123) and illicit drug use was reported in 24% (n = 239) of the patients. In univariate analysis, RR-TB patients were significantly more often in the age group 45-60 years (reference 15-24 years), had the Creole ethnicity, were previously treated for TB, had HIV co-infection and had documented illicit drug use, compared to patients with rifampicinsensitive TB. Pulmonary RR-TB patients had more frequently microscopy-positive sputum smears (97%; 56/58) than pulmonary rifampicin-sensitive-TB patients (88%; 480/548) (p = 0.07). In multivariate analysis, patients with RR-TB were significantly more often of Creole ethnicity (aOR 3.5) ( Table 2). HIV and illicit drug use were confounding factors related to Creole ethnicity: 32% (64/198) of all Xpert-positive Creole TB patients were HIV co-infected and 42% (84/ 198) had documented illicit drug use versus respectively 12% and 23% of patients with other ethnicities. Treatment outcome was successful (cured/completed) in 74.6% of all TB patients, in 77.8% of patients with Xpert rifampicin-sensitive TB and in 78.0% of patients with Xpert diagnosed RR-TB (Table 3). Secondline drugs (ciprofloxacin (n = 15), streptomycin (n = 2), or both ciprofloxacin and streptomycin (n = 20)) were added to the standard firstline treatment regimen in 37 patients; 21 of these patients had rifampicin-sensitive TB (4% of all rifampicin-sensitive TB patients) and 16 had RR-TB (27% of all RR-TB patients). Fifty-two (88%) RR-TB patients were treated with rifampicin-containing first-line regimens (11 out 52 were also treated ciprofloxacin and/or streptomycin), five (8%) patients were treated with the first-line drug regimen without rifampicin but to which was added ciprofloxacin and/or streptomycin, and two (3%) patients did not start treatment. In total, 12 RR-TB patients were previously treated for TB (one person was treated for TB two times and another person three times) ( Table 4). Three of these patients restarted treatment after discontinuing the treatment in the first episode (two had RR-TB in the first episode, the other one had a diagnosis before 2012) and nine completed treatment in the first episode (two diagnosed with RR-TB before, one with rifampicinsensitive TB, five had a TB diagnosis before 2012 with an unknown resistance pattern, and in one patient DST was not done in the previous episode). Whole genome sequencing (WGS) and additional laboratory tests WGS revealed a D435Y mutation in the rpoB gene in all 32 rifampicin-resistant M. tuberculosis isolates. One isolate had an additional resistance mutation in the inhA gene S94A, but not in the more common inhA promoter gene region, and by definition labelled as MDR. None of the remaining 31 isolates had additional first-line drug resistance-associated mutations in the katG, inhA, fabG1, ahpC, embA, embB, pncA or rpsA genes. The 32 isolates were all linked together by WGS in one genetic cluster with ≤ 12 single nucleotide polymorphisms (SNPs) difference to each other. The GenoType MTBDRplus analysis of 14 isolates (from 2015 to 2018) showed loss of hybridization of wild type probes 3 and 4, and no hybridization of the known and most frequently reacting mutation probes. Phenotypic DST, done by the Middlebrook 7H10 agar proportion method [8], indicated a MIC (minimal inhibitory concentration) for rifampicin of 1-5 mg/L, known as low level resistance (cut-off is 1 mg/L) and showed susceptibility to rifabutin for the three isolates tested (all patients diagnosed in 2018). These three isolates were also tested in the MGIT and were found susceptible to rifampicin. D435Y isolates and patients in the Netherlands The Dutch national TB laboratory database contained 15 Abbreviations: TB = tuberculosis * The total number of tuberculosis patients in the year 2012 (January-December) was 135. M. tuberculosis isolates with the same D435Y mutation found in Suriname ( Table 5). The isolates were from patients born in Europe (n = 6), Suriname (n = 5), Africa (n = 3) and Asia (n = 1). The isolates from the 10 non-Suriname-born patients were resistant to isoniazid and 8 out of 10 susceptible to rifabutin; patients were treated accordingly ( Table 5). The isolates from the 5 Suriname-born patients were all susceptible to isoniazid, rifampicin-susceptible in two cases (both tested by MGIT-DST) and low-level rifampicin-resistant in three cases (one patient with recurrent TB was counted twice; all these isolates were tested by Middlebrook 7H10). The two patients with rifampicin-sensitive TB completed standard first-line treatment, including standard dose rifampicin. One RR-TB patient was treated for 6 months with a first-line regimen, containing rifabutin instead of rifampicin, to which the strain was susceptible. The patient diagnosed twice with RR-TB first completed 12 months treatment with isoniazid, ethambutol, moxifloxacin and 2 months pyrazinamide, but developed RR-TB again after ten years. This second time, he was treated for 6 months with the first-line drug regimen, with rifabutin, to which the strain was susceptible, replacing rifampicin. Genotypically, all five M. tuberculosis isolates of the Suriname-born patients in the Netherlands clustered in the WGS with those of the 32 patients diagnosed in Suriname. Discussion Our study shows that rifampicin-resistant TB in Suriname is caused by a M. tuberculosis isolate with a D435Y mutation in the rpoB gene, known to be associated with a low-level resistance for rifampicin. [9][10][11][12] Only one patient had also an uncommon mutation coding for isoniazid resistance and hence had MDR-TB; all other RR-TB patients had rifampicin mono-resistant isolates. RR-TB patients were mainly treated with rifampicin-containing regimens. Treatment outcome was similar in patients with rifampicin-sensitive and RR-TB, with a successful treatment completion rate of 78% in both groups. Patients were not actively followed up after treatment completion and only identified with recurrent TB if they presented with symptoms and were diagnosed. Patients with isolates harbouring the D435Y mutation were also identified in the Netherlands, but only the patients born in Suriname had an infection with a rifampicin mono-resistant strain. These patients had been treated either as rifampicin-sensitive TB, or with first-line regimens including rifabutin. Rifampicin resistance in M. tuberculosis is mostly caused by an undisputed mutation in the rpoB gene, most commonly the S450L mutation. The D435Y mutation described in our study is a mutation with disputed drug-resistance and characterised by a substitution of the amino acid asparagine (Asp/D) by tyrosine (Tyr/Y) in codon 516. [13] In the previous classification with E. coli codon numbering, this mutation was known as D516Y or GAC516TAC and renamed with the H37Rv numbering to D435Y. Studies investigating the accuracy of current laboratory tests to identify resistance in D435Y mutation isolates concluded that low level rifampicin resistance is often missed in the single point concentration MGIT-DST, while it is identified by MIC methods like Middlebrook 7H10 and Löwenstein-Jensen and by molecular test such as Xpert and MTBDRplus [9][10][11]14] Other studies concluded that the rifampicin resistance is often borderline or low level. [12,[15][16][17][18][19] Almost all studies concluded that isolates with a D435Y mutation are susceptible to rifabutin. [10,[15][16][17]20] The Suriname NTP had difficulties in the interpretation of the rifampicin resistance detected by the Xpert test. In 2012, six Xpert rifampicin-resistant M. tuberculosis isolates were transported to an international laboratory, where MGIT-DST and 7H10 Middlebrook MIC method revealed susceptibility to rifampicin in all isolates. In 2016, the supranational reference laboratory of Massachusetts re-analysed these isolates using an agar proportion method and found rifampicin resistance in four of the six isolates. Furthermore, it performed PCRmediated direct DNA sequencing of the rpoB gene on these six isolates and on 10 lysates of Xpert rifampicin-resistant M. tuberculosis isolates from 2014, revealing the D435Y mutation in 14 of the 16 samples. [4] WHO's treatment advise on rifampicin mono-resistant TB has changed over time. In the first guidelines on drug-resistant TB, a 12-18 months regimen with isoniazid, ethambutol and a fluoroquinolone, and Abbreviations: TB = tuberculosis 11 RR-TB patients were treated with first-line drugs including standard dose rifampicin, and in addition with ciprofloxacin (n = 4), streptomycin (n = 1) or ciprofloxacin and streptomycin (n = 6); 6 (55%) had a successful treatment outcome (median days treatment: 289; IQR 280-304). Five RR-TB patients were treated with first-line drugs without rifampicin, and in addition with ciprofloxacin (n = 2) or ciprofloxacin and streptomycin (n = 3); 4 (80%) had a successful treatment outcome (median days treatment: 381; IQR 362-447). Two RR-TB patients did not start treatment: one died before treatment and one was not evaluated. * Not evaluated: patients transferred out and patients whose treatment DST = drug susceptibility test; ND = not determined; RR = rifampicin resistance; TB = tuberculosis pyrazinamide for at least two months, was recommended. [21] These guidelines also advised a retreatment regimen with first-line drugs and streptomycin ("Category 2 regimen") for patients with relapse or return after loss to follow-up. In 2011, WHO published an update stating that the detection of rifampicin resistance by Xpert test usually suffices to prescribe second-line TB regimen [22], but also cautioned for situations in which the Xpert test has a low predictive value and advised that results need to be confirmed by phenotypic DST or line probe assay. The update also noted that a potential harm from placing all rifampicinresistant patients on an MDR-TB regimen, was the exclusion of isoniazid from their treatment, thus depriving them of a safe and useful bactericidal drug. [22] A systematic review in 2016 identified only three studies reporting treatment outcomes for patients with RR-TB, preventing a meta-analysis. [23] Since 2016, WHO guidelines on drugresistant TB recommend similar treatment regimens for patients with RR-TB and MDR-TB. [3,24] The guideline for treatment of TB and RR/ MDR-TB in Suriname was updated following these latest recommendations of the WHO, and does not include ciprofloxacin and streptomycin anymore because of limited evidence of their effectiveness and sterilizing activity. The clinical relevance of disputed mutations with low-level rifampicin resistance has been an issue of debate. One study indicated that disputed mutations had the same poor clinical prognosis as the most frequent undisputed mutations. [25] Williamson et al. suggested to conduct a multi-centre retrospective study to correlate the different types of rpoB mutations with clinical outcomes. [18] They stated that the retrospective nature of such a study would actually become a methodological strength, as it would allow data on treatment outcome to be obtained, and thus allow the clinical relevance of mutations to be assessed. [18] We consider our observational study to be such a study, that contains to our knowledge the largest set of patients infected with M. tuberculosis strains carrying the D435Y mutation. Our observation of good treatment results of patients with this peculiar type of low-level rifampicin resistance and susceptibility for isoniazid, suggests that treatment regimens with isoniazid and rifampicin can still be effective. The relatively high number of RR-TB cases with previous TB treatment is a concern and suggests that a sterilizing effect was not achieved. However, one-quarter of these patients did not complete the previous treatment which obviously contributed to relapse, while reinfection is also possible in an environment with ongoing transmission. Several authors have proposed alternative treatment options for patients infected with a low-level rifampicin resistance, e.g. a higher dose of rifampicin or replacing rifampicin by rifabutin. [10,16,18,20,26] The currently recommended dose of rifampicin has been challenged over the last years. [27,28] Dosage studies indicate that 35 mg/kg rifampicin instead of 10 mg/kg is well tolerated and more effective than the standard dose. [29,30] These authors recommend high dose rifampicin for patients with TB meningitis, co-infection with HIV, diabetes mellitus and serious ill TB patients, since plasma concentrations of rifampicin are often too low. Our hypothesis is that patients with lowlevel rifampicin mono-resistant TB could benefit more from a first-line regimen, including isoniazid and high dose rifampicin (or rifabutin), rather than from a lengthy regimen for RR/MDR-TB treatment or the standard TB treatment. We have developed a study to treat these patients accordingly in Suriname and a protocol has been developed and cleared by the Human Scientific Research Ethic Committee, and includes informed consent of the patient for treatment with high-dose rifampicin (30 mg/kg); timely WGS testing of all RR-isolates; measuring of rifampicin blood levels; close supervision of treatment, including monitoring of side effects; case discussion in a Concilium; and a minimal follow-up of one year after treatment. Our study also revealed several other issues, some beyond the scope of this study. All examined isolates of RR-TB patients, both found in Suriname and the Netherlands, belonged by WGS genetically to one large molecular transatlantic cluster. Transmission of the rifampicinresistant strain is likely to be ongoing in Suriname, and possibly also in the Netherlands. Second, the observed TB/HIV co-infection rate (23%) is one of the highest in the WHO Region of the Americas [1]. More TB/HIV collaborative efforts are needed to control TB and move towards TB elimination in Suriname. RR-TB was more often detected in Creole people, which is also the ethnic group with a very high TB incidence, HIV co-infection rates and illicit drug use. Our data show a high TB case fatality of 14%, which has been associated with higher age and HIV coinfection. [31] Our study had several strengths and limitations. We made use of a detailed electronic database and advanced molecular tests were applied on a subset of rifampicin-resistant M. tuberculosis isolates, although not all rifampicin-resistant isolates were available for additional typing. The 100% concordance of rifampicin resistance found by Xpert in Suriname and by WGS done in the Netherlands, confirmed that these patients actually had RR-TB. The major limitation of our study is that RR-TB patients were not actively followed up for recurrent TB after treatment completion. We will address this issue in the planned prospective study. Our analysis did not include rifampicin-susceptible isolates. Recently, we analysed another 118 lysates, including 18 rifampicin-resistant strains of patients diagnosed in 2018-2020. All rifampicin-resistant isolates had the D435Y mutation; none of the other strains carried mutations in the katG or inhA genes or had other first-line drug resistance- associated mutations. The findings of our study provide clarity to the rifampicin resistance situation in Suriname and possible implications for tailored treatment regimens. Our study shows that an epidemic of RR-TB was driven by a mutation of which the clinical significance is disputed. All but one strains examined revealed low-level rifampicin resistance and isoniazid susceptibility. Treatment results with rifampicin-containing regimens were unexpectedly good. Further research is needed to study alternative treatment options for these patients, such as regimens with high dose rifampicin for these specific group of patients, with systematic follow-up after treatment completion. We state that one size does not fit all for the treatment of RR/MDR-TB, i.e. some people need XL (extra-large) and others XS (extra-small) treatment regimens, depending on the causative strain. Technological advances make it increasingly possible to tailor treatment to the patient and their bacteria. Furthermore, collaborative networks of professionals and countries with different resources can work together to provide the necessary scientific evidence for effective XL or XS RR/MDR-TB treatment regimens. In the end, we want to provide each patient the right treatment, not too much and not too little, a concept of stratified medicine also advocated for the treatment of TB. [32] Ethical statement Ethical approval for this study was obtained from the Human Scientific Research Ethic Committee of the Ministry of Health of Suriname. The Dutch National TB Registration Committee approved the use of the data from the Netherlands for this study.	2021-02-17T05:07:51.215Z	2021-01-29T00:00:00.000	{ "year": 2021, "sha1": "c1d6da028c49b3116f63d16d51d7513bbb2c76d7", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.jctube.2021.100222", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "c1d6da028c49b3116f63d16d51d7513bbb2c76d7", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
73252170	pes2o/s2orc	v3-fos-license	Recording health care and sharing the information - more bureaucracy or a welcome challenge to prevailing practice? The NHS Plan (2000) outlines a number of strategies that respond to the consumer agenda and enhance the notion of patient and public involvement in all aspects of healthcare. While there are considerable changes ahead in the ‘ macro’ whole service-related involvement of patients and public ( The NHS Plan (2000) outlines a number of strategies that respond to the consumer agenda and enhance the notion of patient and public involvement in all aspects of healthcare. While there are considerable changes ahead in the 'macro' whole service-related involvement of patients and public (patient/public involvement strategies, health service scrutiny by local authorities and the development of foundation 'hospitals'), there has been a revolution in the expectations of the patient and their carers regarding choice, access to information about treatments and diagnosis, and rights of access to all clinical information. The NHS Plan (2000) outlines the intention for patients to receive copies of all clinician-clinician correspondence by right if they so wish by April 2004. It is not often that a relatively quiet policy initiative brings in its wake a major change in practice for most clinicians. The last time this happened in mental health care was the introduction of the care programme approach in 1990. It is likely that copying letters to patients will rapidly be followed by a number of related policies, responding to consumer rights and expectations. One possible explanation for this is that in this area, clinical practice has lagged well behind the public will. Clinicians are increasingly facing up to the challenge that everything they record on any aspect of patient care is open to external scrutiny at any time. If it is not by the patient or carer, then it is by others, including Mental Health Act Tribunals, solicitors, the Data Protection Commissioner, the Health Service Ombudsman, health service managers and, alas, the coroner. A case is often made that mental health services are special, and that the consumer agenda is driven by other health services. In certain cases this may be so, but there is little comfort for psychiatrists in following this route and every indication that (except in a few exceptional cases) the rights of access, the involvement of patient and public and the notion of full patient choice is a policy expectation in mental health, as in all other health services. Some mental health practitioners routinely share clinical information with their patients, give good-quality information and have rarely had patients using the formal 'access to healthcare records' route. They may be somewhat puzzled by enshrining what is good, sensible and flexible practice into a bureaucratic programme with targets and monitoring. In this edition of the Bulletin, a number of papers address aspects of sharing of information with patients and with others. Currently, the health record is mostly a paper-based repository (the clinical casenote) for all written communications relating to an individual patient. Health care records should provide a sequential and confidential record of the documentary evidence of the management of care. These health records are regularly being scrutinised by the patient and/or carers, and increasing care needs to be taken in what we write, and how we record both direct and third party information. Patients are gaining access to the notes through 'access to healthcare records' as of right, and it is worth noting that a clinicallyled refusal to divulge all or part of the healthcare record is based solely on the notion of 'significant' harm to self or others. It is important that clinicians are able to justify a decision to refuse access, up to and including a full explanation to the Data Protection Commissioner. Clinical supervision of doctors and other staff provides a vehicle for assisting health care practitioners to develop the skills of record-keeping that stand both the direct scrutiny of the patient and others, and also how to make a series of Roy Recording health care and sharing the information Box 1. Doctors taught the art of writing clearer notes -TheTimes, Monday 9 June 2003. The leader column expands -'A century ago the successful Victorian doctor was said to need four things, A top hat to give him Authority. A paunch to give him Dignity. Piles to give him an Anxious expression. And illegible handwriting to impress his patients with the potency of his Prescriptions, Potions and Pills' . The leader misses the point somewhat in suggesting that there is no chance of remedial writing classes succeeding, suggesting that the leader writer's understanding of the challenge to modern doctors lies only in writing legibly. The challenge is increasingly in the clarity, understandibility, and thoughtfulness of what is written. And perhaps this is occasionally a somewhat tall order for a busy and often harassed psychiatrist? clinical entries encapsulating an open clinician-patient relationship that is at once both respectful and therapeutic. Unfortunately, personal embarrassment because of tone and content of what we (or others) have written in the past is not a valid reason for refusing access. A good set of case notes (or dare one say an electronic health record) should provide easy access to the up-to-date plan of care (including current medication); a full account of the patient's history, including contacts with health (and where fully integrated, social services) and treatments; any relevant risk information that should influence clinical decision making; contact information for all health and social care professionals; and other key contacts such as housing, schooling, family (nearest relative and next of kin) as a minimum. It should also be constructed and presented in such a way that the patient can relate to the entries, and in many cases benefit therapeutically from the experience of reading (and correcting) them. Copying letters to patients (including patient held records) This new NHS-wide initiative poses a significant administrative and practical challenge ahead for the NHS (what's new?). In addition to the copying of letters, patients have to be asked whether they wish to receive the letters at all, and where they would like the letters to be sent. This provides an added sting to the proposal (asking patients whether they would like to receive the copy of the communication at their very next appointment seems a flexible and less costly response to the challenge?) Nandhra et al (2004, this issue) instituted the programme of copying letters to 73 out-patients, and received feedback from them. The results are encouraging. Although only 40 of the 73 patients responded, 33 (83%) valued receiving the letters. The authors make some pertinent comments about the guidance that may be useful for mental health services. They suggest that the process of writing to patients was generally wellreceived by psychiatrists as well, although two psychiatrists in the sample did have particular concerns about the possible distressing impact of the information on their patients. One patient's letter was sent to the wrong address, and this highlights an area of potential risk for services when the practice is introduced widely this year. Lloyd (2004, this issue) reports on a survey of patients attending a liaison psychiatry clinic after copying the correspondence to the referring specialist and general practitioner. A relatively large number responded to the postal survey (52%) and those who responded were overwhelmingly in favour of receiving these letters. He goes on to point out that the copying of letters is in keeping with the greater openness that is being adopted in modern clinical practice. He outlines the need for letters to be written in a particularly clear, jargon-free style, and also the challenge of respecting the patient's wishes in what information is included or not. He suggests that most doctors would support the copying of letters to patients, but that more evaluation is required, adding that perhaps particular problems may be experienced where patients do not share the professionals' views of their problems (community psychiatry, forensic psychiatry) and for older adults services. A number of reports from the pilot sites are now on the Department of Health website (www.doh.gov.uk/patientletters), but there is nothing to suggest that particular services will be exempt. Perhaps Dr Lloyd is being too cautious on behalf of community, forensic and old age psychiatry, and that copying letters to patients even in the most challenging of situations could actually enhance the therapeutic relationship where it has been quite tricky. Child and adolescent mental health services pose a challenge, but probably not an insurmountable one. Even more challenging, more engaging, and providing a solution to notes being lost, is developing patient held records in mental health. Laugharne & Henderson (2004, this issue) provide an overview following a systematic literature search. They describe a number of interesting studies, particularly in community mental health, with patients suffering from psychotic illnesses. The number of patients with psychotic illness using the patient-held record was surprisingly high. However, they do comment that anecdotally the patients who chose to hold their own records were generally easier to engage and collaborated with services. The paper concludes that as yet, there is no research evidence that would support widespread use of patient-held records. However, they make the point that there is more work to be done in understanding why some patients offered them choose not to use them, and looking perhaps more radically at the patient group who do choose them, specifically measuring patient empowerment and patient-professional communication using a more appropriate methodology. As we get closer to the development of the electronic record, it is quite likely that technological advances will have delivered a more effective patient held 'smart card' as an adjunct to the record. The ultimate patient-held record? Confidentiality and sharing of information The expectations of doctors are clearly laid out by the General Medical Council in Confidentiality: Protecting and Providing Information (General Medical Council, 2000). There is a clear and well-established standard that patients will be consulted on the extent to which confidential information will be shared by other health care professionals. In mental health, and any other service where care is delivered extensively via multi-professional clinical teams, it is possibly still rather unusual for patients to be given explicit written information (and choice) that they actually sign up to as to how their personal information is to be used, and the extent of the teambased discussion of their problems. Normally, the issues of confidentiality and disclosure of information fall to the consultant within a clinical team, although obvious exceptions to this rule include psychological therapies and psychotherapies where the clinical psychologist or therapist is being asked to disclose information. In those cases, the Caldicott Guardian would advise. Care clearly needs to be taken when disclosure of information is considered (and without patient consent it is useful to assume that all patient-related information is confidential. The statutory requirements for disclosure are helpfully laid out in Royal College of psychiatrists, 2000) and Dr Dolan, in her overview on disclosure (Dolan, 2003) outlines the responsibilities and limitations (and liabilities) in arriving at such decisions. Whatever decision is taken, and whatever disclosure is decided upon, it goes without saying that clear and accurate records of these decisions should be made, and are best done within the patient health record. Clinicians are perhaps lagging behind public expectation and legal scrutiny in the rigour we apply to record-keeping. We occasionally scapegoat 'the clinical records staff' and 'the notes', rather than working to improve the quality of data entry and communication. In addition, we need to ensure that we are training clinicians, and in particular young psychiatrists, to avoid the jargonridden and often obtuse prose we have often resorted to, both in written and verbal communication with our patients and professional colleagues. Access to records, greater openness and the requirement to copy all correspondence to patients will pose a welcome challenge. Yes, mental health is somewhat different. However, by establishing that mental health patients have the same rights of scrutiny, choice, access, and of course assurance of confidentiality, despite the exceptional difficulties particular to mental health services, it is possible that these new developments could make a significant contribution to reducing stigma and alienation. The traditional options for clinician refusal to divulge information to the patient and/or carer, and to account for clinical decision-making, is rapidly diminishing. This should not be confused with the core role of the psychiatrist in maintaining the confidentiality of patient information on behalf of the patient while society and healthcare delivery is pushing at the information boundary.	2019-03-11T13:07:57.022Z	2004-02-01T00:00:00.000	{ "year": 2004, "sha1": "975599fa1d894de6c6fa399ea62a4a54a1c2b96e", "oa_license": "CCBY", "oa_url": "https://www.cambridge.org/core/services/aop-cambridge-core/content/view/EFF6FD84DD28BFF6AFB79E02079FBAC9/S0955603600069968a.pdf/div-class-title-recording-health-care-and-sharing-the-information-more-bureaucracy-or-a-welcome-challenge-to-prevailing-practice-div.pdf", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "9d1872df2e223179049987779a58357e8c417020", "s2fieldsofstudy": [ "Medicine", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
15854861	pes2o/s2orc	v3-fos-license	Residue Formulation of Chern Character on Smooth Manifolds The Chern character of a complex vector bundle is most conveniently defined as the exponential of a curvature of a connection. It is well known that its cohomology class does not depend on the particular connection chosen. It has been shown by Quillen that a connection may be perturbed by an endomorphism of the vector bundle, such as a symbol of some elliptic differential operator. This point of view, as we intend to show, allows one to relate Chern character to a non-commutative sibling formulated by Connes and Moscovici. The general setup for our problem is purely geometric. Let \sigma be the symbol of a Dirac-type operator acting on sections of a \Z_2-graded vector bundle E. Let \nabla be a connection on E, pulled back to T^M. Suppose also that \nabla respects the Z_2-grading. The object \nabla+\sigma is a superconnection on T^M in the sense of Quillen. We obtain a formula for the H_(M)-valued Poincare dual of Quillen's Chern character ch(D)=trace(exp(\nabla+\sigma)^2) in terms of residues of \Gamma(z)trace(\nabla+\sigma)^{-2z}. We also compute two examples. Introduction. The historical background for noncommutative index theory has two basic parts. The first one dates back to the nineteen fifties, when Israel Gelfand pointed out to Sir Michael Atiyah that the index of a Fredholm operator was stable under small perturbations. The ultimate consequence of this remark is quite famous: the Atiyah-Singer Index Theorem [2,3,4]. The second part is development of noncommutative geometry by Alain Connes [6,8]. In particular, two noncommutative versions of the Chern character were developed. There is one due to Connes [5] and a more recent one due to Connes and Moscovici [7]. We are interested in this more recent version, which is called the residue cocycle. Its individual terms are certain residues which are geometrically interesting, as pointed out by Higson. However, they are not very well understood. In the present paper, we shall prove a formula which resembles the formula of Connes and Moscovici in [7], albeit in a classical setting. Suppose M is a compact smooth manifold with no boundary. Let E → M be a Z 2 -graded complex vector bundle. Let D be an elliptic, odd, first-order, skew-adjoint differential operator on E. Finally, let π : T M → M be the standard projection map of the cotangent bundle. With this setup, we will establish a formula for the Chern character of the symbol of D which resembles the Connes-Moscovici Chern character in noncommutative geometry. It is comprised of finitely many residues of zeta functions constructed from the symbol of D and a connection on E. We shall use Quillen's formalism in which the symbol L of D, together with a connection on E, determines a superconnection on π * E [12]. Quillen's superconnection ∇ + L encodes all the information necessary to define the Chern character in a single object. We shall denote it by ∇ L . The supertrace of exp ∇ 2 L is a mixed differential form which enjoys the major properties of the ordinary Chern character: it is closed and its cohomology class depends only on the underlying vector bundle, not on ∇ or L. But rather than passing to its cohomology class on T * M, we take advantage of the fact that this form is rapidly decreasing on the fibers of T * M. We get this convenient property by sacrificing the traditional −1 2πi factor, an error which we shall also address. Thus, we define a current on Ω * M by the Poincare Duality formula If we expand this dual "Chern character current" in the Taylor series, we obtain: The terms on the right are closed forms and their cohomology classes depend on the isomorphism type of π * E only. However, they are not rapidly decreasing and we cannot form the dual currents by coupling them with the pullback of an arbitrary smooth form on M. We resolve this issue through analytic regularization, to be addressed in section 3, and thus obtain a new formula for the dual Chern character. Briefly, our main result can be stated as follows: Theorem. For R > 0, let Y R be the R-tubular neighborhood of the zero section of T * M. Then: Res\| z Γ(z) The limit arises due to divergence of negative powers of L near zero. We now proceed to briefly describe the Connes-Moscovici local Chern character, i.e., the noncommutative Chern character. The Chern character in [7] is a periodic cyclic cohomology class in HP C * . This cohomology is constructed from spaces of multilinear functionals on A. See [8], chapter 10, for construction of HP C * . The Connes-Moscovici Chern character is represented by a sequence of multilinear functionals on A which we proceed to describe. The n-th term of the sequence is zero for n = 1, 3, 5, . . .; for n = 0, 2, 4 . . ., let a 0 , a 1 , . . . a n be the elements of A. Let Tr s be the supertrace and let k be the variable running through all n-multiindices with nonnegative integer entries. Also, note that the trace T r s (. . . D −2(z+\|k\|+ n 2 ) ) must be replaced by its meromorphic continuation in z, before we take the residues. Existence of such a continuation is also implied by certain axioms and is not trivial at all. Indeed, as it stands, the operators whose trace we are taking typically fail to be bounded, let alone trace class. To us, the most important fact about the Connes-Moscovici formula is that this Chern character is a sum of residues of T r s (. . . D −2(z+\|k\|+ n 2 ) ) times the gamma function. Our main result expresses the Quillen's representative of the (classical) Chern character as a sum of very similar quantities. Much like the proof in [7], our argument hinges on the Mellin transform. Superconnections and Chern Classes. In this section, we give an overview of superconnections according to Quillen [12]. This notion shall be used to define Chern character in the spirit of Chern-Weil theory. Let M be a smooth manifold with no boundary. Let E be a smooth complex Z 2 -graded vector bundle over M. We work with the vector bundle Λ * M ⊗ E. Definition 2.1. (Quillen, [12]) Let a(E) be the space of smooth sections of the vector bundle Λ * M ⊗ End(E). It naturally inherits the Z 2 -grading from the fibers. Definition 2.2. Let ω, ν be homogeneous differential forms on M, let T, S ∈ Γ ∞ End(E) be homogeneous (purely even or purely odd) endomorphisms of E and let s ∈ Γ ∞ (Λ * M ⊗ E) be a homogeneous section. We define the graded multiplication on a(E) by: Lemma 2.1. The equation (1) makes a(E) an associative superalgebra. Also, (2) defines an algebra action of a(E) on Γ ∞ (Λ * M ⊗ E). In fact, this makes a(E) a subalgebra of Γ ∞ End(Λ * M ⊗ E) in the sense that no nonzero element of a(E) kills everything. The condition (2) is called Ω * -linearity. It turns out that Ω * -linear endomorphisms of Λ * M ⊗ E are precisely the elements of a(E). Lemma 2.2. The algebra a(E) is the C-span of homogeneous smooth sections of End ± ( Λ * M ⊗ E) which are in addition Ω * -linear. Remark 2.2. The definition 2.2 resembles the usual definition of multiplication on a tensor product of two algebras. However, it takes the Z 2 -grading into account. Such products of superalgebras are called graded tensor products and are denoted by⊗. Next, observe that the Z 2 -grading on E naturally induces one on the space of sections Γ ∞ (Λ * M ⊗E), which makes it possible to speak of even and odd linear endomorphisms of this space. The even ones preserve the Z 2 grading and the odd ones switch it. We are now ready to define superconnections. In the spirit of [12], we define a superconnection on E to be an odd C-linear map which satisfies the so-called graded Leibniz rule: The curvature of ∇ is defined as ∇ 2 . Lemma 2.3. The curvature is a globally well-defined, even element of a(E). Lemma 2.4. Locally, any superconnection is always of the form d ⊗ id + θ, where d is the de Rham differential and θ is a local odd section of Λ * M ⊗ End(E). Corollary 2.5. Given a superconnection ∇ and an arbitrary odd element L of a(E), ∇ + L is also a superconnection. Lemma 2.6. Any connection which respects the Z 2 -grading is a superconnection. Proof: Such a connection can be locally written as d + θ, where θ = (dx i Γ k ij ) jk is a matrix of 1-forms. Since the connection respects the grading, the matrices (Γ k ij ) ik define even endomorphisms of E. Presence of 1-forms, therefore, makes θ odd. Definition 2.4. We denote the superconnection ∇ + L by ∇ L . Definition 2.5. Suppose W is a finite-dimensional Z 2 -graded complex vector space and A = End(W ). We define the supertrace tr s C : A → C by the following equation: where the traces on the right hand side are the usual traces of operators from W + and W − into themselves. Let R be another Z 2 -graded algebra. A typical simple tensor in R⊗A has the form f = r 11 f 11 r 12 f 12 r 21 f 21 r 22 f 22 . On simple tensors, we define tr s R : R⊗A → R by If W is a fiber of some vector bundle E, this definition extends naturally to sections. We proceed toward the definition of Chern character. To that end, we need the graded version of the trace. Being Z 2 -graded, End(E) has a supertrace tr C s : Definition 2.6. The 2k-th component of the Chern character form is defined as the following differential form: The total Chern character form is We usually write this form as tr s Λ * M (exp(∇ 2 )). Theorem 2.8. ( [12]) ch k is closed and its cohomology class is independent on a choice of the superconnection. This class is an invariant of isomorphism classes of complex vector bundles. Formulation of the Problem. Here we state the main theorems of the paper. The proofs shall be given in subsequent sections. The general geometric setup for our problem is the following. Let M be a compact, n-dimensional, smooth manifold. Let E → M be a smooth, Z 2 -graded complex vector bundle. We assume that E and T M are provided with metrics. Let D be an elliptic, odd, first-order, selfadjoint differential operator on E. Finally, let π : T * M → M be the standard projection map of the cotangent bundle, and let ∇ be a superconnection on π * E which arises as a pullback of some superconnection ∇ ′ from E. Our result is motivated by the Connes-Moscovici formula. We express the right-hand side of the Atiyah-Singer Index Theorem in a way which resembles their residue cocycle. Namely, we shall sum over all the residues of the expression: The Index Theorem using Quillen's Chern charactrer can be stated as: Here we need to prove that the integral (lemma 3.1) converges and that ch(L) may indeed be used in place of the ordinary chern character (theorem 3.3). The factor of ( −1 2πi ) 2n−κ is to correct for the error introduced by leaving the 2πi out of Quillen's definition of Chern chracter. Since M is compact, the integral T * M π * (η)ch(L) converges. The necessary estimates for this lemma are provided in section 3 of [12]. Essentially, it is true because D is selfadjoint, so that L is antiselfadjoint and L 2 is negative-definite, which is where the ellipticity of D comes in. Also, L 2 increases polynomially on the fibers of T * M. When we exponentiate ∇ 2 L = ∇ 2 + [∇, L] + L 2 , the resulting expression decreases as e −Kr 2 . Next consider the Taylor expansion of ch(L): Although the Poincare dual of the left-hand side converges, the duals the individual Taylor terms do not, due to polynomial increase of L along the fibers of T * M. We get around this difficulty by analytic regularization. The general idea is that if we replace the integer power k of ∇ 2 L by a complex number −z, where Re(z) ≫ 1, we also can replace the integral 1 k! T * M π * (η)∇ 2k L with the following expression: Before we can take this residue, though, we need to pass to the meromorphic extension of (4) In particular, we need to prove that such an extension exists (theorem 3.4). However, there is yet another difficulty. Let \| \| denote the fiberwise norm on T * M. Define: The integral in (4) would not really converge, if taken over all of T * M, because L 2 is a symbol of a second-order differential operator. Hence, L 2 is a homogeneous quadratic polynomial in the vertical coordinates ξ of the cotangent bundle. It therefore vanishes at the zero section on T * M. The problem with divergence at infinity shall be resolved by taking Re(z) > 0, meromorphically extending the integral to the whole complex plane and taking residues. We deal with divergence at Y R , in the following way. First, we shall replace the integral over T * M by that over X R . Second, we shall take the residues in z. Third, we shall take the limit as R → 0. That is, we use: lim R→0 z∈C Res\| z Γ(z) It turns out that this quantity is well-defined and is equal to the value of the current P D(tr s exp ∇ 2 L ) on η. We now formally state the main results of the present work. First, we summarize the hypotheses which apply in all the theorems in the sequel: Let M be a compact, smooth n-manifold with no boundary. Let π : T * M → M be the cotangent bundle and let E → M be a Z 2 -graded smooth vector bundle. Suppose also that D is an odd, elliptic, first order selfadjoint differential operator on E. Thus, the symbol L of D is an odd endomorphism of π * E which is invertible everywhere but at the zero section M ⊆ T * M and pointwise anti-selfadjoint. Let ∇ be the pullback of some connection ∇ ′ on E via π. Assume also that ∇ ′ , and hence ∇, respects the Z 2 -grading. Theorem 3.3. Let η be a closed, smooth differential form on M. Consider the following integral: For any R > 0, the following holds on the interior of X R : Thus, the pair (tr s exp ∇ 2 , β) defines a relative cohomology class Hence, I(η) yields the same result as pairing of η with the relative cohomology class defined by (tr s exp ∇ 2 , β). Theorem 3.4. For any R > 0 and for any η ∈ Ω * M and for any z ∈ C with Re(z) ≫ 0, the following integral converges: Further, it has a meromorphic extension to C of the form where A K are constants. In fact, we do have a classification of the poles. There are finitely many of them and they are located at negative integers or half-integers. can only have a nonzero residue at the point (κ − 2n)/2. Further, if n is even and the residue is nonzero, then (κ − 2n)/2 must be an integer. If n is odd, (κ − 2n)/2 must be a half-integer. In either case, nonzero residues occur only for even κ. This lemma says that at each particular point z ∈ C, the residue is a homogeneous current. That is, it vanishes on all the forms except possibly for those of some given degree. Theorem 3.6. Let ∇ be a superconnection which has been pulled back from E via π. For any η ∈ Ω * M All but finitely many residues on the right-hand side vanish as R → 0. Corollary 3.7. For each z, the following defines a closed current on Ω * M: Proof: By theorem 2.8, tr s exp ∇ 2 tL is a closed form. It is also rapidly decreasing on the fibers of T * M, so for an exact form η = dω on M, Stokes theorem yields: The rapid decay property assures that there is no boundary term. Thus, tr s exp ∇ 2 tL induces a closed current on Ω * M. By lemma 3.5, for each κ, R z either vanishes on or agrees with the current induced by tr s exp ∇ 2 tL . In either case, R z is a closed current on Ω κ M. Just as the computation in [7], our proof of theorem 3.6 hinges on Mellin transform. The simplest example of a Mellin transform is the well-known formula, valid for Re(σ) > 0: It says that σ −z Γ(z) is the Mellin transform of e −σt . (See [1] for details). Connes and Moscovici apply the same transform to the socalled JLO cocycles [11] in order to obtain the residue cocycle. Let Σ k be the standard k-simplex in R k+1 with coordinates u 0 , u 1 . . . u k−1 . Using the notation of section 1, the JLO cocycles are comprised of multilinear functionals on a -algebra A given by We, however, apply Mellin transform to Quillen's Chern character, which is an exponential, and obtain complex powers of the curvature. Proof of Theorem 3.3. Theorem 3.3. Let η be a closed differential form on M. Under the hypotheses outlined in section 3, consider the following integral: For any R > 0, the following equation holds on the interior of X R : Thus, the pair (tr s exp ∇ 2 , β) defines a relative cohomology class Hence, I(η) yields the same result as pairing of η with the relative cohomology class defined by (tr s exp ∇ 2 , β). The content of this theorem is really due to [12]. For part (a), assuming η = dω, we compute: The right-hand side vanishes as R → ∞. For (b), the fact that the cohomology class of tr s exp ∇ 2 L is independent on ∇ ′ or L is not enough. We need to prove that if we replace the connection ∇ ′ on E with some∇ ′ , so that on π E we have∇ = π * ∇′ , then there exists a differential form β 1 , rapidly decreasing on the fibers of T * M and such that We present the so-called "homotopy" argument. Suppose first there is some connection ∇ t on π * E which depends on a parameter t in a differentiable way. The example we have in mind is: Here, t is a coordinate on the manifold T * M × R. We take the pullback vector bundle F = pr * 1 (π * E), on T * M × R and the following defines a connection D t on F : Then D 2 t = ∇ 2 t + dt∇ t and more generally: where µ k and ν k are unambiguously defined by the above equation. Let d ′ denote the de Rham differential on T * M × R and denote the one on T * M by simply d. This way, d ′ = d + dt∂ t . By theorem 2.8, tr s D 2k 1 0 νdt, which means that: Or, taking supertraces and keeping in mind that supertraces kill supercommutators: This implies that This equation applies to any ∇ t which depends on t differentiably. In our particular example, and we may define β 1 as 1 0 tr s exp ∇ 2 t∇ t dt. Because exp ∇ 2 t is exponentially decreasing along the fibers of T * M for each fixed t, the rapid decay property of β 1 is easy to prove. To proceed with (c), we change our definition of ∇ t : This does not affect the fact that ∂ t µ k = dν k and we have: or, taking supertraces: Just as in part (b), we obtain: But as long as L is invertible, exp ∇ 2 t → 0 as t → 0 (since, there are non-zeroes among the eigenvalues of L 2 ). Hence, (integrating (11)) yields: So, the above equation holds on X R . Keeping in mind (8), which can be restated here as: we see that on X R : For (d), recall how does one integrate compactly supported cohomology classes defined by pairs. If we have a pair (tr s η exp ∇ 2 , ηβ) as above, β being equal to ∞ 0 tr s exp ∇ 2 tL Ldt, and η being closed, then: Similarly, for β 2 = − ∞ 1 tr s exp ∇ 2 tL Ldt we already have: Combining (12) and (14) we see that these quantities are equal. Taking R → ∞, due to exponential decay, ∂Y R ηβ → 0. It follows that Proof of Theorem 3.4. Theorem 3.4 Under the hypotheses outlined in section 3, for any R > 0 and for any η ∈ Ω * M and for any z ∈ C with Re(z) ≫ 0, the following integral converges: Further, it has a meromorphic extension to C of the form where A K are constants. First, we set up some notation. Over each coordinate chart U α of M, we may define a pullback chart V α = π −1 (U α ) of T * M. It has horizontal coordinates x = (x 1 , . . . , x n ), which are just the coordinates of M, and vertical ones ξ = (ξ 1 , . . . , ξ n ). On the fibers of T * M, let ρ and Ξ be the spherical coordinates. We can take ρ = \|ξ\| to be the coordinate induced by the metric g. Let S * ρ M denote the sphere bundle of T * M of radius ρ. The theorem follows by direct computation from the following proposition. Proposition 5.1. Over each chart X R V α , there exist smooth local sections Θ K (z, x, ρ, Ξ) of Λ * T * M⊗End(π * E) whose coordinate expressions, in fact, do not depend on ρ (i.e., they are pullbacks from the unit sphere bundle via the obvious map X R → S * M), so we write Θ K (z, x, Ξ). For all z with Re(z) ≫ 0, for all R > 0 and η ∈ Ω * M, Figure 1 these sections Θ K (z, x, Ξ) satisfy: The sum in the right-hand side is finite. Further, each Θ K (z, x, Ξ) extends to an entire function in z which is also smooth in x and Ξ. This proposition follows, essentially, by separation of powers of ρ in the coordinate expression of ∇ 2 L . Proof of proposition 5.1. The major steps in the proof are the following. First, for a suitable contour γ, we express (−∇ 2 L (p)) −z at each p ∈ X R by holomorphic functional calculus: Convergence of the integral is obvious and the fact that γ may be used instead of the usual counter-clockwise oriented curve follows by standard argument as in figure 1. Note that γ does not depend on p. Second, working on a single coordinate chart, V α X R , we shall prove that ∇ 2 L is polynomial in ρ. Namely, for certain G 0 , G 1 and G 2 , which are independent of ρ, we show that Indeed, if we let ∇ = d + θ, then: Observe that G 0 and G 1 are nilpotent of degree at most 2n, while G 2 is just L 2 /ρ 2 , hence invertible on X R . Third, by nilpotence of G 0 and G 1 , the integrand in (20) can be expanded in a terminating geometric series: Fourth, we separate the powers in the k-th term of these series and for certain sections Θ k l obtain: The details of this computation are postponed until the end of the proof. Here, the sections Θ k l will depend on the quantity λ/ρ 2 , but otherwise will not depend on ρ explicitly. Also, err represents the terms which may be ignored because they are not multiples of the vertical volume form dρ dΞ and hence do not contribute to the integral over X R in (19). Then, ignoring those error terms, (23) becomes: Since the integrals can be computed using the substitution σ = λ/ρ 2 we can define Θ K (z, x, Ξ) as follows: Finally, observe that the above integrals do not really depend on ρ because the contour γ/ρ 2 can be replaced by a certain contour γ ′ independent of ρ. This basically finishes the proof, except we need to supply the following details: a) The construction of Θ k l and the derivation of (25). b) The exact choice of γ ′ . For (a), we need to work out the details of (25). Fix ρ > 0. Consider some multiindex I k,l = (ι 1 , ι 2 , . . . , ι k ) ∈ {0, 1} k , in which 1 appears l times and 0 appears k − l times. Let G ′ 0 = G 0 and G ′ 1 = G 1 ρ. Then we define G I k,l as Then, recalling that σ = λ/ρ 2 and G 2 = L 2 /ρ 2 : We defineΘ k l as the sum of those G I k,l which are multiples of the vertical volume form dρdΞ 1 . . . dΞ n−1 = ρ n−1 dρdΞ. (All the others do not contribute to the integral over X R in (18) and we ignore them). Therefore we can pull both ρ −2k+l and dρdΞ out ofΘ k l and define Θ k l through the equation:Θ k l = ρ −2k+l Θ k l dρdΞ. (24) and (25) become clear. The only possible issue is that as we change the variable of integration from λ to σ in the Cauchy integral, the contour of integration shifts: Now both Part (b)takes care of this issue. We choose γ ′ to be the vertical contour in C, which is parameterized by T − iχ, (χ ∈ R) for a suitable T . We are about to show that there exists T such that ). Here, the notation ∇ 2 L (p) reminds us that ∇ 2 L is a section of End(π * E) which depends on p ∈ T * M, and sp denotes the spectrum over each point p. Proof: Indeed, ∇ 2 L equals L 2 plus the nilpotent term [∇, L] + ∇ 2 . So, λ+∇ 2 L is invertible whenever λ+L 2 is invertible. This is apparent from the geometric series (23). Thus, sp(−∇ 2 L ) ⊆ sp(−L 2 ), so it is enough to find T such that But by compactness of S * M, there exists T such that: Appealing to homogeneity of L 2 in ρ, one sees that T satisfies the assertion of the lemma. This finishes the proof of the lemma and the proposition. Elaborating on this proof, and retaining the notation therein, we can obtain an estimate which shall be useful later: Suppose η is compactly supported in a single chart U α of M. Then there exist constants K, K ′ such that for every local section Θ k l of Λ * T * M⊗End(E) as in the proof of proposition 5.1, and for all complex z: Proof: By compactness of S * M, there exists a closed loop γ ′′ which simultaneously surrounds all the pointwise spectra of −L 2 (p) for all p ∈ S * M. Such a loop may be chosen strictly to the right of the imaginary axis. This loop can be used in place of the contour γ ′ in the above integral without affecting its value. The advantage is that γ ′′ is compact. Then \|σ −z \| ≤ Ke −K ′ Re(z) for all σ ∈ γ ′′ and some constants K, K ′ , Also, π * (η)Θ k l is bounded on the compact set γ ′′ × S * M π −1 supp(η) by some K ′′ (with the appropriate choice of charts U α and V α , as assumed). Then, integrating out the variables σ, x, and Ξ over this set we see that: where K ′′′ comes from the supertrace. Lemma 3.5.Let η ∈ Ω κ (T * M). Then I(z, η) can only have a nonzero residue at the point (κ − 2n)/2. Further, if n is even and the residue is nonzero, then (κ − n)/2 must be an integer. If n is odd, (κ − 2n)/2 must be a half-integer. In either case, nonzero residues occur only for even κ. Proof: Utilizing the proof of proposition 5.1 we reason out the case of even n, the odd case being treated similarly. Let η be a κ-form. Recall that in in the said proposition, We restrict our attention to a single chart V α . We have seen that Θ k l is expanded into the sum of terms G I k,l using (27). The only such terms that could possibly contribute to I(z, η) are the ones of differential-form degree 2n−κ, because η multiplied by them must produce a 2n-form on T * M. Thus, we need to collect all the appropriate (i.e., contributing) terms of the form: The remainder of the proof is but an exercise in counting the differential form degrees. They are products of l copies of G 1 (which is locally a matrix of 1-forms), and k −l copies of G 0 , which is ∇ 2 +d ρ L. Thus, any contributing term requires k − l ≥ 1, because at least one copy of G 0 is needed to supply the differential dρ for the 2n-form. Each additional copy of G 0 can only supply the curvature ∇ 2 , which is a matrix of 2-forms. Therefore, each additional copy of G 0 may be replaced by two copies of G 1 without changing the total degree of (30). So, all the contributing terms satisfy: which means that the quantity 2k − l is the same for all of them. But one can see from (25) that the location of the residue which arises from each contributing term of (27) depends only on that quantity. It is apparent from (27) that I(z, η) can have at most one nonzero residue, whose location must be 1 2 (l − 2k) = κ−2n 2 . This location does not depend on the topological information about M (other than its dimension). Neither does it depend on the vector bundle E, on the curvature, etc. If κ is even, then by (31), l must be odd. Similarly, if κ odd, then l must be even. Now, suppose we equip Λ * T * M⊗End(π * E) with the Z 2 -grading inherited from End(π * E), rather than the total one. The supertrace vanishes on the sections of Λ * T * M⊗End(π * E) which are odd in that inherited grading. We say that such sections are of an odd profile. The term even profile is defined similarly. Hence, if l is even, we get a term γ λ −z G I k,l dλ which involves some number of the even-profile sections (σ + L 2 ) −1 and ∇ 2 . Also, it involves d ρ L and an even number of copies of G 1 . Such a term will be of odd profile and its supertrace is zero. Thus, only if κ is even can one hope to get a non-zero residue. Combining (31) with the fact that the residue is located at (l − 2k)/2, we get l − 2k = −2n + κ. Proof of Theorem 3.6. Theorem 3.6 Under the hypotheses outlined in section 3, for any η ∈ Ω * (M) Further, all but finitely many residues on the right-hand side vanish as R → 0. In fact, we shall prove that for any R > 0: Taking limits of both sides as R → 0, we get theorem 3.6. The outline of our proof is the following. 1) We introduce a parameter t ≥ 0 and express exp ∇ 2 tL using a Cauchy integral. Thus, for a suitable vertical contour γ in C, Strictly speaking, (32) is incorrect, because the integral over γ does not converge. Still, the formula holds in a weak sense. That is, for all R > 0 and η ∈ Ω * M, where the integral over γ converges. This is proven using the geometric-series expansion of e −λ (λ + ∇ 2 tL ) −1 very similar to that in (19). We show that the above integral γ converges at least for those terms of the expansion which do contribute to (33). See lemma 3.5 for discussion of contributing and noncontributing terms. Thus, (33) is an equality of currents on Ω * M induced by tr s exp ∇ 2 tL and by γ e −λ (λ + ∇ 2 tL ) −1 dλ via Poincare duality, as discussed in section 3. 2) Assuming Re(z) ≫ 1, we show that in the same weak sense, which means that: This is an application of the so-called Mellin transform which is is given by where C is a suitable vertical contour C. See [1] for details. Roughly, the computation for (35) is the following: Interchanging the integrals ∞ 0 . . . dt and X R is easy, because L 2 is negative definite and invertible, so exp ∇ 2 tL is rapidly decreasing and absolutely integrable on R × X R . We will need to prove that we can interchange ∞ 0 . . . dt and γ . . . dλ, at least for Re(z) ≫ 1 and for the relevant terms of the geometric series expansion of e −λ (λ + ∇ 2 tL ) −1 . Also, notice that the exponential is e −λ , not e −tλ , as one might expect from the well-known identity: The algebra behind this will be explained. Also, observe that by theorem 3.4 the integral X R . . . in the right-hand side has a meromorphic extension to C with at most simple poles. 3) Next, we simply restate (36) in terms of the inverse Mellin transform. For a certain vertical C ⊂ C, The integral X R tr s π * (η)(−∇ 2 tL ) −z dzwill be abbreviated by I t (z, η). Since the meromorphic extension of I t (z, η) is defined for all z except at a certain discrete set, we can choose C to pass through the domain where I t (z, η) is defined. We will need to check the convergence of the integral C . . . dz. Observe that t has reappeared in the subscript tL on the right-hand side. This will require clarification. 4) After taking the meromorphic extension of I t (z, η) in the righthand side of (37), we "collect" the residues by translating the contour C to the left. We will need to prove that as the contour translates, This is an application of the residue theorem (Fig. 2). We proceed with the proof. In order to make the equation (33) in step (1) less bulky, we introduce the following notation. Definition 6.1. Let µ, ν be smooth forms on T * M. We say that they are equal in the weak sense and we write µ = w ν if for all η ∈ Ω * (M) In other words, this means that µ and ν are equal as currents on Ω * (M). With this kind of equivalence relation, it is possible to write exp(∇ 2 tL ) as a Cauchy integral, similarly to (20). But first, we introduce some more notation. We expand ∇ 2 tL , similarly to (23): , and let γ τ denote a vertical line {Re(λ) = τ }, oriented downward. (E.g., γ 0 is just the imaginary axis). Since locally, Θ t is a matrix of differential forms of positive degree, and T * M is 2n-dimensional, we have the following geometric series: Lemma 6.1. Fix p ∈ T * M. Choose ǫ ≥ 0 such that γ ǫ lies to the left of the pointwise spectrum sp(−t 2 L 2 (p)) of −t 2 L 2 (p). Then the following equality holds and the integral on the right-hand side converges: In view of (38), this comes close to Proof: The contributing terms (see the proof of lemma 3.5) have enough negative powers of λ to assure convergence. Note that we start the series at k = 1 which explains the weak equality: the integral of the term with k = 0 diverges. Fortunately, that term does not involve vertical differentials dΞ i or dρ. Thus, by the proof of lemma 3.5, it does not contribute to any current. In step (2), we need to prove some version of (35) (it is not true literally). To do this, we shall: a) Apply the geometric-series expansion (38) to On the k-th term of that expansion, perform a secondary expansion into terms which will be denoted by Φ k l . This will separate the powers of t: c) For each term Φ k l , take the Mellin transform of the integral γ e −λ t l−2(k+1) Φ k l dγ. Homogeneity in t makes this task possible. This will yield a formula much like (35), for each of a collection of separate terms. At a certain later point, we shall reassemble the Mellin transforms of these terms into the quantity Γ(z)(−∇ 2 L ) −z . We accomplish a), b) and c) in the following proposition. Also, our earlier warning about interchanging the integrals ∞ 0 . . . dt and γ . . . dλ in (35) receives due attention here. Proposition 6.2. There exist smooth sections Φ k l of Λ * T * M⊗End(π * E) such that the following expansion holds for each k > 0: These sections depend on the quantity λ t 2 , but they do not depend on t in any other way. Further, there exists τ > 0 such that for Re(z) ≫ 0, Here, γ 0 is the imaginary axis. Also, note that we are using 2z instead of z in the Mellin transform. Proof: This proof is very similar to that of proposition 5.1. Each term (λ + t 2 L 2 ) −1 Θ t k is a non-commutative polynomial in the quantities ∇ 2 and t[L, ∇], which are globally well-defined sections of the vector bundle Λ * T * M⊗End(π * E). Therefore, each term can be further further expanded as follows: where the quantityΦ k l is the sum of all the monomials which are products of l copies of t[L, ∇], k − l copies of ∇ 2 , and k + 1 copies of (λ + t 2 L 2 ) −1 . This is similar to the construction of Θ k l in the proof of proposition 5.1. But (λ + t 2 L 2 ) −1 = t −2 ( λ t 2 + L 2 ) −1 , so, we can pull t −2(k+1)+l out ofΦ k l to obtain Φ k l : So (41) holds and we can prove (43.) Just as in the lemma 6.1, the weak equality is there because we start the geometric series at k = 1. In what follows, by sp(t 2 L 2 ) we always mean the pointwise spectrum over a point p ∈ T * M. Define τ by Such τ exists by lemma 5.2. Then for each t > 0, the pointwise spectrum of t 2 L 2 is to the right of γ τ t 2 . Fixing one such t for the moment, we see that the vertical γ τ t 2 is a suitable contour for the Cauchy integral expression of exp ∇ 2 tL (by lemma 6.1) and we can compute, for the k-th term: Since k > 0, Φ k l involves at least 2 factors of (σ + L 2 ) −1 . Therefore it is absolutely integrable with respect to σ, while t 2(z−k)+l−1 e −t 2 σ is absolutely integrable in t for nonnegative Re(z). By Fubini's theorem, we may interchange the integrals and finish the computation: This proves that and the integrals X R and ∞ 0 . . . dt in the left-hand side can be interchanged, as remarked in our discussion after (36). This equation is as close as we get to (36). Our next step is to apply the inverse Mellin transform to the right-hand side. Our estimate from lemma 5.3 comes in here. The inverse Mellin transform of (48) is By theorem 3.4, the vertical line C may be chosen very far to the right so the residues of Γ(z)I t (z, η) are nowhere near. Convergence of the integral over C is assured by the estimate very similar to lemma 5.3 and by the fact that Γ(z) is rapidly decreasing on the vertical lines. Introducing the variables s = z + l/2 − k and the verticals C l,k = C + l/2 − k, we may rewrite (50) as: In view of the next lemma, we may replace all the contours C k,l with C. Lemma 6.3. Fix some p ∈ T * M. Let C and C ′ be two vertical lines in C with the same orientation. If the expression Γ(s) γ σ −s Φ k l dσds has no singularities between them, then: Proof: First, we join C and C ′ by horizontal line segments ab and cd, located below and above the real axis, as in Fig. 3. Then: Because on the vertical lines Γ(s) is rapidly decreasing and the rest of the integrand is bounded in s, the integrals over ab and cd vanish as those line segments move away from the real axis. The result follows. This lemma allows us to continue the computation, using C instead of C k,l , provided that C were originally chosen far enough to the right. We are about to reassemble the individual terms γτ σ −s Φ k l dλ into the quantity (−∇ 2 L ) −z , as promised earlier. We now invoke theorem 3.4 and lift our standing assumption that Re(z) ≫ 0. Hence, the integral X R . . . in (57) may be replaced by its meromorphic extension. We can now finish the proof, by moving the vertical C to the left and "picking up" all the residues. The procedure is explained in the following lemma. Lemma 6.4. For any r > 0 such that (C − r) does not intersect the real axis at any of the residues, the following holds: Further, substituting r m = 2m+1 2 instead of r in the above expression, the integral on the right-hand side tends to zero as m → ∞, so that Proof: The first equation follows by the argument similar to that in lemma 6.3 (Fig. 4). Next, because of the identity zΓ(z) = Γ(z + 1), the quantity sup y∈R \|Γ(x + iy)\| decays superexponentially as x → −∞. Therefore, by our estimate in corollary (5.4) on the integral I t (η, z) = X R π * (η)(−∇ 2 tL ) −s ds, (59) follows. Finally, we need to prove that as R → 0, only finitely many residues survive. By theorem 3.4, I t (η, z) has only finitely many poles and they are at most simple. Also, Γ(z) has at most simple poles, at z = 0, −1, −2 . . .. So, the residues of Γ(z)I t (η, z) are due to either one of these factors. But the theorem 3.4, provides us with some knowledge of the general algebraic form of I t (η, z). It implies that for m >> 0, the residue at z = −m will be a multiple of a positive power of R, so it will vanish as R → 0. The de Rham operator on Riemanian surfaces. We consider the complexified vector bundle E ⊗ C = Λ * M ⊗ C of exterior forms over a compact manifold M with no boundary. The grading decomposition is that into differential forms of even and odd degree. Given a Riemanian metric g on M, the associated de Rham operator D = d + d * has a well-known symbol L = −ρ 2 . In order to apply the theorem 3.6, we need to compute: The argument of the function ν → −ν −z can be viewed as −ρ 2 plus some commuting perturbation which is nilpotent. It follows that we may just use the Taylor series expansion instead of the Cauchy integrals: For example, we consider the case when M is a 2-manifold and η ≡ 1. Since that is exactly the todd class for any 2-surface, by the Atiyah-Singer index theorem both sides of theorem 3.6 should give us the euler characteristic. The left-hand side of theorem 3.6 yields: Thus, the left-hand side of theorem 3.6 reads: (61) Similar remarks apply on the right-hand side and we obtain: Since t r s (∇ 2 [L,∇] 2 +[L,∇] 2 ∇ 2 +[L,∇]∇ 2 [L,∇]) = ωρdρ for some differential form ω, it is enough to see that: 8. The Chern character for a general spinor bundle. We apply our results to the Chern character of a spinor bundle S → M associated to a vector bundle π : F → M, as computed by Mathai and Quillen [10]. The role of the cotangent bundle π : T * M → M is played by F in this example, and the role of E → M is played by S. So, theorem 3.6, strictly speaking does not apply, though we could have proven it in a more general setting. The reason we stated our theorem for T * M is that we have the Atiyah-Singer index theorem in mind, for future applications. Rather than applying theorem 3.6, we will go through its proof. Namely, we shall repeat steps (2) and (3) in a simpler way. We proceed to outline the result of [10]. Some familiarity with spin structures is assumed here. The reader can consult, e.g., Spin Geometry by Lawson and Michelsohn for details [9]. We also warn that the notation of [10] is quite a bit different from our own. We will briefly explain the differences in the end of this section. Let π : F → M be a complex even-dimensional vector bundle with a spin structure. In particular, this means that there is a fiberwise metric µ on F . Let S → M be the associated spinor bundle. The assumption of spin structure implies that S can be split into a direct sum of even and odd subbundles: S = S + ⊕ S − , where the fibers S + x and S − x of S + and S − are the only two irreducible representations for the spin group of the fiber F x . Thus, the spin structure induces a Z 2 -grading of S. In order to form a Chern character we need a connection ∇ ′ on S which respects that Z 2 -grading. We also need an odd antiselfadjoint endomorphism L of π * S. The spinor bundle setup in [10] requires, among other things, that: • L be homogeneous of degree 1 in the radial coordinate ρ of the fibers of F . Much as in the proof of theorems 3.4 and 3.6, ρ is induced by the metric µ and is given by ρ(p) = µ(p, p) for all p in F . • ∇ ′ preserve the fiberwise metric (·, ·) which is induced on S by the metric µ of F . This means that for any two sections α and β of S, d(α, β) = (∇ ′ α, β) + (α, ∇ ′ β). The coordinate notation is the same as in the proof of theorem 3.4. The local coordinates on F are the vertical (fiberwise) cartesian coordinates are ξ 1 , . . . , ξ m , and the horizontal coordinates x 1 , . . . x n , which are also coordinates of M. In fact, it makes sense to choose the µ-orthonormal local frame e 1 , . . . e m of F and to choose coordinates ξ j associated to that frame. They may be replaced by spherical coordinates ρ and Ξ 1 , . . . , Ξ m−1 at our convenience. To describe L we recall that the spin structure of F stems from the fiberwise metric µ. To begin with, we have the Clifford action of F on S which is a fiberwise R-linear bundle map c : F → End(S), such that for any (x, ξ) in F x , c(x, ξ) 2 = −µ x (ξ, ξ). It is one of the standard axioms for a Clifford actions that c(x, ξ) be fiberwise anti-selfadjoint endomorphism. Thus, each fiber F x is contained in a clifford algebra Clif f (F x , µ x ), which is a fiber of the bundle Clif f (F, µ). Also, there is a map Clif f (F, µ) → End(S), which is an isomorphism of bundles and fiberwise an isomorphism of algebras. Now, the pullback π * F to the total space of F is equipped with the Clifford action on π * S which we shall also denote c instead of π * c. Let τ : F → π * F be the tautological section. Then the endomorphism L = c(τ (x, ξ)) has all the required properties. Its homogeneity in ρ is obvious and it is antiselfadjoint by hypothesis. Abbreviating c(τ (e j )) by γ j , we may write L = j ξ j γ j . Since the construction of the clifford action on spinors using an orthonormal basis is completely canonical, the coordinate expression for L does not involve the x-variables. Since, it follows that L 2 = µ(ξ, ξ) = −ρ 2 . Next, to pick a connection on ∇ ′ , we start with a connection on F which is locally given by d + θ. The connection ∇ ′ on S is constructed from it (see [10] and [9]). In order to describe the construction, we adopt the summation notation: we reserve the right to write any index as an upper or a lower index. (Since we have chosen an orthonormal local frame, there is no difference at all). Repetition of the same index on the top and on the bottom implies summation. Repetition on the top only or on the bottom only does not. The connection on π * S is given by where θ ij are just the matrix entries of the endomorphism-valued1-form θ. The connection ∇ = π * ∇ ′ on π * S therefore makes sense. Observe that since γ j are odd, the local endomorphism θ ij γ i γ j of π * S is even. Moreover, it only depends on the variables x i and horizontal differentials dx i , just as before. Therefore, the curvature of the connection ∇ L may be written as: Here, just as in the previous example, the fact that L 2 is a scalar is a tremendous simplification. We may use Taylor × tr s (π −m/2 e −ρ 2 I ε(I,I ′ )P f (∇ 2 /2) I j∈I ′ ((dξ j )γ j + ξ j 4 θ i j γ i )), where: • I, I ′ are complementary (strictly increasing) multiindices over the set {1, 2, . . . m} and ε(I, I ′ ) is a certain combinatorial ±1valued function of them, which shall not be relevant here. • P f (∇ 2 /2) I is the Pfaffian of the submatrix of ∇ 2 /2 determined by the multiindex I. For an unfamiliar reader, it suffices to know that it is a certain polynomial in the matrix entries of ∇ 2 which is just 1 if I is the empty multiindex. For us, (65) is greatly simplified by the fact that we are only interested in the currents induced by this Chern character on Ω * M. Therefore, as observed in theorem 3.4 and lemma 3.5, we need only those terms of (65) which involve all the differentials dξ j , so the only relevant multiindex is I ′ = (1, 2, . . . , m), I being empty and P f (∇ 2 /2) I being 1. Integrating this over any fiber of F , we see that for any η ∈ Ω * M, where by P µ,ν (A, B) we denote the homogeneous non-commutative polynomial in A and B obtained by summing all the words comprised of µ copies of A and ν copies of B. We now recall (65). It involves the sum over multiindices I ′ and the only relevant multiindex was determined to be I ′ = (1, 2 . . . , m), where m is the fiberwise dimension of F . This means that in (68) only the terms with l = m contribute to the current induced by Chern character on Ω * M. We have seen a special case of this in the previous example, where a normal coordinates argument was used to show that only the terms which involve two copies of [∇, L] are relevant. (Recall from lemma 3.5 that such terms were called contributing.) In particular, it means that [∇, L] contributes the vertical differentials and no other differentials. Coming back to our computation, the right-hand side of (68) is readily seen to be the Taylor series for Γ(z)(−∇ 2 L ) −2z . The discussion in the previous paragraph implies that the contributing part of P k−l,l (∇ 2 , [∇, L]) is a multiple of the vertical volume form: Thus, it supplies m − 1 powers of ρ. It remains to determine the residues, using the procedure from theorem 3.4. Just as in that theorem, we set X R = def {p ∈ F : ρ(p) ≥ R}, and integrate from R to ∞ with respect to ρ. This, as we shall see, produces the residue at (m − 2k)/2. Let η ∈ Ω κ M, and express (−∇ 2 L ) −z as a sum of two differential forms: (−∇ 2 L ) −z = ν z + ω z dρ, where neither ν z nor ω z involve dρ. We obtain: R −2(z+k)+m 2(z + k) + m S * M π * (η)ω z . Counting the differential form degrees, we see that if deg(η) = κ, then the only contributing term of (68) is the (k, m)-th term. Here k satisfies 2k −m = m+n−κ. This term produces a residue at m/2−k which is a current on κ-forms. But according to (67), the same current is induced by the (n − κ)-component of the differential form det sinh(∇ 2 /2) (∇ 2 /2) Warning: In [10], the relevant computation is in section 4, where F is denoted by E, ∇ 2 is denoted by Ω and the fiberwise coordinates ξ j are denoted by x j .	2014-10-01T00:00:00.000Z	2005-05-07T00:00:00.000	{ "year": 2005, "sha1": "a6f8c8d7b8bd7b73cfeff536863e051c503787e8", "oa_license": "elsevier-specific: oa user license", "oa_url": "https://doi.org/10.1016/j.topol.2007.03.004", "oa_status": "BRONZE", "pdf_src": "Arxiv", "pdf_hash": "a6f8c8d7b8bd7b73cfeff536863e051c503787e8", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
201844863	pes2o/s2orc	v3-fos-license	Racial/Ethnic Disparities in Pregnancy-Related Deaths — United States, 2007–2016 Approximately 700 women die in the United States each year as a result of pregnancy or its complications, and significant racial/ethnic disparities in pregnancy-related mortality exist (1). Data from CDC's Pregnancy Mortality Surveillance System (PMSS) for 2007-2016 were analyzed. Pregnancy-related mortality ratios (PRMRs) (i.e., pregnancy-related deaths per 100,000 live births) were analyzed by demographic characteristics and state PRMR tertiles (i.e., states with lowest, middle, and highest PRMR); cause-specific proportionate mortality by race/ethnicity also was calculated. Over the period analyzed, the U.S. overall PRMR was 16.7 pregnancy-related deaths per 100,000 births. Non-Hispanic black (black) and non-Hispanic American Indian/Alaska Native (AI/AN) women experienced higher PRMRs (40.8 and 29.7, respectively) than did all other racial/ethnic groups. This disparity persisted over time and across age groups. The PRMR for black and AI/AN women aged ≥30 years was approximately four to five times that for their white counterparts. PRMRs for black and AI/AN women with at least some college education were higher than those for all other racial/ethnic groups with less than a high school diploma. Among state PRMR tertiles, the PRMRs for black and AI/AN women were 2.8-3.3 and 1.7-3.3 times as high, respectively, as those for non-Hispanic white (white) women. Significant differences in cause-specific proportionate mortality were observed among racial/ethnic populations. Strategies to address racial/ethnic disparities in pregnancy-related deaths, including improving women's health and access to quality care in the preconception, pregnancy, and postpartum periods, can be implemented through coordination at the community, health facility, patient, provider, and system levels. chain of events initiated by pregnancy, or aggravation of an unrelated condition by the physiologic effects of pregnancy. U.S. natality files were the source of live birth data (3). PRMRs were analyzed by age group, highest level of education, and calendar year for women who were non-Hispanic white, black, AI/AN, Asian or Pacific Islander (A/PI), and Hispanic. Per the PMSS assurance of confidentiality, statespecific data are not authorized to be released. States were anonymously classified by PRMR and grouped into lowest, middle, and highest tertiles by PRMR; the PRMR was calculated by race/ethnicity per state tertile. Disparity ratios (comparisons of PRMR between two racial/ethnic groups) were calculated by five 2-year intervals, demographic characteristics, and state PRMR tertiles. White decedents were the referent group because they represented the largest racial/ethnic group. Cause-specific proportionate mortality was classified in 10 mutually exclusive categories,* and differences by race/ ethnicity were identified using chi-squared tests. SAS statistical software (version 9.4; SAS Institute) was used for the analyses. During 2007-2016, a total of 6,765 pregnancy-related deaths occurred in the United States (PRMR = 16.7 per 100,000 births). PRMRs were highest among black (40.8) and AI/AN (29.7) women; these rates were 3.2 and 2.3 times the PRMR for white women (12.7) ( Table 1). From 2007-2008 to 2015-2016, the overall PRMR increased slightly from 15.0 to 17.0. The disparity ratios did not change significantly over time. PRMR increased with maternal age; the black:white disparity was lowest among women aged <20 years (1.5) and highest among those aged 30-34 years (4.3); the AI/AN:white disparity was lowest among women aged 20-24 years (1.2) and was * Cause of death coding includes the following 10 mutually exclusive categories: hemorrhage; infection; amniotic fluid embolism; thrombotic pulmonary or other embolism (i.e., air, septic, or fat); hypertensive disorders of pregnancy (i.e., preeclampsia or eclampsia); anesthesia complications; cerebrovascular accidents; cardiomyopathy; other cardiovascular conditions (e.g., congenital heart disease, ischemic heart disease, cardiac valvular disease, hypertensive heart disease, and congestive heart failure); and other noncardiovascular medical conditions (e.g., endocrine, hematologic, immunologic, and renal). Deaths caused by hypertension that were not preeclampsia, eclampsia, or gestational hypertension were categorized in the "other cardiovascular conditions" category. Deaths caused by cerebrovascular accidents that were a result of preeclampsia or eclampsia were classified in the "hypertensive disorders of pregnancy" category; otherwise, deaths were classified in the "cerebrovascular accidents" category. Abbreviations: AI/AN = American Indian/Alaska Native; A/PI = Asian/Pacific Islander. * Blacks, whites, AI/AN, and A/PI were non-Hispanic; Hispanic women might be of any race. † 25 pregnancy-related deaths with unknown race/ethnicity were included in the total analyses but not presented in an individual column; two pregnancy-related deaths with unknown age were excluded from age analyses; 687 pregnancy-related deaths with unknown educational levels were excluded from education analyses. § Dashes indicate fewer than 10 deaths; these results were suppressed because ratios might be unreliable. highest among women aged 35-39 years (5.1). Racial/ethnic disparities were present at all education levels. The PRMR among black women with a completed college education or higher was 1.6 times that of white women with less than a high school diploma. Among women with a college education or higher, the PRMR for black women was 5.2 times that of their white counterparts. The black:white disparity ratio in the PRMR for the states in the lowest, middle, and highest tertiles was 3.0, 3.3, and 2.8, respectively. Cardiovascular conditions (including cardiomyopathy, other cardiovascular conditions, and cerebrovascular accidents), other noncardiovascular medical conditions, and infection were leading causes of pregnancy-related deaths. The proportion of pregnancy-related deaths attributed to each of 10 mutually exclusive causes varied by race/ethnicity (Table 2). Cardiomyopathy, thrombotic pulmonary embolism, and hypertensive disorders of pregnancy contributed to a significantly higher proportion of pregnancy-related deaths among black women than among white women. Hemorrhage and hypertensive disorders of pregnancy contributed to a higher proportion of pregnancy-related deaths among AI/AN women than among white women. Discussion Racial/ethnic disparities in pregnancy-related mortality were evident in 2007 and continued through 2016, with significantly higher PRMRs among black and AI/AN women than among white, A/PI, and Hispanic women. The PRMR for black and AI/AN women aged ≥30 years was approximately four to five times that of their white counterparts. Even in states with the lowest PRMRs, and among groups with higher levels of education, significant disparities persisted, demonstrating that the disparity in pregnancy-related mortality for black and AI/AN women is a complex national problem. Multiple factors contribute to pregnancy-related mortality and to racial/ethnic disparities. Previous analyses found that for each pregnancy-related death, an average of three to four contributing factors were identified at multiple levels, including community, health facility, patient/family, provider, and system (1). Thirteen state maternal mortality review committees reported 60% of pregnancy-related deaths were preventable, and there were no significant differences in preventability by race/ethnicity (1). Differences in proportionate causes of death among black and AI/AN women might reflect differences in access to care, quality of care, and prevalence of chronic diseases (4). Chronic diseases associated with increased risk for pregnancy-related mortality (e.g., hypertension) are more prevalent and less well controlled in black women (5). Ensuring access to quality care, including specialist providers, during preconception, pregnancy, and the postpartum period is crucial for all women to identify and manage chronic medical conditions (4). Systemic factors (e.g., gaps in health care coverage and preventive care, lack of coordinated health care, and social services) and community factors (e.g., securing transportation for medical visits and inadequate housing) have also been identified as contributors to pregnancy-related deaths (1). Addressing these factors and ensuring that pregnant women at high risk for complications receive care in facilities prepared to provide the required level of specialized care can improve outcomes. †, § In addition, innovative delivery of care models in the preconception, pregnancy, and postpartum periods might be further evaluated for their potential to reduce maternal disparities. Quality of care likely has a role in pregnancy-related deaths and associated racial disparities. A national study of five specific pregnancy complications found a similar prevalence of complications among black and white women, but a significantly higher case-fatality rate among black women (6). Studies have suggested that black women are more likely than are white women to receive obstetric care in hospitals that provide lower quality of care (7). Hospitals and health care systems † https://www.cdc.gov/reproductivehealth/maternalinfanthealth/cdc-locate/ index.html. can implement standardized protocols and training in quality improvement initiatives, ensuring implementation in facilities that serve disproportionately affected communities. Quality improvement efforts, such as perinatal quality collaboratives ¶ that facilitate a change in the culture of care provision, implement standards of care,** and rapidly use data to identify opportunities for improvement, can improve the quality of care received by all pregnant and postpartum women. Implicit racial bias has been reported in the health care system and can affect patient-provider interactions, treatment decisions, patient adherence to recommendations, and patient health outcomes (8). This report's findings demonstrate that black and AI/AN women have a more accelerated trajectory in age-specific PRMRs compared with white women. This might be related to the "weathering" hypothesis, which proposed that black women experience earlier deterioration of health because of the cumulative impact of exposure to psychosocial, economic, and environmental stressors (9). Identifying and addressing implicit bias and structural racism in health care and community settings, engaging communities in prevention efforts, and supporting community-based programs that build social support and resiliency would likely improve patient-provider interactions, health communication, and health outcomes (4). Summary What is already known about this topic? Approximately 700 women die annually in the United States as a result of pregnancy or its complications; racial/ethnic disparities exist. What is added by this report? During 2007-2016, black and American Indian/Alaska Native women had significantly more pregnancy-related deaths per 100,000 births than did white, Hispanic, and Asian/Pacific Islander women. Disparities persisted over time and across age groups and were present even in states with the lowest pregnancy-related mortality ratios and among groups with higher levels of education. The cause-specific proportion of pregnancy-related deaths varied by race/ethnicity. What are the implications for public health practice? Identifying factors that drive differences in pregnancy-related deaths and implementing prevention strategies to address them could reduce racial/ethnic disparities in pregnancyrelated mortality. Strategies to address racial/ethnic disparities in pregnancy-related deaths, including improving women's health and access to quality care in the preconception, pregnancy, and postpartum periods, can be implemented through coordination at the community, health facility, patient and family, health care provider, and system levels. state and local maternal mortality review committees, which thoroughly review pregnancy-related deaths and make actionable prevention recommendations, offer the best opportunity for identifying priority strategies to reduce disparities in pregnancy-related mortality. † † The findings in this report are subject to at least three limitations. First, PMSS predominantly uses death certificates and linked birth or fetal death certificates to determine the pregnancy-relatedness of each death. Errors in reported pregnancy status on death certificates have been described, potentially leading to overestimation of the number of pregnancy-related deaths (10). Second, pregnancy-relatedness cannot generally be determined in PMSS for cancer-related deaths or injury deaths such as drug overdoses, suicides, or homicides, and thus, these are often not included in the PRMR calculated from PMSS data. Finally, small cohort sizes precluded the reporting of some factors by race/ethnicity; in addition, there might be inconsistencies in the reporting of race/ethnicity when death certificates were used for classification. § § Most pregnancy-related deaths can be prevented, and significant racial/ethnic disparities in pregnancy-related mortality	2019-09-07T13:14:58.164Z	2019-09-06T00:00:00.000	{ "year": 2019, "sha1": "c411c42fcad549e3ad6ed1769e3f4d8cc237f441", "oa_license": "CCBY", "oa_url": "https://www.cdc.gov/mmwr/volumes/68/wr/pdfs/mm6835a3-H.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "cc370e5477feb47842f57676d972623898706b01", "s2fieldsofstudy": [ "Medicine", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
114874042	pes2o/s2orc	v3-fos-license	Multi-objective MDVRP solution considering route balance and cost using the ILS metaheuristic Article history: Received January 15 2017 Received in Revised Format April 1 2017 Accepted May 7 2017 Available online May 8 2017 The multi-objective problem of multi-depot vehicle routing (MOMDVRP) is proposed by considering the minimization of the traveled arc costs and the balance of routes. Seven mathematical models were reviewed to determine the route balance equation and the bestperforming model is selected for this purpose. The solution methodology consists of three stages; in the first one, beginning solutions are built up by means of a constructive heuristic. In the second stage, fronts are constructed from each starting solution using the iterated local search multi-objective metaheuristics (ILSMO). In the third stage, we obtain a single front by using concepts of dominance, taking as a base the fronts of the previous stage. Thus, the first two fronts are taken and a single front is formed that corresponds to the current solution of the problem; next the third front is added to the current Pareto front of the problem, the procedure is repeated until exhaustion of the list of the fronts initially obtained. The resulting front is the solution to the problem. To validate the methodology we use instances from the specialized literature, which have been used for the multi-depot routing problem (MDVRP). The results obtained provide very good quality. Finally, decision criteria are used to select the most appropriate solution for the front, both from the point of view of the balance and the route cost. © 2018 Growing Science Ltd. All rights reserved Introduction The Multi-Depot Vehicle Routing Problem (MDVRP) is a variant of the classical Vehicle Routing Problem (VRP), which consists of designing a set of routes with a set of clients which consume a determined demand.A fleet of vehicles attends each client´s demands with a capacity already defined from a depot. The objective is to minimize the total distance traveled (Toth, 2014).The MDVRP considers several depots from which a set of vehicles attends a number of clients; once the tour is completed, they return to the same depot.Both the MDVRP and the VRP are NP-hard combinatorial problems (Cordeau et al., 1997;Ho et al., 2008).Keskinturk and Yildirim (2011) propose that each driver´s workload is defined according to the length of each route, the volume carried during a time frame (including charging and discharging times) and the number of clients that need to be visited.Schwarze and Voß (2013) propose six different types of objective functions related to the workload balancing, and they take into account three types of indicators included in the objective function.First, the route length, which refers to the distance, time or cost to carry out a route; second, the time invested in the discharging operation in each client and third, the demand of each client that implies the transportation volume. To solve the MDVRP, several exact and metaheuristic techniques have been suggested.In the case of the exact-technique approach, the MDVRP is formulated as a Mixed-Integer Linear Programming (MILP) problem, as described by Kulkarni and Bhave (1985) and Montoya et al. (2015).However, these techniques converge into optimal solutions for small-size problems (less than 50 clients).On the other hand, the metaheuristic techniques have been widely used to solve efficiently both the mono-objective MDVRP and the Multi-Objective Multi-Depot Vehicle Routing Problem (MOMDVRP). Regarding the MOMDVRP, very little has been researched as only about 12% of the papers reviewed address the MOMDVRP, and only about 4% take into account the workload balancing, as shown by Montoya et al. (2015).Geiger (2008) proposes a concept denominated as Pareto Iterated Local Search (PILS), that combines intensification and diversification in one algorithm to generate a set of solutions traditionally called population, which starts from an initial solution x1, from which an approximated Pareto set is obtained applying VNS iterative searches.From this set, the non-dominated solutions (Pareto front) are computed, from which a unique solution is selected x2 and from which diversification is applied by using a perturbation operator obtaining x3 as a perturbed solution.This procedure is performed to solve the Multi-Objective Flow Shop Scheduling problem. The concept of Multi-Objective Local Search based on the dominance concept (DMLS) is explained by Liefooghe et al. (2012); besides, the following strategies are described in detail: dominance relation, selection of current set of solutions, neighborhood exploration and stopping criteria.The strategy is tested in two combinatorial optimization problems with several objectives: the Flow Shop Problem (FPS) and the Traveling Salesman Problem (TSP) from which, a DMLS model is proposed and a comparative study of different strategies for the DMLS to solve FSP and TSP variants is presented. On the other hand, in Duarte et al. (2015), the VNS metaheuristic adaptation is explored along with its extensions to solve multi-objective combinatorial problems.To achieve the objective, the solution concept is redefined and adapted to the multi-objective context, where a set of solutions called approximated set of efficient solutions is taken.This new definition also allows redefining the meaning of improvement i.e. an improvement is given when a new solution is added to the approximated set of efficient solutions.Under these considerations, a procedure is developed for solving multi-objective combinatorial optimization problems, considering that this approach may require a high computational effort. In both the MDVRP and the VRP, it is generally aimed to minimize the operation cost; that is, the total distance traveled by the vehicle fleet without exceeding its load capacity; however, there are other objectives that might be optimized such as the environmental and social impact.In this regard, the social matter is approached by balancing the drivers' workload through the minimization of the objective function, which is calculated from all the length-routes standard deviation.However, since the calculation of this objective function requires an important computational effort, it offers better results and no additional parameters in the objective function are required.This paper proposes a new multi-objective methodology for solving the workload balance and cost in the MDVRP, where the metaheuristic is used based on the trajectory called Iterated Local Search (ILS) that includes the Variable Neighborhood Search (VNS).The ILS is composed of two stages that keep the diversification and intensification in the search space.The diversification stage is performed through a perturbation mechanism that allows exploring promissory regions in the solution space.On the other hand, the intensification stage is implemented with the VNS, which consists of specialized operators responsible for reducing the solution space by making the search in nearby surroundings (neighborhoods) the current solution.In the present work, the VNS is implemented by using two types of operators: The Inter-route operators that look for a better solution between two routes and the Intra-route operators that look for a better solution into an only one route.Both operators are based on shift, swap and 2-opt strategies. The proposed methodology includes multi-objective optimization, with which the approximated Pareto front is obtained, based on the non-dominated solutions generated by the ILS.The methodology is validated with instances from the literature taken from Cordeau et al. (1997).The results obtained are of good quality and allow concluding about the relationship between cost minimization and route balance, which is of interest for the academic community. Finally, the rest of the article is organized as follows: Section 2 presents the model for the multi-objective multi-depot vehicle rout problem (MOMDVRP), where two objectives are defined: the solution cost and the standard deviation of the total distance traveled in each route.In Section 3, the new methodology proposed is described to solve the MOMDVRP using the ILS-VNS.In Section 4, the results are analyzed comparing them with some existing instances for the MDVRP.Section 5 presents the conclusions, considerations and guidelines for future works. MOMDVRP Proposed Model The MDVRP is an extension of the VRP that determines a set of routes traveled by specific vehicles.(i) every vehicle starts and ends its trip in the same depot, (ii) every client is attended by a single one vehicle once only, (iii) the total demand of every route does not exceed the vehicle capacity and (iv) the routes traveled are minimized (Montoya et al., 2015).Kulkarni and Bhave (1985) propose a three-index mathematical model that requires the definition of a binary decision variable xijk that takes the value of "1" when two nodes i and j are in the vehicle route k and take the "0" value otherwise.The model is formulated as a generalized TSP problem. Objective function for route balancing To define the objective function whose purpose is balancing the routes in Halvorsen and Savelsbergh (2016) and Schwarze and Voß (2013), different approaches available in the literature are describe that include load-balance VRP modifications.In all the cases, lr, lt and lu are the lengths of the routes r, t and u, that belong to the set of routes T, being \|T\| the number of routes in the solution and l the average route length. In Eq. ( 1), the maximum route length is minimized. In Eq. ( 2), the difference between the maximum and minimum route length is minimized. In Eq. ( 3), the accumulated difference between each route length and the shortest one of these is minimized. min ( min ) In Eq. ( 4), the variance of the route length is minimized. In Eq. ( 5), the relative deviation of the lengths, regarding the maximum length is minimized. In Eq. ( 6) the summation of the absolute deviation of the length is minimized from an average already known in advanced (parameter). min \| \| Eq. ( 7) minimizes the summation of the absolute deviation of the length from an average already known in advanced. The above equations show an approximated value of the route-balance measurement; however, the objective function ( 7), which measures the standard deviation for the length of the routes, is the most accurate to observe the route balancing even though it implies a greater computational effort. The objective function ( 7) is chosen, since the standard deviation is the measure of better behavior around the average value (Ribeiro & Ramalhinho, 2001).The objective function ( 1) is the easiest implementation; however, the results obtained are of low quality.In ( 2), ( 3) and ( 5) the length of the shortest route is subtracted, presenting undesirable behavior (Schwarze & Voß, 2013).In objective function (6), a predefined average value must be assumed which makes difficult the calculation. Objective functions above display an approximated value of the route-balance measurement.Objective functions ( 4) and ( 7), which measure the variance and standard deviation respectively for the length of the routes, are the most accurate to observe the route balancing even though they are quadratic functions and greater computational effort is required.Finally, the objective function ( 7) is chosen, since the standard deviation is the measure of better behavior around the average value (Ribeiro, Ramalhinho, 2001). Mathematical model The equations of the model are shown below Nomenclature Parameters Distance between nodes i and j. Cost associated with the trajectory between nodes i and j.Quantity of the product to deliver to every client ∈ . Variables Binary variable which indicates whether the path between clients i,j ∈ V is traveled, both belonging to depot k.Auxiliary binary variable which indicates whether the path between clients i,j ∈ V is traveled, both belonging to depot k.Quantity of merchandise carried between nodes i and j. Equations of the mathematical model are shown as follows, subject to The multi-objective model has two objective functions; the objective function (8) minimizes the total distance traveled by the vehicles from the k depots. The objective function ( 9) is formulated considering (Halvorsen, Savelsbergh, 2016) and the minimization of the standard deviation of the distance traveled by every route in the solution, where µ is the average distance of every route in the solution and lr is the length of every route.A greater computational effort is required to calculate the mean due to the need of knowing the length of all the routes in the solution; however, better results are obtained. The constraints ( 10) and ( 11) guarantee that all the arcs arriving to a node and leaving a node must be equal to one.The constraint (12) guarantees that the number of vehicles arriving and leaving a depot is the same.A client i assigned to a unique depot k is assured by constraint (13), thus sets of clients assigned to determined depots are obtained.The arrival and departure of a single arc to node i assigned to node k is guaranteed in constraint ( 14).The connection between a node and its respective depot is guaranteed by constraints ( 15) and ( 16).The constraints ( 17) and ( 18) show the flow equations in which the demand for each client is guaranteed and that the demand in the depot is equals to 0. Constraint (19) guarantees that the amount of resources leaving node i, is equal to the difference between the amount of resources entering node i and the resources delivered to node i.The restrictions (20) guarantees that the flow sij between nodes i and j is considered if and only if arc xijk is active.Depot k capacity is restricted in ( 21).The type of variables used in the mathematical model are shown in constraints ( 22), ( 23) and ( 24). Methodology In general, the multi-objective problems have been solved using metaheuristics based on sets of solutions called population, and evolutionary algorithms such as the NSGA-II (Non-Dominated Sorting Generic Algorithm), where a genetic algorithm is used for generating a dominance-based population and ordering of solutions.The NSGA-II, uses selection and mutation operators to create half of the population following the selection of the best solutions (according to the function and the diversity adaptation).For most problems, the results show that NSGA-II is capable of finding diverse solutions and good convergence close to the optimal Pareto front, in comparison to the multi-objective evolutionary algorithms (MOEAs) (Deb et al., 2002). Given the above, Geiger (2008), explains a metaheuristic for solving multi-objective optimization problems denominated as Pareto Iterated Local Search (PILS).PILS combines proper characteristics of how metaheuristic algorithms operate, whose development is based on two stages: intensification and diversification.The intensification is done by applying the VNS explained by Mladenovic and Hansen (1997).On the other hand, the diversification is performed by applying a perturbation which uses operators to avoid getting stuck in local optima. An adaptation of the method previously explained is presented in this work, where a front of nondominated solutions of constant size F is created.On each solution, s that belongs to the front, a local search and a perturbation is made.The new s0 solutions are evaluated and selected according to their non-dominance front F. The local search is performed by using a modified VNS to evaluate the two objectives of the problem.The procedure is explained in Algorithm 1 named MOILS (Muti-Objective ILS). The procedure starts by obtaining an initial solution s0 using the algorithm cited by Paessens (1988) (step 2 of Algorithm 1).From this solution, a search in the neighborhood of the initial solution s0 using interroute operators denominates as Inter_Ruta is performed.Initially, the operators list is enable to be used during the iterative process (steps 6 and 7).As long as there are non-explored neighborhoods, a neighborhood v is randomly selected (steps 8 to 10).Then, from the randomly selected neighborhood v, the set of non-dominated solutions for every solution s of the front F is searched.The new set of nondominated solutions is stored into F' (steps 11 and 12).The sets F and F' are blended and the front is updated (step 14).If during the former process, there was at least one non-dominated solution that became part of the front F, the search is performed over a set of neighborhoods with only modified routes Intra-Ruta operations (steps 16 to 20).F ← F ∪ F' 28: iter ← iter + 1 The non-dominated solutions obtained during this process are stored in F' and after this; F is updated (step 21).In the case that no non-dominated solution in the neighborhood v is found, it is excluded from the list (step 23).On the current front F, a perturbation operation is performed on each of the front solutions, the new solutions are stored in F' (steps 24 to 26).Finally, the front is updated and a new iteration starts (steps 27 and 28). 1: Algorithm 2: V(S) 2: for rx ∈ S do 3: for ry ∈ S do 4: if ri 6= rj then 5: for i ∈ rx do 6: for j ∈ ry do 7: S' ← set(S,i,j) 8: dominated ← false 9: for S ∈ F do 10: if dominated(S',S) then 11: dominated ← true 12: Break 13: if ¬dominated then 14: Every one of the neighborhoods v in Inter-Route and w in Intra-Route operates in a general way, as described in Algorithm 2. The search in the neighborhood S is performed exhaustively (steps 2 to 6).For every route rx and ry, different movements of clients i and j are made.According to the selected neighborhood, the objective functions are evaluated and a new solution s' is created (step 7).If the solution s' is not dominated by the current front F, it is added to the front F' (steps 9 to 14).In Table 1 and 2 a list of inter-route and intraroute neighborhoods is shown. Table 1 Table 2 List of inter-route neighborhoods List of intra-route neighborhoods The perturbation is the mechanism that allows escaping from local optima.In this work, the perturbation mechanism is applied on the current front of solutions F, which consists in randomly applying a neighborhood operator considering the objective function of costs.To define the solution size belonging to the front F, the crowding distance explained by Deb et al. (2000) is used. Computational Results For the analysis, benchmark instances from Cordeau et al. (1997) were used.For each instance the Pareto front was obtained and from every front, three solutions were selected for later analysis.Two of them correspond to the solutions placed in the extremes of the front; the third one corresponds to the solution obtained by using the min-max metric, detailed by López et al. (2011).This metric normalizes the solutions for objective function values regarding to the extreme points; from these, the minimum value is selected and finally, the maximum value among the values previously chosen corresponding to a solution of equilibrium between both objectives is selected.The results were obtained starting from a solution with which a front is generated; a second front is generated from another initial solution, these solutions generate a Pareto front using the non-dominance concept.A third front is generated from another initial solution, which uses again the current Pareto front, the process continues until 10 fronts have been generated by updating the Pareto front in each iteration.The time presented in Table IV corresponds to the average time = total Time//10.The algorithm was run in an Intel Core i3, 3.3 GHz and 4 GB of RAM memory and implemented in C++.In Table 3, three different solutions of the Pareto front from the instance P01 are shown: two solutions correspond to the extreme points and the third one is obtained using the min-max metric.Fig. 1 presents an extreme point of the front, whose value for fo1 corresponds to the optimal operation cost and fo2 is the standard deviation measured among the solution routes.For this case, the maximum and the minimum route length is of 81.39 and 23.49 units of distance respectively.A second solution is shown in Fig. 2, which was selected according to the min-max criterion where intermediate values for both objective functions were chosen.fo1 is worse than the solution mentioned earlier, and fo2 corresponds to a solution of better quality with maximum and minimum length for the routes of 81.87 and 38.47.The third solution is shown in Fig. 3, with a value for fo2 equivalent to the standard deviation of 2.02 with a route length between 72.76 and 64.65 showing an appropriate route balancing.As opposed to point 1, the fo1 presents a high value, apparently moving away from the optimal value.In brief, the optimal operation cost value is presented in Fig. 1 and the optimal route balance value is presented in Fig. 3.The intermediate value between the two optimal values is shown in Fig. 2; that is, one objective function is deteriorated to benefit the other one until a position of equilibrium is achieved.In all the cases studied, a conflict between both objectives was observed.The results using the benchmark instances proposed by (Cordeau et al., 1997), excluding those with a length constraint are shown in Table 4.In fo2, low values indicate proper route balances that would have similar length in the case under study.However, this condition in the fo1 expresses elevated operation costs that would make the implementation infeasible.Thus, it is necessary to select a point of equilibrium for both the economic and the social, for which the min-max concept is applied in this study.Nonetheless, this decision may be considered according to the decision-making criterion.This table also presents the solutions of the front of each one of the instances studied, the maximum and minimum length of the routes for the extreme points and that by using the min-max criterion.In the minimum cost point, a big difference between the minimum and the maximum route length is shown.On the contrary in the extreme point of balance, high costs and a relatively low difference between the minimum and the maximum route length are evidenced.The solution selected using the min-max criterion depicts a proper relation between costs and route balance.The time of the previous table corresponds to the average of the 10 cases studied with different seeds is, where a front is generated for each case.The fronts that are being generated update the incumbent front using the non-dominance criterion.The objectives behavior in the studied instances is explained through Fig. 4. In general, the routing problems have been studied so far using different models and solution methods whose objective is the cost minimization.In all the studied solutions, it is observed that a great imbalance is presented when the cost is optimized, which entails social inequality.On the other hand, if the problems were studied with an objective function for route balance, solutions socially equitable will be obtained but with operative costs excessively high, not suitable for a real-life operation.Given the conflict of interests associated, it is intended to stablish a methodology that considers a balance between both objectives.The methodology is focused on identifying a common region for both objectives.Table 5 presents the results considered as acceptable since they intersect minimum cost and route balance as presented in Fig. 4. To calculate Table 5 the minimum cost solutions are used as reference, which are generally used in the decision making.From this value, the percetage variation is obtained according to the obtaind with the min-max criterion that corresponds to columns fo1 and fo2.In the previous table, it is possible to establish that with small deteorations in the operation-cost, great social improvements are obtained.The solutions presented in Table 5 are considered acceptable as it is shown in Fig. 4.These solutions may be determined by some criteria according to decision-maker considerations. Conclusions and Future Work A multi-objective methodology based on the ILS metaheuristic was proposed for solving the MOMDVRP considering two objective functions.The first one consists in minimizing the operation cost, the second one is the route balancing using the standard deviation among the routes as a criterion for achieving the aforementioned objective.It was observed that in all instances the objective functions are in conflict.In the solutions of the front extreme points, a big difference is observed not only in cost but also in the route balance, with the biggest differences found in the latter.It is also observed that with relatively small deteriorations of the cost function great benefits in the route balance are achieved.The model was tested with the instances proposed by Cordeau et al. (1997), obtaining excellent quality results.The ILS operators were efficiently adapted to the multi-objective obtaining great-quality fronts with a high-diversity degree among them.With the objective of improving the computational effort, it is necessary to define strategies in the following aspects: (i) the front size, (ii) solutions in the front to perform the VNS, (iii) efficient computation of the objective functions, (iv) multi-objective specialized operators and (v) parallel solution methods.The social objective is an issue that has been little treated in the studies that refer to load transportation.The study set forth in this article, shows the conflict between operative cost and route balance.It was also noticed that points of equilibrium between both objectives might be obtained using relatively low deteriorations in the cost function. Table 3 Solutions selected for the instance P01 Table 5 Variations with respect to the minimum cost solution	2018-12-21T01:18:13.909Z	2018-01-01T00:00:00.000	{ "year": 2018, "sha1": "f36a4d26431062f69e158eaf8d642c804dfc8e6e", "oa_license": "CCBY", "oa_url": "https://doi.org/10.5267/j.ijiec.2017.5.002", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "f36a4d26431062f69e158eaf8d642c804dfc8e6e", "s2fieldsofstudy": [ "Engineering", "Computer Science" ], "extfieldsofstudy": [ "Engineering" ] }
234373969	pes2o/s2orc	v3-fos-license	Chemical inducers, nutrient management, guava intercropping and insecticides can reduce Huanglongbing incidence and severity in Sweet orange Received: 28 September 2020 Revised received: 02 December 2020 Accepted: 10 December 2020 Huanglongbing (HLB) or citrus greening is the most economically devastating disease of citrus in the world. HLB is a vector-borne disease and transmitted by Asian Citrus psyllid (ACP). HLB is now a serious threat to the cultivation and expansion of Sweet orange and Mandarin in Bangladesh. As no suitable cure is available against the disease, inducing plant immunity by chemical inducers or nutrient management and intercropping could be an effective way to combat this challenge. In this study, two inducers viz., Bion (Acibenzolar S-methyl) and Bactroban (Bismerthizol), nutrients formulations SICOGREEN® (soil application) and foliar spray, intercropping with guava, spraying guava leaf extract (10%), foliar spray with insect growth regulators (IGR) such as Heron (Lufenuron), insecticides such as Neonicotinoids/ Imidachloropid + Thiomethoxam and foliar spray of Beauveria bassiana (Commercial formulation) showed comparatively better performance as compared to untreated control considering both HLB incidence and severity in both locations (Haluaghat and Bhaluka) of Sweet orange orchards. All these treatments reduced HLB incidence by 57.5 to 89.44% and HLB severity by 54.16 to 80.35% in Sweet orange considering both Haluaghat and Bhaluka orchards. The results revealed that Bion (Acibenzolar S-methyl), nutrients formulations SICOGREEN® (soil and foliar application), intercropping with guava, spraying guava leaf extract, foliar spray of insecticides can be integrated to reduce HLB incidence and severity in Sweet orange. Some of these treatments have also some positive effects on plant growth and yield parameters of Sweet orange as compared to control. These results comprehensively suggest that chemical inducers and nutrient management seem a better alternative to control HLB aimed to increase tree lifespan and productivity. INTRODUCTION Citrus huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus and has been rapidly spreading worldwide, resulting in significant economic losses (Bové, 2006;FAO, 2012;Khan and Razi, 2018). HLB has been known in East Asia for over a century and is currently wide-spread in most citrus areas of Asia, Africa, and the Americas (Gottwald et al., 2007). HLB has been reported previously in Sweet orange and Mandarin in Bangladesh (Tipu et al., 2017, The program highlights controlling psyllid vectors with insecticides, reducing inoculum through the removal of HLBsymptomatic trees, propagating and using pathogen-free budwood and nursery trees. In Florida, foliar nutrition programs coupled with vector control are often used to slow down HLB's spread and reduce the devastating effects of the disease (Gottwald, 2010). These control practices have shown a limited impact on preventing the further spread of HLB. Recently, various treatment strategies including applications of penicillin and streptomycin (Zhang et al., 2011), enhanced nutrient program (Gottwald et al., 2012), thermotherapy (Hoffman et al., 2013), soil-conditioners (Xu et al., 2013), and small molecules targeting Las virulence traits including osmotic stress tolerance (Pagliai et al., 2014), have been examined for HLB disease management and some showed promising progress. However, no effective approach has been established to control HLB and stop it from spreading to new citrus-production areas. Induced resistance, either locally or systemically, may confer long-lasting protection against a broad spectrum of plant diseases (Durrant and Dong, 2004;Walters et al., 2013). The plant defense mechanisms can be activated by pathogens (Durrant and Dong, 2004), beneficial microorganisms (Weller et al., 2012;Zamioudis and Pieterse, 2012;Tang et al., 2018), or by chemical inducers (Walters et al., 2013;Hu et al., 2018). Tremendous effort has been put into the development of agents that can mimic natural inducers of resistance. These include acibenzolar-S-methyl (ASM), benzothiadiazole (BTH), 2,6dichloroisonicotinic acid (INA), β-aminobutyric acid (BABA), oligosaccharide from plant and fungal cell walls, and probenazole. These agents could induce plant resistance effectively against a wide range of pathogens including bacteria, fungi, viruses, nematodes, and parasitic weeds (Beckers and Conrath, 2007), even though effects varied with concentrations pathosystems (Vallad and Goodman, 2004;Walters et al., 2005). For example, soil applications of systemic acquired resistance (SAR) elicitors induced systemic resistance against canker under greenhouse conditions and showed season-long control of canker epidemics on young citrus trees (Francis et al., 2009). In addition, BABA induced citrus resistance against psyllids in the greenhouse , suggesting BABA's potential for the management of HLB. In certain nutrient/SAR programs, salicylic acid (SA) and/or its analogs were applied as foliar amendments to act against the HLB pathogen by activating the SAR pathway and the effects on disease expression of HLBinfected trees and fruit yield remain to be demonstrated (Stansly et al., 2014). Overall, no conclusive study has been conducted regarding how to control HLB by inducing plant defense. HLB symptoms could be reduced by foliar applications of micronutrients, especially Mn, Zn, B, and Mg, (Pustika et al., 2008;Shen et al., 2013;Stansly et al., 2014). Some other studies have shown the benefits of enhanced nutritional programs for HLB management (Pustika et al., 2008;Shen et al., 2013;Stansly et al., 2014). HLB causes declines in the canopy and root system (Graham et al., 2013) and since leaves typically persist up to 2 years in citrus, it would be expected that alleviation of disease symptoms would require a 2-year window to rebuild the root system and canopy before yield would recover. It appears that longer-term studies with foliar applications of Mn, Zn, B, and Mg are required with the twin goals of first aiding the recovery of the root systems and the canopy followed by recovery of yield. Some growers in the Florida citrus industry are using phosphites as the anionic complement to the cationic micronutrients in the salts used to apply essential nutrients to the foliage, such as Mn 3 (PO 3 ) 2 and Zn 3 (PO 3 ) 2 . Early studies of phosphites use in agriculture involved evaluating their nutritional (Rickard, 2000) and other horticultural benefits, including increased citrus flowering, fruit set, yield, and higher fruit quality (Rickard, 2000). Elicitors are compounds that stimulate any type of defense in the plant and favor the synthesis of secondary metabolites under both abiotic and biotic stress conditions and can be applied through foliar sprays. Within these compounds, carbohydrates, lipids, (glyco)peptides, (glyco)proteins, vitamins, and phytohormones can be found (Wallace et al., 1954;Angelova et al., 2006;Boubakri et al., 2016;Thakur and Sohal, 2013;Tsagkarakis et al., 2012). Among the compounds studied to activate defense mechanisms in plants against the attack of biotic agents (macro-and microorganisms) are SA, phytohormones, chitosan, and thiamine (vitamin B1) (Ahn et al., 2005;El-Hadrami et al., 2010;Thakur and Sohal, 2013). In the present study, the effects of chemical inducer, elicitor, nutrient element, insecticide and entomopathogenic fungus on HLB progression under field conditions were investigated to determine the feasibility of the integrated use of these control approach in controlling citrus HLB. Experimental locations Field experiments were conducted in two in situ Sweet orange orchards located in Bhaluka and Haluaghat, Mymensingh from 2017 to 2020. The age of Sweet orange plants in Haluaghat was 2 years and the age of Bhaluka Sweet orange plants is three years at the time of experiment setting. Standard citrus fertilization, minimum weed and pest control measures were taken for all the plants in both Orchards. Application of the treatments The following treatments were applied in situ orchards two times before the rainy season and two times after the rainy season. Chitosan was dissolved in 1% acetic solution and was sprayed with a concentration of 1000 ppm on the surface of the citrus plants. The bio-activator, bion [acibenzolar-S-methyl (ASM)] was applied as foliar spray @ 200 mL (500 ppm) per tree at an interval of 90 days. The chemical activator, Bismerthizol was applied as both foliar spray and soil drench @ 200 mL per tree at a concentration of 200 ppm at an interval of 60 days. The micronutrient was applied as both foliar spray and soil drench at 200 mL per tree (@ 0.2%) at an interval of 60 days. One guava seedling was planted in the middle of four citrus plants to assess the survival efficacy of citrus psyllid bug, the insect vector carrying the Las. Heron 5 EC (Lufenuron), (Haychem Bangladesh Ltd.) was applied @ 200 mL (75 ppm) per tree at an interval of 60 days six times in a year. Two insecticides viz., Confider (Neonicotinoids) and Actara (Thiomethoxam) were sprayed @ 100 mL (0.5%) per tree at one-month intervals during the rainy season (July to October) because the availability of the Asian citrus psyllid is high at that time. Data collection Data were collected on the following parameters-citrus greening incidence, citrus greening severity, plant height (cm), number of branches and number of fruits Monitoring disease progress To address whether the treatments have any effect on the disease progress, the citrus trees were evaluated based on disease symptoms and presence of the Las at 6 and 12 months in a year. For disease symptoms, the trees were visually examined for the presence of typical citrus greening symptoms, such as asymmetric mottling and thickening of veins in mature leaves. Further, the presence of Las was determined based on the polymerase chain reaction (PCR) test using specific primers for the detection of Las (Tipu et al., 2017). Assessment of HLB infection Leaf sampling: Leaf samples for the detection of Candidatus Liberibacter asiaticus (Las) were collected as visual ratings. For each target tree, samples containing 3-4 green twigs of 6 to 8 inches long with approximately twenty leaves, preferably with the petiole still attached and with well recognizable symptoms, were collected. Samples were kept in double bags in cool conditions, preferably in plastic containers containing silica gel. The leaf samples without symptoms were collected as the symptomless carrier of Las. PCR based confirmation or detection of Las Extraction of genomic DNA: Collected leaf samples were washed with sterile distilled water and 70% ethanol and dried on blotting paper to remove excess water. Leaf midribs were ripped off and chopped with sterilized scissors. Approximately 60mg of leaf tissues were processed by freezing with liquid nitrogen in a microcentrifuge tube and crushed into a fine powder using a micro pestle. The Las genomic DNA was extracted using the Wizard Genomic DNA purification Kit (Promega, Madison, WI, USA) following the manufacturer's instructions. Briefly, approximately 60mg of midrib tissues were processed by freezing with liquid nitrogen in a microcentrifuge tube and crushed into a fine powder using a micro pestle. For digestion, 600 µl of Nuclei Lysis Solution was added. It was vortexed for 1-3 seconds to wet the tissue and incubated after 15 minutes in a water bath. Then 3 µl of RNase Solution was added to the cell lysate. The sample was mix by inverting the tube 2-5 times. The samples have then incubated the mixture are 37ºC for 15 minutes in a water bath. The example was allowed to cool to room temperature for 5 minutes before proceeding to the next step. 200 µl of protein Precipitation Solution was added. It was then vortex vigorously at high speed for 20 seconds. The mixture was centrifuged for 3 minutes at 15,000×g. The precipitated proteins were formed a tight pellet. The supernatant containing the DNA (leaving the protein pellet behind) was removed carefully. It was then transferred to a clean microcentrifuge tube containing 600 µl of room temperature isopropanol. The solution was mixed gently by inversion until thread-like strands of DNA from a visible mass. Then it was centrifuged at 15,000×g for 1 minute at room temperature. The supernatant was decanted carefully. 600 µl of room temperature 70% ethanol was added. The tube was inverted gently several times to wash the DNA. It was then centrifuged at 15,000×g for 1 minute at room temperature. The ethanol was aspirated carefully. The tube was then inverted onto clean absorbent paper, and the pellet air-dried for 15 minutes. Five µl of DNA Rehydration Solution was added. The rehydrated DNA was incubated at 4ºC for overnight. Finally, the DNA was stored at -20ºC. Visualization of PCR products: The PCR products were visualized in 1% agarose gel containing 0.2 µg ethidium bromide per 100 ml gel from the stock solution. After electrophoresis, the gel was placed under a UV transilluminator (GelView Master, Dynamics, UK) for visualization of DNA bands. The UV light of the apparatus was switched on. The image of the desired bands on the gel was viewed on the monitor and saved on the computer disc (CD-R) for taking photographs. Statistical analysis RCBD design was followed for field experiments and Mstat-C statistical program was used for data analyses. DMRT was used to compare the treatment means. Effect of different treatments on the citrus greening incidence (% citrus greening infected plant) and severity Citrus greening incidence: Experiments were set up in situ Sweet orange Orchards in Haluaghat and Bhaluka, Mymensingh. The greening suspected trees were confirmed by PCR using Las specific primers. The results showed that all suspected trees yielded a fragment size 500bp which confirmed trees were infected with Las. In Haluaghat orchard, the highest (33%) citrus greening incidence was recorded in T 0 (Control) while the lowest (3.7%) citrus greening T 3 = Bactroban (Bismerthizol, a chemical inducer) followed by T 2 [Bion (Acibenzolar S-methyl, a chemical inducer)] and T 8 [Foliar spray with insect growth regulator (IGR) such as Heron (Lufenuron) Figures 1A and 1B). PCR based confirmation of HLB infection: Symptomatic trees were confirmed by PCR using Las specific primers Las606 and LSS. The results revealed that all suspected trees were positive by PCR. An amplicon size 500bp confirmed the presence of Las in samples collected from suspected trees of the respective treatment ( Figure 2). (Table 2). (Table 2). It is well documented that a wide range of biotic and abiotic agents are able to induce resistance to pathogen infection in various plants (Durrant and Dong, 2004;Van-Loon et al., 2006;Walters et al., 2013). In this study, we observed that Bion (Acibenzolar S-methyl, a chemical inducer), T 3 (Bactroban (Bismerthizol, a chemical inducer), T 4 (Balanced nutrition with micronutrients formulations SICOGREEN® (soil application), T 5 (Balanced nutrition with micronutrients formulations SICOGREEN® (foliar spray) and intercropped with guava] These results are in accordance with a recent report which showed that application of several plant defense inducers, such as ß-aminobutyric acid (BABA), 2,1,3-benzothiadiazole (BTH), and 2,6-dichloroisonicotinic acid (INA), singly or in combination suppressed Las growth in planta and the progress of citrus greening symptoms (Li et al., 2016). Another inducer, Bactroban (Bismerthizol) reduced the citrus greening incidence by 88 and 90% in Haluaghat and Bhaluka, respectively. The citrus greening severity has been reduced by 77 and 83%, respectively by the application of Bactroban. Bismerthiazol has been widely used to control X. oryzae pv. oryzae and X. oryzae pv. oryzicola infections in China (Liang et al., 2015a(Liang et al., , 2015b. Bismerthiazol has also been demonstrated to control effectively citrus canker by both inhibiting the growth of X. citri ssp. citri and triggering the plant's host defense response through the expression of several pathogenesis-related genes and the non-expresser of PR genes in 'Duncan' grapefruit, especially at early treatment times (Yu et al., 2016). However, in the present study, reduced levels of citrus greening incidence and severity by SICOGREEN primarily hinted effect of macro and micro-nutrient in reducing citrus greening incidence and severity. Foliar micro-nutrient deficiencies are a noted symptom of citrus greening affected citrus trees (Spann and Schumann, 2009). Therefore, foliar applications of micronutrients have been used by an increasing number of citrus growers in Florida to help mitigate citrus greening-induced deficiencies and counter the debilitating effects of the disease. Asymptomatic leaves from citrus greening-infected trees showed a significant decrease in K which is linked to plant pathogen susceptibility of those tissues. The decreases in Ca, Mg, and B are from restrictions of nutrient uptake, transport, or metabolism induced by citrus greening infection (Span and Schumann, 2009). Foliar nutrition for reducing citrus greening severity is promising in Florida, USA (Spann et al., 2011). A growing trend in Florida is the manipulation of nutrition and irrigation regimes to reduce the effects of citrus greening on tree health, fruit production and fruit quality and nutrients, the trees may show less severe disease symptoms, including milder effects on fruit production and yield. The use of intensive fertigation practices that promote water and nutrient uptake within a limited root zone would be appropriate to minimize nutrient leaching and accelerate tree development particularly during the time of spring flush (Kadyampakeni et al., 2014). Research that demonstrated that citrus greening symptoms could be reduced by foliar applications of micronutrients, especially Mn, Zn, B, and Mg, and other physiologically active compounds, such as salicylic acid and phosphite (Pustika et al., 2008;Shen et al., 2013;Stansly et al., 2014). Combined soil and/ or foliar application of the above nutrients to stimulate FRLD and improve root lifespan on citrus greening affected Sweet oranges with emphasis on root-zone soil pH (Atta et al., 2020). Moreover, two insecticides such as IGR [Heron (Lufenuron)] and Neonicotinoids performed better as compared to control and some other treatments. The IGR, Lufenuron reduced HLB incidence and severity by 80.22 and 68.05%, respectively considering both Haluaghat and Bhaluka Sweet Orange orchards. IGRs are known to be highly effective in killing immatures, especially nymphs, of several sucking insect pests, including ACP. The majority of the data on the efficacy of IGRs against ACP comes from laboratory studies (Boina et al., 2010;Tiwari et al., 2012) while few studies evaluated their efficacy in the field (Qureshi and Stanslay, 2007;Abbaszadeh et al., 2011;Rao and Shivankar, 2011). Three IGRs, pyriproxyfen (juvenile hormone mimic), buprofezin and diflubenzuron (both are chitin synthesis inhibitors), showed promising ovicidal and nymphicidal activities against ACP, as well as adverse effects on reproduction (both fecundity and egg viability) and morphology of adults emerging from treated older nymphs (Boina et al., 2010;Tiwari et al., 2012). Under field conditions, the protection offered by IGRs, (diflubenzuron, flufenoxuron, lufenuron, novaluron and pyriproxyfen) ranged from 3 days (diflubenzuron) to 4-6 weeks (lufenuron and flufenoxuron) (Qureshi and Stanslay, 2007;Abbaszadeh et al., 2011;Shivankar, 2011 andFarmanullah andGul, 2005). Given the potential of IGRs to reduce adult fecundity and control immatures, IGRs are an important and promising rotational tool in insecticide resistance management (IRM) programs for ACP. The Neonicotinoids/Imidachloropid + Thiomethoxam reduced HLB incidence and severity by 72.77 and 62.5%, respectively considering both Haluaghat and Bhaluka Sweet Orange orchards. Field studies conducted on young King mandarin trees in Vietnam and mature (five-year-old) Valencia orange trees in the United States, however, found that foliar application of neonicotinoids (clothianidin, imidacloprid and thiamethoxam) and an anthranilic diamide (cyantraniliprole) gave the longestlasting protection (8-9 weeks), and among the three neonicotinoids, imidacloprid provided maximum control of adults (50-90%) (Ichinose et al., 2012;. In general, broad-spectrum insecticides, such as OPs, carbamates and SPs, exhibited more rapid killing of both adults and nymphs of ACP than systemic neonicotinoids, but neonicotinoids showed longer-lasting residual activity (Yasuda et al., 2006;Hayashikawa et al., 2006). As a result, broad-spectrum insecticides need more frequent applications than neonicotinoids. Neonicotinoids, being systemic in nature and with long-lasting residual activity, can protect the adults from disease transmission (30 min-7 h) (Capoor et al., 1974;Xu et al., 1988;Roistacher, 1991) could be disrupted with antifeedants. In support of this idea, pymetrozine applied at the labeled rate significantly reduced the feeding activities of adults on treated plants or directly treated adults, as measured by electrical penetration graphs (EPGs) (Biona et al., 2013). This effect resulted in reduced disease transmission by ACP adults feeding on treated plants compared with controls (Biona et al., 2013). Intercropping with guava in Sweet orange orchards reduced citrus greening incidence by 66 and 90% and citrus greening severity by 66 and 83% in Haluaghat and Bhaluka orchards, respectively. Hall et al. (2008) reported the effect of guava on adult ACP. They found that adult ACP released into cages containing the only citrus generally moved faster to citrus than when either guava or cotton was present. They also observed a greater number of adults were consistently observed on citrus over time in cages with only citrus as compared to in cages with citrus in the presence of guava or cotton. They explained that this might be due to differences in the total plant surface area in cages with citrus alone compared to citrus caged with another plant. Mortality rates of adults were increased in cages containing both citrus and guava in one of two studies. While significant reductions in infestations of adults on young grapefruit sometimes occurred in cages containing both citrus and guava in the greenhouse, the reductions were not enough to verify the Vietnamese guava effect. These results are also supported by Beattie et al. (2006) and Gottwald et al. (2010). They reported that infestations of ACP and, consequently, incidences of citrus greening disease in citrus are greatly reduced when citrus is interplanted with guava, Psidium guajava L. (plant family Myrtaceae). The authors speculated that guava volatiles or phytotoxins might be responsible for reducing infestations of the psyllid on citrus. Putative guava volatiles may interfere with the psyllid's ability to locate and infest citrus grown next to guava, or they might repel psyllids away from citrus. Putative guava toxins might negatively affect the biology of the psyllid, interfering with psyllid reproduction in citrus. The reports from Vietnam prompted greenhouse investigations in Florida. Conclusion It can be concluded Bion (Acibenzolar S-methyl), nutrients formulations SICOGREEN® (soil and foliar application), intercropping with guava, spraying guava leaf extract, foliar spray of insecticides can be integrated to reduce HLB incidence and severity in Sweet orange. Some of these treatments have also some positive effects on plant growth and yield parameters of Sweet orange as compared to control. These findings collectively suggest that chemical inducers and nutrient management can pose a better alternative to control HLB sustainably to increase tree lifespan and productivity.	2020-12-31T09:05:53.073Z	2020-12-25T00:00:00.000	{ "year": 2020, "sha1": "a09598c7d9b12beb4ce61e30df9bfc92d68d8691", "oa_license": "CCBYNC", "oa_url": "https://journals.aesacademy.org/index.php/aaes/article/download/05-04-01/pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "84a1209828df11cad9f2ab0c1a17b32937fe8f52", "s2fieldsofstudy": [ "Environmental Science", "Agricultural and Food Sciences" ], "extfieldsofstudy": [ "Biology" ] }
265500358	pes2o/s2orc	v3-fos-license	Effect of the res2 transcription factor gene deletion on protein secretion and stress response in the hyperproducer strain Trichoderma reesei Rut-C30 Background The fungus Trichoderma reesei is one of the most used industrial cellulase producers due to its high capacity of protein secretion. Strains of T. reesei with enhanced protein secretion capacity, such as Rut-C30, have been obtained after several rounds of random mutagenesis. The strain was shown to possess an expanded endoplasmic reticulum, but the genetic factors responsible for this phenotype remain still unidentified. Recently, three new transcription factors were described in Neurospora crassa which were demonstrated to be involved in protein secretion. One of them, RES2, was involved in upregulation of secretion-related genes. The aim of our present study was therefore to analyze the role of RES2, on protein secretion in the T. reesei Rut-C30 strain. Result Deletion of the res2 gene in Rut-C30 resulted in slightly slower growth on all substrates tested, and lower germination rate as well as lower protein secretion compared to the parental strain Rut-C30. Transcriptomic analysis of the Rut-C30 and the Δres2 mutant strain in secretion stress conditions showed remarkably few differences : 971 genes were differentially expressed (DE) in both strains while 192 genes out of 1163 (~ 16.5%) were DE in Rut-C30 only and 693 out of 1664 genes (~ 41.6%) displayed differential expression solely in Δres2. Notably, induction of protein secretion by cultivating on lactose and addition of secretion stress inducer DTT induced many genes of the secretion pathway similarly in both strains. Among the differentially expressed genes, those coding for amino acid biosynthesis genes, transporters and genes involved in lipid metabolism were found to be enriched specifically in the Δres2 strain upon exposure to lactose or DTT. Besides, redox homeostasis and DNA repair genes were specifically upregulated in the Δres2 strain, indicating an altered stress response. Conclusion These results indicate that in the T. reesei Rut-C30 strain, RES2 does not act as a master regulator of the secretion pathway, but it contributes to a higher protein secretion by adjusting the expression of genes involved in different steps of protein synthesis and the secretion pathway. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-023-03125-z. Background The filamentous fungus Trichoderma reesei is a wellknown enzyme producer, and hypersecreting strains are industrially used to produce cellulases.One of the best studied hyperproducing strains is Rut-C30 that has been derived from the wild type (WT) strain QM6a by several rounds of mutagenesis.Sequencing of both strains has revealed a total of 269 single nucleotide polymorphisms (SNPs), three chromosomal translocations, one inversion, eight small insertions or deletions (indels), and five large deletions in Rut-C30 [1][2][3].Moreover, cre1 is truncated at its 3' end with a loss of 2478 base pairs resulting in a low basal cellulase expression in the presence of D-glucose [4].Rut-C30 is also unable to efficiently grow and produce enzymes on α-linked glucans [5]. In addition to having efficient systems for transporting carbon sources and highly inducible cellulase genes, mutant T. reesei strains, such as Rut-C30 also possess a well-developed secretion system.Rut-C30 was found to secrete 2.7 times more proteins than QM6a in 6% (w/v) roll-milled cotton [6].Using transmission electron microscopy (TEM), several studies investigated the ultrastructure of T. reesei [7][8][9].They found a link between the high protein secretion capacity of Rut-C30 and the changes in its ultrastructural characteristics in relation to the wild type QM6a.On a microcrystalline cellulose culture, an approximately seven-fold higher ER content in Rut-C30 than in QM6a was observed during the secretory phase, i.e. during the third and sixth day of culturing.Unlike QM6a that had short and vesicular or narrow intracisternal ER profile, Rut-C30 was distinguished by its long and stacked ER whose cisternal space was wide and filled with amorphous material [7].Similarly, a culture of QM6a and Rut-C30 on microcrystalline cellulose resulted in an about three-fold transient increase in the ER surface area in Rut-C30 after 96 h of culture in comparison to QM6a [8].In addition, [9] demonstrated that distinct ultrastructural features of Rut-C30 in the early secretory pathway (24 h of cellulase induction) are inherent to the strain rather than a response to increased protein secretion.Despite the sequencing of Rut-C30 and QM6a, there is still no straightforward explanation for these ultrastructural characteristics of Rut-C30. The secretion system has been well studied in other organisms such as S. cerevisiae, Aspergillus species and higher eukaryotes [10].In fungi, most secreted proteins follow the ER-Golgi secretion pathway.After polypeptides are synthesized in the ribosomes, they are processed in the endoplasmic reticulum (ER) and then in the Golgi-apparatus before being secreted into the extracellular medium via secretory vesicles [10,11].Certain conditions, however, such as high load of protein production, inhibit the proper folding of proteins resulting in secretion stress.In response to secretion stress, several mechanisms are activated, mainly the unfolded protein response (UPR), UPR-linked ER-associated degradation (ERAD) pathway, and repression under secretion stress (RESS) response [12].UPR is a highly conserved intracellular signaling pathway between the ER and the nucleus, and it involves two main regulators: ER-transmembrane protein IRE1 and a bZIP transcription factor HAC1. Upon secretion stress, HAC1 is activated, leading to its migration to the nucleus and binding to the unfolded protein response elements (UPREs) found in the promoters of certain genes, such as the ER-chaperon bip1 and the foldase pdi1, which reinforce ER folding capacity [13].Interestingly, various mechanisms exist for the regulation of UPR in fungi.Some of the mechanisms are HAC1independent, as found in A. niger, [14] while others are IRE1-independent, as it is the case in yeast [15,16] where TFs different than HAC1, such as Gcn4p, might induce the expression of UPR target genes [17].In T. reesei, the activation of UPR in an IRE1/HAC1-dependent manner has been evidenced upon treatment with DTT and in heterologous protein producing strains 13].DTT induces UPR by preventing the formation of disulfide bonds which is required for the proper folding of proteins and their transport from the ER [12].UPR was also triggered during cellulase secretion in lactose containing media [18].In cDNA subtraction libraries, more than 400 genes including previously known UPR-related genes were found to be induced under secretion stress [13].But it is presently not known if also other regulators are involved in the control of this secretion stress response, and if these could be responsible for the hyperproducing phenotype of Rut-C30. In N. crassa, three TFs (RES-1, RES2, and RRG-2) have been identified to play a role in cellulase and hemicellulase secretion.Unlike Δres-1, Δres2 and Δrrg-2 strains showed nearly 30% decrease of cellulase secretion in comparison to the WT [19].Three-to fourfold reduction in expression of genes coding for hemicellulases was observed in addition to 1.6-1.8-folddecrease in cbh-1 and cbh-2 genes.Some of the differentially expressed genes in the Δres2 strain relative to the WT have roles in cellular export and secretion in addition to cellular import (endocytosis) which contribute to hyphal tip growth and tip secretion.It was thus suggested that RES2 regulates secretory pathways in N. crassa [19]. To elucidate the role of the RES2 homolog in T. reesei in secretion and the secretion stress response, the corresponding gene was deleted in the hyperproducing strain Rut-C30.The effect on growth and protein secretion was assessed and a transcriptomic analysis was conducted under two secretion stress conditions: cellulase induction by lactose and addition of dithiothreitol (DTT).Results show that RES2 does not have the same importance for protein secretion in T. reesei Rut-C30 as in N. crassa but exerts a more subtle regulatory function on protein synthesis, the secretion pathway and stress response. The T. reesei Δ res2 mutant shows growth and germination defects The res2 ortholog in T. reesei was identified with fun-giDB.Pair-wise sequence alignment with the N. crassa RES2 showed a 60% sequence identity on the amino acid level between both sequences.To study the role of the res2 orthologue of T. reesei, the gene was deleted using CRISPR-Cas9.After genetic validation by PCR and sequencing, confirming the correct insertion of the deletion cassette, three of the validated transformants were randomly kept for further analysis.The absence of offtarget integrations was also confirmed in these mutants by qPCR. Although deletants showed no phenotype on transformation plates, we first wanted to investigate if the res2 deletion impacts basic functions such as growth and germination.To address this question, Rut-C30 and the three deletion transformants were plated on solid minimal medium containing glucose or a cellulase secretion-inducing substrate such as lactose in the presence or absence of 1 or 5 mM DTT. Figure 1A shows that growth of both Rut-C30 and the mutant was faster on glucose and slower on lactose.However, growth of the ∆res2 mutant was clearly reduced compared to that in the Rut-C30 parental strain on both substrates, but especially on lactose, Fig. 1A.It is interesting to note that the mutant tended to grow better in the presence of glucose + DTT, reducing the difference with Rut-C30 (Fig. 1B).One hypothesis to explain this observation is that the reducing activity of DTT enhances the activity of some enzymes, as it is the case for cellobiase [20], which leads to increased availability of nutrients and increased fungal growth. To investigate if growth was already affected at the germination step, we assessed the germination rate for both Rut-C30 and the Δres2 transformant 2 on three media: minimal medium with glucose as a non-inducing condition, and minimal medium containing lactose or soluble hydroxyethyl cellulose (HEC) as inducing substrates.Hyphal growth was detected after 14 h on glucose and lactose for the Rut-C30 strain (Additional file 1 and Fig. 2).Δres2 showed slower germination on the three substrates.On HEC, hyphae appeared after 21 h for both strains with a slight delay for the mutant strain (Fig. 2).However, even if the germination rate was not or not much reduced for the Δres2 after 21 h on the three substrates, it can clearly be seen on Additional file 1 that the hyphal length of the mutant was much shorter. Deletion of res2 leads to reduced protein production Protein secretion of the two strains was quantitatively measured after growing them in shake flasks in the presence of either glucose or cellulose + lactose.As expected, higher protein secretion was observed in the cellulaseinducing condition than with glucose for both strains.However, protein secretion in the three ∆res2 mutants was lower compared to that in the Rut-C30 parental strain (Fig. 3A).The biomass of the mutants and the parental strain were very similar or even slightly higher in the mutants, leading also to a reduced protein production yield in the ∆res2 strains (Fig. 3B).These results are in agreement with those of [19] where protein secretion decreased approximately by 30% in N. crassa ∆res2 strains grown on 1% w/v crystalline cellulose supporting the hypothesis that the TF Res2 is involved the regulation of protein secretion. Impact of secretion stress on gene expression in Rut-C30 and ∆res2 To determine if RES2 plays a role in the secretion pathway regulation or the secretion stress response in the hyperproducer Rut-C30, a transcriptomic study was conducted in conditions inducing secretion stress.It had previously been demonstrated that growth of Rut-C30 in lactose fed-batch culture [18] and addition of DTT [12] to T. reesei culture induce UPR.To choose appropriate stress conditions, preliminary fed-batch cultures on lactose and glucose with or without addition of DTT were conducted and the transcript levels of the UPR marker gene bip1 determined at different time points by qPCR.Fed-batch fermentation allows to obtain a higher protein production in lactose culture and to control the pH, thus reducing variables that can affect gene regulation and mimicking industrial like conditions.The results showed that induction of cellulase production by lactose feeding and exposure to 10 mM DTT led to an increase in the UPR marker gene bip1 (Fig. 4).The highest increase of bip1 transcript levels was detected at 2 h after addition of DTT.In the lactose cultures, there was only a low increase of bip1 mRNA levels compared to the glucose cultures, indicating a moderate secretion stress.Similarly to bip1, transcript levels of the UPR marker genes pdi1 and hac1 (spliced version) were also highest 2 h after DTT addition (Additional file 2).In addition, lactose induced the transcription of cellulases as expected, evidenced by an increase of the transcription levels of the cbh1 gene at all time points.However, the cbh1 gene was repressed after 4 and 6 h in the glucose culture with DTT, and much less induced in lactose cultures with DTT (Additional file 2).Taking into account these results, we decided to analyze gene expression by RNA-seq 2 h after exposure (D) or not (C) to 10 mM DTT in the reference strain Rut-C30 (R) and one representative transformant of the Δres2 mutant (∆r) in glucose (G) and lactose (L) fed-batch cultures, leading to four culture conditions : Fig. 1 Growth of three ∆res2 transformants and Rut-C30 on different substrates.A Rut-C30 and Δres2 transformants were grown on minimal medium supplemented with 2% non-inducing substrate (glucose) or 2% inducing substrate (lactose), with or without addition of DTT.Photos and measurements were done on the fifth day after inoculation.For each strain, three biological replicates were assayed, but only one replicate is shown representatively.B Size of colonies of the ∆res2 mutant compared to its parental strain Rut-C30.The error bars indicate the standard deviation of three biological replicates.Significance of the difference between the Rut-C30 and the mutant strains was calculated with the two-tailed t-test with independent variables (*P < 0.01) Fig. 2 Germination rate of Rut-C30 and Δres2 glucose, lactose and HEC.The error bars indicate the standard deviation of three biological replicates of one representative transformant of Δres2 Fig. 3 A Concentration of secreted proteins of ∆res2 mutant and its parental strain Rut-C30.B Biomass and yield of ∆res2 and Rut-C30 in glucose cultures.Strains were cultured for seven daysin 25 mL of BTCA medium in the presence of either glucose or cellulose and lactose.All cultures were inoculated with the same number of spores (~2x10 5 spores).The error bars indicate the standard deviation of three biological replicates for glucose cultures and two biological replicates for cellulose + lactose cultures.Significance of the difference between the Rut-C30 and the mutant strains was calculated with the two-tailed t-test with independent variables (P < 0.05, P < 0.01,*P < 0.001) Glucose Control (GC), Glucose DTT (GD), Lactose Control (LC), Lactose DTT (LD).Transcriptome data were used for pair-wise comparison of cultures within each strain (GD/GC, LD/LC, LC/GC, or LD/GD) and between strains for the same conditions.The results of the fedbatch fermentations (protein concentration and biomass) are presented in Additional file 3. A total of 1163 and 1664 genes (~ 10% of the genome) were differentially expressed (DE) in at least one of the four comparisons in the Rut-C30 and the Δres2 strains, Additional files 4 and 6, respectively.Exposure to 10 mM DTT led to a much higher change in gene expression in Rut-C30: 851 and 849 Differentially Expressed Genes (DEGs) in GD/GC and LD/LC compared to 171 and 129 in lactose-induced stress conditions LC/GC and LD/GD, respectively.Similarly, 1188 and 1172 genes were DE in Δres2 in GD/GC and LD/LC compared to 318 and 259 in LC/GC and LD/GD, respectively.Remarkably, in Δres2 more genes were differentially expressed genes than in Rut-C30.Whereas approximately as many genes were up -and downregulated due to the addition of DTT, there were much more upregulated genes in the lactose compared to the glucose cultures for both strains.The only exception is the LD/GD of the Δres2 strain with more down-than upregulated genes (Fig. 5A). Out of 1163 DEGs, only 192 (~ 16.5%) were uniquely DE in Rut-C30 while 693 (~ 41.6%) out of 1664 DEGs were uniquely DE in Δres2.971 DEGs were common between the two strains.934 genes (~ 56%) of the DEGs in Δres2 were common with Rut-C30 in DTT-induced stress conditions (GD/GC and LD/LC) (Fig. 5B).What is intriguing is that only few DEGs (141, ~ 8.5%) were common between the two strains in the lactose-induced stress conditions (LC/GC and LD/GD) (Fig. 5C).Most of them code for CAZymes (e.g.CEL3D), and a smaller number code for transporters (e.g.TrSTR1) or redox regulators (e.g.AOD1).Interestingly, a much higher number of genes, i.e. 336 were differentially regulated in the Δres2 strain only, but only 175 were unique to Rut-C30 in the lactose conditions. res2 and Rut-C30 have similar clusters of DE genes A clustering analysis of DEGs of both strains was performed.Five different expression profiles were found for Rut-C30 and Δres2, but Δres2 had a sixth cluster of unassigned genes (Fig. 6).The first five clusters display very similar profiles. Gene ontology enrichment analysis (Additional files 5 and 7) revealed that the first cluster was enriched in genes encoding secreted proteins such as cellulases and other lignocellulose-degrading enzymes (e.g.TRIRE-DRAFT_123989 cel7a/cbh1, TRIREDRAFT_74223 xyn1, TRIREDRAFT_76672 cel3a/bgl1) in both strains.Transcript levels were greatly reduced in the presence of DTT, both with lactose and glucose as carbon source, and significantly increased in the presence of lactose compared to the glucose control culture.Rut-C30 had an additional enriched GO term cell wall polysaccharide metabolic process (e.g.TRIREDRAFT_120229 xyn3 and TRIRE-DRAFT_121127 bxl1).Thus, the res2 deletion did not impact lactose induction of genes involved in biomass degradation.The second cluster contained genes involved in carbohydrate metabolic process for both strains.These genes (e.g.TRIREDRAFT_104797 bgl3j and TRIRE-DRAFT_46816 cel3d) showed moderately lower expression levels in the presence of DTT and either no change in expression in the presence of lactose or some increase in transcript levels.In addition, small molecule metabolic process genes (e.g.TRIREDRAFT_110414 uga1 and TRI-REDRAFT_123288 xki1) and transmembrane transport (e.g.TRIREDRAFT_104072 xlt1) were also enriched.Some GO terms were specifically enriched in Δres2 with 430 genes in this cluster, compared to only 118 in Rut-C30: carboxylic acid metabolic process (e.g.TRIRE-DRAFT_102382 glo2 and TRIREDRAFT_121449 his3) and branched chain amino acid metabolism (e.g.TRIRE-DRAFT_122868 hom6 and TRIREDRAFT_51499 ilv5) were found to be enriched. The third cluster containing approximately a hundred genes in both strains was characterized by a high upregulation of genes (e.g.TRIREDRAFT_106245 cta1 and cytochrome P450-encoding genes) acting against oxidative stress in conditions where DTT was present.An increase in transcript levels of Redox active genes is unsurprising as DTT is a reductant that has a variety of stress effects on fungal cells. The fourth cluster grouped genes which displayed upregulation in the presence of DTT, but at a lower level than genes in cluster 3. Additionally, they were also slightly upregulated on LC/GC.Genes in this cluster have functions in protein targeting, secretion and lipid metabolism.Also, UPR genes such as TRI-REDRAFT_122920 bip1 and TRIREDRAFT_122415 pdi1, as well as genes of the ERAD pathway like TRIRE-DRAFT_50647 hrd1 and TRIREDRAFT_47330 lcl2 fall in this group.In Rut-C30, protein glycosylation related genes were also enriched, whereas the GO terms DNA repair and cellular response to stress were more specifically enriched in the mutant. Although the fifth cluster grouped genes with similar gene expression profiles, i.e. a moderate downregulation with DTT and in LC/GC, the most enriched GO terms in each strain were different.The three most enriched GO terms in RUT-C30 were ribosome biogenesis (e.g.TRIREDRAFT_104595 snu13 and TRIRE-DRAFT_124149 nhp2), purine-containing compound metabolic process (e.g.TRIREDRAFT_120568 eno1 and TRIREDRAFT_47221 ynk1) and branched-chain amino acid biosynthetic process, while in Δres2, genes related to amide transport (e.g.TRIREDRAFT_59364 opt2) were enriched.For the sixth cluster of unassigned genes in Δres2, no specific enrichment of functions was found, but it contained genes coding for proteins with putative functions in pseudouridine synthesis (e.g.TRIREDRAFT_3671 gar1 and TRIREDRAFT_44449 cbf5) or carbohydrate catabolic process (e.g.TRIRE-DRAFT_121735 cel3b and TRIREDRAFT_55319 abf2). Function of differentially regulated genes in the Δres2 strain Although clustering did not reveal major differences in global the gene expression patterns between Rut-C30 and the res2 deletion strain, we looked for individual genes that were differentially regulated in both strains.To potentially identify genes related to the protein secretion pathway, we focused on the fourth cluster grouping many of these genes of which 443 and 622 (~ 5% of the genome) were DE in Rut-C30 and Δres2, respectively.However, genes involved in secretion or secretion stress response such as UPR or ERAD were not differentially expressed in the Δres2 strain compared to Rut-C30 under any condition implying that RES2 is not involved in the regulation of the secretion stress response.But several other genes related to the secretion pathway and to other metabolic functions displayed differential expression in the two strains in various clusters and are described in more detail below. As DTT might impact a lot of cellular functions and lead to a high number of DEGs, we first concentrated on lactose-induced stress conditions (LC/GC or LD/GD).This condition more specifically highlights DEGs that are potentially involved in protein secretion rather than other kinds of biological processes.For example, some MFS (Major facilitator superfamily) and ABC transporters were found to be upregulated in the condition LC/GC in Δres2 but not regulated in Rut-C30, such as low-affinity glucose transporter TRIREDRAFT_106556 (hxt13), TRIREDRAFT_62747, and TRIREDRAFT_47897 which is involved in the oxidative stress response.One MFS transporter in this cluster is downregulated in the mutant (TRIREDRAFT_58561).On the other hand, TRIRE-DRAFT_61278 encoding a putative high affinity glucose transporter was upregulated specifically in Rut-C30 in the LD/LC condition (Table 1).Another gene, TRIRE-DRAFT_124198 coding for a putative secreted protein of unknown function, displayed a Log2 fold change (L2FC) that increased from 1.56 in Rut-C30 to 4.3 in Δres2 in the condition LC/GC. Other upregulated genes in LC/GC in Δres2 but not DE in Rut-C30 include TRIREDRAFT_46285 encoding the chaperon HSP30, the putative alcohol dehydrogenase involved in redox reactions TRIREDRAFT_77770 and pks2 (TRIREDRAFT_65891) having roles in secretion and secretion metabolism.Also, a G-proteincoupled receptor involved in cellulose sensing csg1 (TRIREDRAFT_27948) is upregulated in GD/GC and LC/GC in in Δres2 only and falls in cluster 4. Interestingly, several lipid metabolism genes of this cluster were specifically DE in the lactose culture in the mutant strain, such as phospholipase D (pldB, TRI-REDRAFT_22331) and TRIREDRAFT_45980, a gene encoding a protein putatively involved in phospholipid translocation.They are both upregulated in the in Δres2 strain in this condition (Table 1).The ple gene (TRIRE-DRAFT_21960) encoding phospholipase E showed a similar upregulation in LC/GC but was grouped in the unassigned genes cluster of Δres2.On the other hand, the acyltransferase CST26 gene (TRIREDRAFT_109980) was induced in Rut-C30 only in the LD/LC condition.This Candida albicans orthologue encodes a transferase involved in phospholipid synthesis and its lack of induction suggests that its expression could be regulated by RES2 (fungiDB). An important component of fungal membranes is ergosterol.But only two genes of the ergosterol pathway were found to be differentially regulated in the Δres2 strain compared to Rut-C30: the are2 gene encoding a sterol-O-acyltransferase (TRIREDRAFT_50607) and erg3, a putative C-14 sterol reductase (TRIRE-DRAFT_81049).The former was specifically induced in LC/GC whereas the latter was repressed in the presence of both DTT and lactose (LD/LC) in the mutant strain.Therefore, biosynthesis of ergosterol did not seem to be significantly affected by the lack of RES2. In yeast and filamentous fungi, UPR is linked to cell wall integrity [21] and in yeast, the two pathways are coordinately regulated.In Rut-C30 and the Δres2 mutant, UPR is induced with DTT and cellulase induction by lactose.Even if UPR related genes were not DE in the two strains, we verified if the expression of genes coding for putative cell wall modifying enzymes were differentially affected.Indeed, a chitinase (TRIREDRAFT_120953), two β-1,3 glucanases belonging to family GH55 (TRIRE-DRAFT_73248 and TRIREDRAFT_ 121746) and a GH71 α-1,3 glucanase were differentially regulated in the two strains in either of the two secretion stress conditions.The GH18 gene was induced in LC/GC in the Δres2 strain only whereas both GH55 genes were repressed in the same strain in the presence of either lactose or DTT.GH71 gene is downregulated in LC/GC, but only in Rut-C30 (Table 1).All four proteins are predicted to be secreted or cell wall located. We also analyzed the effect of the res2 deletion on media containing the redox stress agent DTT in more detail.Again, genes encoding proteins involved in the translocation or intracellular transport of phospholipids were found to be upregulated uniquely in the Δres2 mutant (ept1, TRIREDRAFT_55627 and Sec14, TRIRE-DRAFT_8192).These results suggest that the deletion of res2 impacts the expression of genes involved in the ER membrane synthesis or homeostasis, which is particularly important in conditions of secretion stress. Another functional group of genes for which the expression was found to be impacted by the deletion of res2 encode glycosyltransferases (GT).Among other functions, these enzymes are catalyzing several protein glycosylation steps during maturation in the ER and Golgi.Three GT (TRIREDRAFT_72788, TRIRE-DRAFT_77283 and TRIREDRAFT_120923) belonging to CAZy families GT2, GT31 and GT32 were upregulated in the presence of DTT, but in the Δres2 mutant only.Three other GT (TRIREDRAFT_66687, TRIREDRAFT_64925 and TRIREDRAFT_122992) were upregulated with DTT uniquely in Rut-C30.It is noteworthy that all the mentioned GT whose genes were DE are predicted, by localization prediction online tools, to be located in the ER or Golgi apparatus.Even if it is not possible at this stage to evaluate the real impact on glycosyl side chains of secreted proteins, glycosylation is probably altered in the Δres2 mutant in the presence of DTT which could interfere with normal secretion and/or activity of secreted enzymes.These results suggest that RES2 is, probably among other functions, somehow involved in the regulation of protein synthesis and protein fate and of pathways related to secretion.It is not known if RES2 acts directly on the regulated genes or if its action involves other factors.Therefore, we analyzed the data for differentially regulated TFs.In Rut-C30, only two genes encoding putative transcriptional regulators were DE compared to the Δres2 strain, one up-and the other downregulated (Table 2).In contrast, in the latter, six genes were specifically DE in the deletion strain and four of them were downregulated in the presence of DTT.TRIRE-DRAFT_109538 belonging to cluster 4 was upregulated in the GD/GC condition whereas the sixth one, TRIRE-DRAFT_12107, a homeodomain-containing protein, was upregulated in the lactose culture in the mutant only.Interestingly, the ortholog of this gene in Penicillium oxalicum was found to be involved in cellulase and xylanase production [22,23].Finally, the VIB1 TF (TRI-REDRAFT_54675) which belongs to cluster 2 was downregulated in both strains in the presence of DTT (GD/GC and LD/LC) but upregulated by Log2fold change factor of 1.85 in the presence of lactose in the Δres2 strain only.This points to an eventual interaction between the two transcription factors in cellulase inducing conditions. Discussion In this study, the role of the TF RES2 in secretion and the secretion stress response in T. reesei Rut-C30 was investigated.We could show that deletion of the res2 gene resulted in reduced radial growth on glucose and lactose and slower germination as well as in decreased productivity in both non-inducing and cellulase secretion inducing conditions.The phenotype observed resembles the one obtained in the N. crassa res2 deletion mutant but is not identical.In N. crassa, growth of the Δres2 mutants on minimal media was indistinguishable from the WT strain on minimal media and reduced only at concentrations > 5 mM DTT [19].In our case, we could not find a negative effect of DTT on the mutant up to 5 mM DTT, the highest concentration tested.Addition of DTT even seemed to promote growth in the mutant strain, an unexpected result.However, growth of the deletion strain was clearly reduced in the absence of DTT on all carbon sources compared to Rut-C30. In this context, it is important to mention that despite the visibly slower growth in solid cultures, biomass production in liquid cultures was not reduced in the mutant and the lower protein secretion could thus not be explained by a reduced growth.The difference in extracellular protein concentration was therefore due to a less efficient secretion, both in non-inducing and inducing conditions. When comparing T. reesei to N. crassa, one must keep in mind that regulation of cellulase gene expression implies different transcription factors in these species.Whereas in N. crassa, CLR-1 and CLR-2 are essential for cellulase gene activation [24], XYR1 and ACE3 are the main regulators in T. reesei [25,26].Indeed, no orthologous genes of clr-1 and clr-2 are present in the latter.Another important fact is that the present study was done on the hyperproducing strain Rut-C30 in which cellulase genes are highly inducible and where the role of RES2 might be altered compared to the wild type strain QM6a.However, it is also worth mentioning that there are no mutations in the sequence of res2 gene in the hyperproducing strain Rut-C30 compared to that in the wild-type strain QM6a and that the expression of this gene is similar in the QM6a strain and the Rut-C30 both in glucose-and lactose-containing media (F.Bidard-Michelot, unpublished results). In accordance with previous studies, we observed downregulation of transcription of many secreted proteins (mainly cellulases) in DTT stress conditions.This can be explained by repression under secretion stress (RESS), since induction of this stress response mechanism by DTT in Rut-C30 was already evidenced before [12].The reduction of cellulase gene expression by DTT is the same in Rut-C30 and Δres2, which indicates that RES2 is not involved in the regulation of this mechanism.It does not seem to play a significant role in triggering the UPR or ERAD response either, as pdi1 and bip1 as well as most ERAD-related genes, such as hrd1, hrd3, lcl2, and cpr1 were not DE between the two strains.This is in contrast to the results obtained in N. crassa, where RES2 was proposed to be involved in secretion and the secretion stress response [19].Although the res2 gene was moderately upregulated in T. reesei Rut-C30 in the presence of DTT, similarly to N. crassa (Log2 fold change 1,38 and 1,6 respectively), the role of this transcriptional regulator under these stress conditions is apparently different N. crassa and in the hypersecreting T. reesei strain. Most oxidoreductases, catalases, dehydrogenases and cytochrome-P450 were upregulated only in DTT conditions.This is expected due to the higher stress induction by the reducing effect of DTT.However, several redox regulators (e.g.TRIREDRAFT_80659 and TRI-REDRAFT_77770) were upregulated in Δres2 uniquely implying that RES2 plays a role in maintaining a stressfree cellular environment in Rut-C30, and its deletion evokes the release of reactive oxygen species. As in N. crassa, the res2 deletion was found to decrease secretion of cellulolytic enzymes.However, whereas hemicellulase and cellulase transcription was downregulated in Avicel cultures of the N. crassa Δres2 strain, the mRNA levels of these genes in the T. reesei Δres2 were identical to those in the Rut-C30 strain upon induction by lactose.This indicates that RES2 does not contribute significantly to the transcriptional induction of cellulase genes by lactose in Rut-C30.In fact, in this hyper producing strain, the gene of the major transcriptional regulator of lignocellulase genes, XYR-1 is highly expressed, and the high induction mediated by this TF might override more subtle regulations by other TFs such as RES2.Despite the induction of cellulase encoding genes by lactose in the deletion strain, protein secretion was lower in this condition compared to the parental strain.There are different explanations for this observation.First, it could be the result of a regulation at the translational level.Even if inhibition of translation does not play an important role in ER stress in Trichoderma [12], the observed impact of the res2 deletion on the amino acid biosynthesis pathway could slow down protein elongation.The decrease could also be related to modification steps at the post-translational level such as folding and maturation of the polypeptides in the ER and the secretory pathway.Among the genes specifically DE in the Δres2 strain in the presence of lactose we could identify genes involved in phospholipid metabolism (e.g.phospholipase E), a chaperone (TRIREDRAFT_46285), both upregulated, but also genes putatively involved in the modification of the protein glycans (TRIRE-DRAFT_73248, TRIREDRAFT_77547, both downregulated).It is therefore possible that changes in the ER membrane or the protein glycan side chains lead to less efficient protein secretion.Alternatively, enhanced protein degradation in the extracellular space might also explain the decreased protein concentration.TRIRE-DRAFT_82623, a putative secreted subtilisin, was specifically induced in the mutant in the presence of lactose, even if the p-value of the LC/GC condition was above 0.005 (0.03).As some lipid metabolism genes were regulated, it is possible that RES2 contributes to the highly developed ER structure in Rut-C30.Analysis of the ER structure of the Δres2 strain by electron microscopy could verify this hypothesis. Approximately 5% (459) of the genome of T. reesei encode proteins having transport functions (T.reesei transporters annotated in the genome portal http:// genome.jgi-psf.org/ Trire2/ Trire2.home.html).Among these genes, there are around 50-100 sugar transporters of which the majority are uncharacterized [27,28].Concerning transporter-coding genes that were DE uniquely in Δres2 in this study, many of them belong to the MFS superfamily that comprises 16 different families with 89 subfamilies [29] of which each can transport essential nutrients and ions [30] and excrete end products of metabolism [31].The MFS transporters STR1, CRT1 and STP1 can induce the expression of CAZymes as demonstrated by previous studies [32][33][34].CRT1 was demonstrated to have a direct regulatory role on cellulase gene expression, independently of its transporting activity [32].In our study, the stp1 gene was not DE in any condition, while str1 and crt1 were induced in the presence of lactose but downregulated in the presence of DTT.These lower transcript levels could also have contributed to the downregulation of cellulase gene transcripts in the presence of DTT, in addition to the RESS mechanism.In the Δres2 strain, the functional category "transmembrane transport" of uniquely DE genes was enriched (Table 3), and many sugar and amino acid transporters, especially those belonging to cluster 3, were upregulated in GD/ GC in this strain only.Others were downregulated in the presence of DTT.The downregulation of transporters in the presence of stress signals was already reported before [35].As transporters must often be addressed to the plasma membrane, they also pass the ER/Golgi pathway, and it is not surprising that their regulation follows those of secreted proteins. We could show that the res2 deletion led to differential expression of several TFs compared to Rut-C30.All but two of them were differentially regulated (up-or down) in response to DTT in the medium, pointing to a role in the stress response of the fungus to this toxic compound, rather than in protein secretion. One of them is the cross-pathway control encoding gene cpc1 which was found to be induced with DTT in both Rut-C30 and Δres2 mutant (log2 fold change of 3.3 and 2.6, respectively).This was already observed in other fungi [19,36] as well as in the work of [13] where also many potential target genes involved in amino acid biosynthesis were induced by DTT.In our study, amino acid synthesis genes were found to be DE in both strains, but with some differences: tryptophane biosynthesis genes were enriched uniquely in Rut-C30 and were upregulated, while valine biosynthesis genes were enriched in the mutant, but downregulated (Table 3).A hypothesis is that RES2 interacts with CPC1 to finetune the expression of AA metabolic genes.But as cpc1 transcript levels were identical in the mutant and Rut-C30, RES2 does apparently not control the expression of cpc1.Yet, it would be interesting to investigate if CPC1 has a role in the regulation of the res2 gene. Conclusions In an effort to decipher the regulatory mechanism of protein secretion in the hyperproducer T. reesei Rut-C30, the role of the RES2 transcription factor was investigated.We could show that the deletion of res2 impacts fungal growth, germination, and protein secretion.Transcriptomic analysis revealed that CAZyme gene expression was not dependent on the action of RES2 which had no regulatory role on most elements of the stress response mechanisms UPR, ERAD and RESS either.The results are therefore rather in favor of an indirect action of RES2 on protein secretion by modulating the expression of transporters, lipid metabolism and protein modification genes.A major regulatory role of RES2 in amino acid synthesis was also evidenced.As a consequence, it would be interesting to dedicate future studies to the elucidation of a potential interplay between RES2 with other transcription factors such as CPC1.A comparison with the regulatory network involving RES2 in the wild-type strain QM6a could also be interesting and deliver some clues for understanding the hypersecreting phenotype of Rut-C30. Strains and media Rut-C30 (ATCC 56,765) strain was used in this study, and the Δres2 mutant was derived from this strain.Butanetetracarboxylic acid (BTCA) medium, which was used to culture Δres2 and Rut-C30 for protein measurement experiments, was composed of 5. g/l agar, the pH was adjusted to 6.To examine the germination of the mutant strain Δres2 and its parental strain Rut-C30, 2 × 10 5 spores were diluted 200x and spread on plates containing 2% carbon source and solid minimal media.Hyphal growth and branching were observed using microscope ZEISS Imager.M2. Fed-flask experiments Fed-flask experiments were performed in an Infors incubator and using peristaltic pumps (Dasgip MP8) as described by [37] Measurement of relative gene expression by RT-qPCR Total extracted RNA was reverse transcribed into complementary DNA (cDNA) using iScript ™ cDNA synthesis kit (Bio-Rad, Hercules, USA) and random primers following the instructions of the manufacturer. To measure relative expression of the secretion stress biomarkers bip1, pdi1 and hac1, as well as the cellobiohydrolase gene cbh1, qPCRs were done using the iQSYBR Green Supermix (Bio-Rad) and 1 µl of five-fold diluted cDNA in a total volume of 20 µl.The sequence of the primers used are indicated in Additional file 8. Normalization was based on two reference genes sar1 (Y08636.1)and glk1 (DQ068384.1)which code for SAR/ARF type small GTPase and glucokinase, respectively [38], and the relative transcript level of each gene was calculated with the Pfaffl method (EGOI) � CtGOI GeoMean[(EREF) � CtREF [39]. RNA-seq library preparation and bioinformatic analyses Library preparation and Illumina sequencing were performed at the Ecole normale supérieure Genomique core facility (ENS Paris, France).Messenger (polyA+) RNAs were purified from 1 µg of total RNA using oligo(dT).Libraries were prepared using the strand specific RNA-Seq library preparation Stranded mRNA Prep kit (Illumina).A 118 bp single read sequencing was performed on a NextSeq 2000 device (Illumina).A mean of 42 ± 6 million sequences passing the Illumina quality filter reads was obtained for each of the sixteen samples (including a biological duplicate for each of the eight samples).Bioinformatic analyses were performed using the Eoulsan (version 2.5) pipeline [40], including read filtering, mapping, alignment filtering, read quantification, normalization and differential analysis: Before mapping, poly N read tails were trimmed, reads ≤ 40 bases were removed, and reads with quality mean ≤ 30 were discarded.Reads were then aligned to the T. reesei QM6a genome (JGI version) using STAR (version 2.7.8a) [41].Alignments from reads matching more than once on the reference genome were removed using htsjdk 1.118 [42].To compute gene expression, a custom T. reesei QM6a annotation file was used.All overlapping regions between alignments and referenced CDS were counted using HTSeq-count 0.5.3 [43].The sample counts were normalized using DESeq2 1.8.1 [44].Statistical treatments and differential analyses were also performed with DESeq2 1.8.1.Log2 fold changes were calculated after count normalization, and genes with a mean of at least 100 reads in the respective conditions and displaying a log2fold change > \|2\| and p-value < 0,005 were considered to be differentially expressed. RNAseq expression data and raw fastq files were deposited in the GEO repository (accession number GSE233738).After filtering DEGs from a total of 10,008 genes, clustering of the DEGs was done using MeV (Multiexperiment Viewer).Gene ontology (GO) of each cluster was obtained using the biological process gene ontology enrichment analysis tool in the fungidb database (https:// fungi db.org).Venn diagrams were drawn with the help of an online bioinformatics tool [45].Intracellular localization was predicted using SignalP − 6.0 [46] and Euk-mPLoc 2.0 [47]. Construction of the deletion strain Gene deletions were performed using CRISPR-Cas9.A mix of guide RNA, Cas9 protein forming the ribonucleoprotein complex (RNP) as well as the donor DNA was set up.The Cas9 protein used was purchased from the NEB group (EnGen ® Spy Cas9 NLS).Guide RNAs were designed using chopchop online tool [48] and Geneious ® Prime bioinformatics software [49].The deletion cassettes containing the resistance gene hygromycin hph flanked by 200 bp homologous to regions on either side of the locus of the target genes were synthesized by Twist Bioscience and PCR amplified.The sequence of gRNAs and Primers can be found in Additional file 8. 10 µL RNP complex containing 1 µl Cas9 (20 µM), 6 µl guide RNA (30 µM), 2 µl of H 2 O DEPC, and 1 µl of 10x Cas9 buffer (NEBuffer ™ 3.1) was incubated 10 min at room temperature, then mixed with 50 µl of protoplasts at a concentration of 2.10 8 ml −1 and 5 µg donor DNA (deletion cassette) and incubated for another ten minutes at room temperature.1 ml of 60% PEG 4000 solution was added and after gentle stirring at room temperature for 20 min, 900 µl of CTS50 (0.4 M saccharose, 0.1 M Tris HCl, 50 mM CaCl 2 , pH 7.5) were added.The transformation reaction was mixed with 40 ml of liquid PDA medium supplemented with 50 µg ml −1 hygromycin and 0.8 M of sucrose and poured into five Petri dishes with selective medium containing 50 µg ml −1 hygromycin.Single transformants were purified in three successive steps as described by [50]. Genetic validation of Δres2 deletion DNA was extracted from mycelia after culturing the strains on PD medium for two to four days.DNA extraction was performed using the Nucleospin Soil Genomic DNA kit (Macherey Nagel) and following the soil protocol provided by the manufacturer.PCR was conducted using the Q5 High-Fidelity DNA Polymerase (NEB) and the primers indicated in Additional file 8. 1 ng -1 µg genomic DNA was used for a 50 µl reaction.In addition to PCRs, the PCR amplicons were sequenced to verify that there were no mutations in the integrated cassette. To verify the absence of off-targets in Δres2 transformants, qPCRs were done on the gDNA of Δres2 and two reference strains, each containing two hygromycin cassettes in its genome.Primers targeting the hygromycin resistance gene and the reference gene bgl2 are indicated in Additional file 8.The copy number of deletion cassettes integrated into the genome of T. reesei was calculated with the Livak method (2 −ΔΔCT ) [51]. Protein concentration measurement Protein concentrations of culture supernatants were determined using the Quick Start Bradford protein assay kit (Bio-Rad) and bovine serum albumin (BSA) as a standard.The assay was done on a Freedom Evo robot (TECAN). Biomass measurement To measure the biomass, the mycelium was recovered by filtering 6 mL of the liquid culture on a cellulose filter which has been previously placed at 105 °C for 24 h.After drying the filter with the mycelium at 105 °C for 24 h, the filter was weighed again, and the biomass dry weight was calculated by subtracting the tare. Fig. 4 Fig. 4 Relative gene expression of the secretion stress biomarker bip1 in Rut-C30 in fed-batch cultures.RNA samples were taken at three timepoints (2h, 4h, 6h) after the addition of DTT.The relative expression of bip1 with respect to that on glucose at 2h is shown.The error bars indicate standard deviation of two biological replicates Fig. 5 Fig. 5 DEGs of Rut-C30 (R) and Δres2 (Δr) in two stress conditions.A Bar graph displaying the number of up and down-regulated genes in Rut-C30 and Δres2 in each condition using a 0.5% false discovery rate cut-off and with an absolute log2 fold change greater than 2. Venn diagrams showing both unique and common DEGs in Rut-C30 and Δres2 in B the presence vs. absence of DTT and C glucose vs. lactose cultures Table 1 Significantly differentially regulated genes in only one of the strains in the indicated condition(s) Table 1 (continued) PM Plasma membrane Table 2 Genes encoding putative transcriptional regulators differentially regulated in only one strain in the indicated condition Table 3 Enriched functions of uniquely DE genes in Rut-C30 and Δres2	2023-11-30T15:08:24.820Z	2023-11-30T00:00:00.000	{ "year": 2023, "sha1": "3aad0f8260506422a597ef27e4c33135ee0f0b26", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Springer", "pdf_hash": "3aad0f8260506422a597ef27e4c33135ee0f0b26", "s2fieldsofstudy": [ "Biology", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
235427846	pes2o/s2orc	v3-fos-license	Antimicrobial Peptides: A New Hope in Biomedical and Pharmaceutical Fields Antibiotics are essential drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. Antibiotic resistance is a serious challenge and has led to the need for new alternative molecules less prone to bacterial resistance. Antimicrobial peptides (AMPs) have aroused great interest as potential next-generation antibiotics, since they are bioactive small proteins, naturally produced by all living organisms, and representing the first line of defense against fungi, viruses and bacteria. AMPs are commonly classified according to their sources, which are represented by microorganisms, plants and animals, as well as to their secondary structure, their biosynthesis and their mechanism of action. They find application in different fields such as agriculture, food industry and medicine, on which we focused our attention in this review. Particularly, we examined AMP potential applicability in wound healing, skin infections and metabolic syndrome, considering their ability to act as potential Angiotensin-Converting Enzyme I and pancreatic lipase inhibitory peptides as well as antioxidant peptides. Moreover, we argued about the pharmacokinetic and pharmacodynamic approaches to develop new antibiotics, the drug development strategies and the formulation approaches which need to be taken into account in developing clinically suitable AMP applications. INTRODUCTION A wide variety of antimicrobial agents are available today and they are broadly applied to treat different types of human infections. Specifically, antibiotics are powerful drugs used for treatments of pathogenic bacteria (Lei et al., 2019). However, their indiscriminate and prolonged use, especially in developing countries, in both human and veterinary medicine, as well as in agriculture have contributed to the development and spread of drug-resistant microorganisms (Huan et al., 2020). As the World Health Organization (WHO) has extensively announced, the alarming rise globally in resistance towards conventional antimicrobials represents a potential and serious risk to public health (Luong et al., 2020). Therefore, the antibiotic resistance issue has made it urgent to search for alternatives to conventional antibiotics, with novel modes of action and less predisposed to bacterial resistance. In the quest of new antibiotics, the antimicrobial peptides (AMPs), also known as host defense peptides, have recently raised great interest (Haney et al., 2019;Bhattacharjya and Straus, 2020;Mahlapuu et al., 2020). Current research is focused on these natural compounds as innovative anti-infective drugs and novel immunomodulatory candidates (Luong et al., 2020;Mahlapuu et al., 2020). AMPs are bioactive small proteins, naturally produced by all living organisms as important and indispensable components of their innate immune system, becoming the first-line defense against microbial attacks in Eukaryotes, or produced as a competition strategy in Prokaryotes, to limit the growth of other microorganisms (Lei et al., 2019;Magana et al., 2020). Natural AMPs have potent and broad-spectrum activity against multiple classes of bacteria, yeasts, fungi, viruses and parasites (Huan et al., 2020;Luong et al., 2020), displaying bacteriostatic, microbicidal and cytolytic properties (Pasupuleti et al., 2012). Moreover, the interest in AMPs has recently increased during the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic in the search of new antiviral molecules to counteract COVID-19 disease (Kurpe et al., 2020). AMPs were discovered in 1939, when the microbiologist ReneD ubos isolated from a soil Bacillus strain, an antimicrobial agent, named gramicidin, which was demonstrated to protect mice from pneumococcal infection (Van Epps, 2006). Afterwards, several AMPs have been discovered from both the prokaryotic and eukaryotic kingdom (Boparai and Sharma, 2020), including the tyrocidine, produced by the bacteria Bacillus brevis, with activity against bacteria, and the purothionin, identified in the plant Triticum aestivum, active against fungi and bacteria (Ohtani et al., 1977). The first described animal-originated AMP is defensin, which was isolated from rabbit leukocytes (Hirsch, 1956); subsequently lactoferrin was identified in cow milk (Groves et al., 1965) and it was demonstrated that lysosomes of human leukocytes (Zeya and Spitznagel, 1966) and human female reproductive tract contain low molecular weight AMPs (Sharma et al., 2011). To date, more than 3,000 AMPs have been discovered, characterized and annotated in the AMP database (APD3) (Huan et al., 2020), just considering that frog skin alone is a reservoir of more than 300 different AMPs (Boparai and Sharma, 2020). AMP Properties and Biosynthesis Natural AMPs are evolutionary conserved gene-encoded molecules with structural and functional diversity, which is responsible for their wide range of activities against different pathogens in various organisms (Zhang and Gallo, 2016). However, although displaying considerable diversity in their physio-chemical and structural properties, origins and mechanisms of action, AMPs share some common features (Moravej et al., 2018). Indeed, they are mostly short molecules (<100 amino acids) (Pasupuleti et al., 2012), typically with a positive net charge (generally ranging from +2 to +11) and a notable proportion of hydrophobic residues (typically 50%) . They display an amphipathic structure, as they contain both hydrophobic and hydrophilic regions, that enable them to be soluble in aqueous environments (Boparai and Sharma, 2020). A less common class of AMPs is represented by the anionic AMPs, which have a negative net charge ranging from -1 to -7 and have been identified in vertebrates, invertebrates and plants (Harris et al., 2009). They include many negatively charged aspartic and glutamic acid residues, and in animals are found in various vital organs, including the brain, the epidermis, the respiratory and gastrointestinal tracts (Lakshmaiah Narayana and Chen, 2015). They show a different mechanism of action than the cationic ones. In order to facilitate their interaction with the target organism, some anionic AMPs use metal ions to form cationic salt bridges with negatively charged constituents of microbial membranes, allowing their penetration into the cell. When they reach the cytoplasm, they may attach to ribosomes or inhibit ribonuclease activity (Jezȯwska-Bojczuk and Stokowa-Sołtys, 2018). Some anionic AMPs, such as theromyzin from Theromyzon tessulatum (Tasiemski et al., 2004), require zinc as a functional cofactor and it was found that the complex with zinc has stronger antimicrobial activity . Despite their relative similarity in biophysical characteristics, AMP sequences are rarely similar among closely related or distinct species/organisms (Pasupuleti et al., 2012). However, for some AMPs, a certain degree of identity is found either in the pro-region (the inactive sequence that is deleted by posttranslational modifications) or in the amino acid patterns. This event could be due to species adaptation to the unique microbial environment that characterize the niche occupied by specific species (Pasupuleti et al., 2012). The amphiphilic nature of the majority of AMPs is responsible for their structural flexibility. AMPs are commonly classified into four categories based on their secondary structure, including linear a-helical peptides, b-sheet peptides with the presence of 2 or more disulfide bonds, b-hairpin or loop peptides with the presence of a single disulfide bond and/or cyclization of peptide chain, and, finally, extended structures (Boparai and Sharma, 2020). Most AMPs belong to the first two categories. ahelical peptides display an unstructured conformation in aqueous solution but adopt an amphipathic helical structure in contact with biological membranes. However, a relevant feature is linked to the possible interactions with bacterial structures, such as lipopolysaccharides (LPS), that provoke conformational changes, influencing membrane permeabilization and the correct passage into the cytosol. Indeed, this interaction could change AMP tertiary structure, and AMP molecules could assume different conformations, such as monomeric helical or helixloop-helix structures ( Figure 1) (Bhunia et al., 2011). For example, the contact with LPS induces oligomerization of specific AMPs, such as temporines, through the interaction among hydrophobic N and C terminal residues, preventing the correct movement throughout the membrane and the correct antimicrobial action (Bhunia et al., 2011). A particular amino acids composition could prevent this oligomerization, enhancing temporin activity. This is the case of temporin-1Tl, which is rich in aromatic residues with two positively charged amino acids (Bhunia et al., 2011). The synergy of temporin-1Tl with other temporins (Temporin A and Temporin B), prevent their oligomerization and facilitate the correct crossing of the bacterial membrane (Bhunia et al., 2011). Exceptions are related to some AMPs with particular structural characteristics, including the peptide MSI-594 (an analogue of magainin), that is unstructured in free solution, but have a folded helical hairpin structure when interact with LPS (Bhattacharjya, 2016). The interactions between two helical segments, facilitated by the fifth phenylalanine residue, allows the acquisition of the hairpin structure, implicating its very high activity against bacteria, fungi, and viruses (Domadia et al., 2010;Bhattacharjya, 2016). Another example of change in conformation after the interaction with LPS, is the b-hairpin structures of Tachyplesin I, that becomes more ordered and compact when interacting with LPS (Saravanan et al., 2012;Kushibiki et al., 2014). Another interesting example is linked to the human LL-37 AMP, one of the best studied peptides of this group, present in neutrophils and epithelial cells (Mahlapuu et al., 2016). It has been demonstrated that aromatic-aromatic interactions stabilize protein structure in correlation with lipids (Li et al., 2006) and that LL-37 could undergo a re-orientation depending on the concentration, suggesting also in this case an oligomerization process (Ding et al., 2013). On the contrary, b-sheet peptides are more ordered in aqueous solution because of their rigid structure and do not undergo radical conformational changes as helical peptides upon membrane interaction (Mahlapuu et al., 2016). It is not easy to clarify the structural conformations of bsheet AMPs in membranes, because of the potential micelle aggregations; indeed, a recent report on thanatin peptide, isolated from insect Podisus maculiventris, showed dimerization of b-sheet structures (Sinha et al., 2017). These dimeric structures could facilitate the bond with LPS molecules, also at the distal ends, fostering bacterial cell associations and agglutination (Sinha et al., 2017). Defensins, a large group of AMPs, which are produced in macrophages, neutrophils and epithelial cells belong to this class (Mahlapuu et al., 2016). It was observed that the right combination of hydrophobicity, charge density and peptide length influence the antimicrobial activity of AMPs. Changing the amino acids position in the peptide chain or increasing the number of positively charged residues affect the secondary structure of AMPs, and consequently their biological activity against pathogens (Wu Q. et al., 2018). Besides the principle that the amino acid sequence determines the function of a peptide, it was found that the amino acid composition (in terms of abundance of residues with specific phyco-chemical properties) also affects AMP activity as clearly documented for a novel class of cationic AMPs known as "cationic intrinsically disordered antimicrobial peptides'' or "CIDAMPs" since they are characterized by an intrinsically disordered structure. CIDAMPs have been detected in human skin and other barrier organs (Gerstel et al., 2018;Latendorf et al., 2019) and, carrying a positive net charge, have a low percentage of order-promoting amino acids (mostly hydrophobic residues commonly located within the hydrophobic core of foldable proteins) and a high percentage of disorder-promoting amino acids (mostly charged and polar residues, typically found at the surface of foldable proteins). They show microbicidal activity against several microbes, including Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa (Gerstel et al., 2018). The protein hornerin, expressed in the cornified epithelium, seems to be the main source of CIDAMPs, which act as disinfectants, helping to keep the surface of healthy skin free of infections (Gerstel et al., 2018). AMP biosynthesis can occur in three different ways: classical ribosomal synthesis, non-ribosomal synthesis and proteolytic digestion of proteins (Buda De Cesare et al., 2020). Ribosomally synthesized AMPs, such as histatins and human b-defensins, are produced by ribosomal translation of specific mRNAs into the biologically active amino acid sequences in vertebrates, insects, plants, and bacteria. Non-ribosomally synthesized peptides are produced by large enzymes referred to as non-ribosomal peptide synthases, which incorporate nonproteinogenic amino acids into the sequence, and are found in filamentous fungi and bacteria (Actinomycetes and Bacilli). Finally, some AMPs, called cryptic peptides, are generated by proteolytic cleavage of bigger proteins with other functions. For example, the histone H2A of the Asian toad (Duttaphrynus melanostictus) is processed by the enzymatic activity of pepsin C producing buforin I, which in turn is processed by an endopeptidase to generate buforin II (Buda De Cesare et al., 2020). Interestingly, many AMPs are produced as inactive precursors and are active after proteolytic cleavage. Therefore, their activity is not only dependent on their own expression but also on the presence of appropriate proteases (Mahlapuu et al., 2016). The expression of AMPs can be constitutive or inducible by specific external factors (Mahlapuu et al., 2016;Lei et al., 2019). Some AMPs are expressed during the whole cellular lifetime but are stored at high concentration as precursors in granules and are released upon infection in the site of infection or inflammation (Mahlapuu et al., 2016). P9A and P9B are examples of inducible peptides, whose expression can be induced in silkmoth (Bombyx mori) hemolymph by vaccination with Enterobacter cloacae, as demonstrated by Hultmark and colleagues (Hultmark et al., 1980). In addition, Bals et al. (1999) reported that defensin production from epithelial cells of multiple mouse organs increases upon infection with P. aeruginosa PAO1. Insights Into the Mechanisms of Action of AMPs The prerequisite to develop efficient AMPs as novel candidate drugs is the understanding of their mode of action. AMPs exert their activity by interaction with microbial cell membranes and this interaction is strongly affected by the lipid composition of biological membranes (Wu Q. et al., 2018). Since microbial membranes are the primary targets of AMPs, it is difficult for bacteria to develop resistance to AMPs as easily as to conventional antibiotics (Boparai and Sharma, 2020). Membrane interactions are mediated by electrostatic forces between positively charged AMPs and negatively charged microbial surfaces. The teichoic acids in the cell wall of Grampositive bacteria and the LPS in the outer membrane of Gramnegative bacteria supply electronegative charge to the microbial surfaces, strengthening the interaction with AMPs (Boparai and Sharma, 2020). On the contrary, the outer layer of eukaryotic membranes is composed by zwitterionic phosphatidylcholine and sphingomyelin, which do not favor AMP interaction because of their neutral charge at physiological pH. Based on their mode of action, AMPs are divided into "membrane acting peptides", which destabilize bacterial membranes causing their disruption, and "non-membrane acting peptides", which are able to translocate across the membranes without damaging them but destabilizing normal cell functions (Boparai and Sharma, 2020) ( Figure 2). Three models have been proposed to explain the permeabilization of bacterial membranes by AMPs: barrelstave model, toroidal-pore model and carpet model (Raheem and Straus, 2019). Thanks to their positive net charge, AMPs are able to interact with components of bacterial membranes, resulting in the disruption of the lipidic bilayer with cell death. AMP insertion can be perpendicular, as in the barrel-stave model, or perpendicular with the interaction with the head groups of lipids that provokes a deflection in the membrane (toroidal model) (Brogden, 2005). AMPs can also dispose A B C FIGURE 2 \| Antimicrobial peptides can act through a membranolytic and non-membranolytic mechanism. In the membranolytic mechanism AMPs can lead to (A) pore formation on the cell membrane or (B) micelle formation on the cell membrane. In the non-membranolytic mechanism, (C) AMPs can penetrate cell membranes and interact with intracellular targets, such as DNA and proteins. Figure created with Biorender.com and UCSF CHIMERA software (Pettersen et al., 2004). parallel to the membrane, covering it completely, and forming, at the same time, micelles with the starting broken membranes (carpet model), as proposed by colleagues in 1996 (Gazit et al., 1996). Moreover, defensins interact with LPS in Gram-negative bacteria and peptidoglycan in Gram-positive bacteria (Pachoń-Ibañez et al., 2017). Defensins have LPSneutralizing activity in different bacteria (Lee et al., 2010) despite the chemical structure of LPS varies among them. LPS can self-aggregate forming oligomers above a Critical Micelle Concentration (CMC) because of its amphiphilic nature, a concentration of LPS, or any surfactant, above which it aggregates in micelles. It has been demonstrated that the association of defensin analogues and other peptides, such as gramicidin A, melittin, LL-37 and polymyxin B, with LPS leads to the disintegration of LPS aggregates. Moreover, it was observed that defensins amino acids (such as Arg, Trp, and Tyr) are involved in the stabilization of the peptide-pathogen surface complexes . The interaction with LPS has been demonstrated to be essential for AMPs like gramicidin S and polymyxin B to exert their mechanism of action for bacterial killing (Zhang et al., 2000). Bhunia and colleagues studied the structure of MSI-594 peptide in LPS micelles. They observed that the peptide is unstructured in solution, while it adopts a helix-loop-helix structure in complex with LPS, suggesting how AMPs could overcome the LPS barrier (Bhunia et al., 2009). A mutant form of MSI-594 peptide, substituting Phe5 with Ala amino acid, displays a limited permeabilization through the LPS layer suggesting that peptide conformation is essential to disrupt LPS (Domadia et al., 2010). Other examples of AMPs acting by perturbation of microbial membrane structure are the fungal peptide alamethicin, the amphibian AMP aurein 1.2, and several defensins (Machado and Ottolini, 2015;Shahmiri et al., 2017;Su et al., 2018) AMPs acting through a non-membranolytic mechanism, thus displaying intracellular activities (such as inhibition of nucleic acids, proteins or cell wall synthesis), include buforin II and indolicidin that bind to DNA (Scocchi et al., 2016), teixobactin that binds to peptidoglycan precursor lipid II (Chiorean et al., 2020), Bac5 that interacts with ribosomes (Mardirossian et al., 2018) and Temporin-L, which binds FtsZ protein inhibiting Escherichia coli cell division . A recent study performed by Moura et al. demonstrated that the AMP thanatin interacts with LptC-LptA proteins, which belong to the Lpt complex, involved in the LPS transport, exploiting an inhibitory activity (Moura et al., 2020). Thanatin interaction with Lpt complex prevents LPS translocation to the outer membrane, modifying its stability and permeability and favoring the cell agglutination process (Dash and Bhattacharjya, 2021). Sources of AMPs and Their Potential Applications in Clinical Practice The survival of organisms in an environment where pathogens are widely distributed, solely depends on their defense mechanisms. The inborn immunity of organisms involves endogenic peptides which supply a quick and viable method for safeguard against microbial attacks (Borah et al., 2020) AMPs are universal and essential components of the defense systems of all life forms, from bacteria to plants and invertebrate and vertebrate species, including mammals (Jenssen et al., 2006;Borah et al., 2020). They are naturally produced in the body of both lower and higher organisms and their production is cell specific and may be constitutive or inducible in response to pathogenic challenges (Borah et al., 2020). In multicellular organisms, AMPs are mostly localized to specific sites that are normally more exposed to microbes, such as the skin and mucosa epithelia (Jenssen et al., 2006). The primary role of these defense peptides is the killing of invading pathogens; however, in higher organisms they act also as modulators of the innate immune response (Jenssen et al., 2006). AMPs are commonly classified according to their sources, which are represented by microorganisms, plants, and animals. Below, we give an overview of various naturally occurring AMPs and the potential clinical application of some of them. Microorganisms as Source of AMPs Bacteria and fungi are reservoirs of AMPs (Huan et al., 2020). Among the numerous AMPs, the first isolated and characterized were those produced by bacteria (Jenssen et al., 2006). AMPs from bacteria are not produced for the purpose to protect against infections, but rather as a competition strategy (Jenssen et al., 2006). With their activity they kill other microbes competing for nutrients in the same niches, ensuring the survival of individual bacterial cells (Jenssen et al., 2006). Bacterial AMPs, also called bacteriocins, are represented by a heterogeneous family of small ribosomally synthesized molecules with strong antimicrobial activity at specific concentrations (Soltani et al., 2021). These molecules, produced by Gram-positive and Gram-negative bacteria, are effective against many pathogenic bacteria and are extraordinarily active compared to their eukaryotic counterparts (Jenssen et al., 2006;Soltani et al., 2021). For example, AMPs isolated from Pseudomonas spp display activity against several bacterial species, such as S. aureus, E. coli, Salmonella, Shigella, showing both general antibacterial and specific antibiofilm activity (Fontoura et al., 2008;Mohammadi-Barzelighi et al., 2019). Mersacidin, isolated by Bacillus spp, shows in vivo bactericidal activity against Methicillin-resistant S. aureus (MRSA) equivalent to that of vancomycin (Jenssen et al., 2006). AMPs are also produced by human microbiota. Hostmicrobiota crosstalk is based on AMPs secretion by phagocytic and epithelial cells and microbiota of the human gut, skin, and oral cavity; these peptides contribute to microbial and ecological balance (Magana et al., 2020). An example of these human microbiota AMPs is the thiopeptide lactocillin produced by the vaginal commensal Lactobacillus gasseri and acting against Gram-positive bacteria, including S. aureus and Gardnerella vaginalis (He et al., 2020). Several filamentous fungi produce AMPs which are similar to plant and animal defensins. Examples of cysteine-rich defensinlike AMPs in ascomycetes are AFP from Aspergillus giganteus, PAF from Penicillium chrysogenum, ANAFP from Aspergillus niger, AcAFP and AcAMP from Aspergillus clavatus (Montesinos, 2007;Hegedüs and Marx, 2013). All these fungal peptides have antifungal activity against filamentous ascomycetes, including animal and plant opportunistic and pathogens, such as Aspergillus fumigatus, Fusarium sp., and Botrytis sp. (Hegedüs and Marx, 2013). On the basis of their antimicrobial properties and their safety and tolerability, some of these natural AMPs have potential therapeutic applications. The bacteriocin nisin, produced by Lactococcus lactis, has been extensively studied being used as food preservative (Soltani et al., 2021). Nisin is the only bacteriocin legally approved as biopreservative and is used in the dairy industry to control contamination from Listeria strains (Soltani et al., 2021). Because of its broad-spectrum activity against both Gram-positive and Gram-negative pathogens, nisin is approved for clinical use as an alternative to antibiotics (Dijksteel et al., 2021). Several studies have reported the suitability of nisin in the treatment of several infection diseases, such as mastitis (Cao et al., 2007;Fernańdez et al., 2008), oral (Shin et al., 2015;Mitra et al., 2019), respiratory (De Kwaadsteniet et al., 2009) and skin (Heunis et al., 2013) infections. Johnson et al. (1978) have been the first to demonstrate that there were fewer numbers of streptococci in the dental plaque of monkeys that received nisin in their foods. Moreover, more recent studies support the antimicrobial abilities of nisin against oral pathogenic bacteria relevant to periodontal diseases and caries. Indeed, Tong et al. (2010) showed that nisin A is able to inhibit the growth of cariogenic bacteria. Cao et al. (2007) demonstrated that a nisin-based formulation was effective in the treatment of clinical mastitis in lactating dairy cows caused by different mastitis pathogens. Mastitis is a common inflammatory disease in lactating women, which causes breastfeeding cessation (Foxman et al., 2002). S. aureus and Staphylococcus epidermidis are two common agents that cause mastitis-associated infections (Foxman et al., 2002). Nisin peptide causes bacterial growth inhibition by membrane pores formation and by interrupting the cell wall biosynthesis through specific lipid II interaction (Prince et al., 2016). Another example of bacterially derived AMPs used in clinics as alternative to antibiotics is gramicidin, which is a mix of gramicidin A, B and C. They are AMPs naturally produced by Bacillus brevis, with activity against several Gram-positive bacteria, inducing membrane depolarization and consequently cell lysis (David and Rajasekaran, 2015;Yang and Yourself, 2018). Gramicidin is a constituent of Neosporin ® , a triple antibiotic used in ophthalmic and topical preparations (Hallett et al., 1956). Gramicidin S is used in the treatment of wound infection and of the root canal of teeth due to the tetracycline resistant Enterococcus faecalis biofilms formation (Berditsch et al., 2016). The bacterium Streptomyces roseosporus is a rich source of the anionic AMP daptomycin, which shows bactericidal activity against Gram-positive pathogens (Ball et al., 2004). Daptomycin exerts its bactericidal action by formation of membrane pores, membrane depolarization and inhibition of cell wall synthesis (Taylor and Palmer, 2016). This peptide has been approved and marketed as anionic AMP for the treatment of skin infections caused by Gram-positive bacteria . Considering the great variety of AMPs existing in nature, it has to be expected that other novel nature-inspired peptides, pharmacological active, might find clinical applications in the future. Plants as Source of AMPs Bioactive peptides are essential components of plants defense mechanisms, with extraordinary physiological importance, providing fast protection against bacterial and fungal infections (Jenssen et al., 2006;Loṕez-Meza et al., 2011;Salas et al., 2015). Plant AMPs not only display microbicide activities but are also involved in cellular signaling (Salas et al., 2015). Several active peptides have been extracted and isolated from roots, flowers, seeds, stems and leaves and are classified based on their amino acids sequence, position and number of cysteine residues involved in the disulfide bridge formation (Loṕez-Meza et al., 2011). Ten families of plant AMPs have been described (Loṕez-Meza et al., 2011) and the best-studied groups are defensins, thionins and snakins (Jenssen et al., 2006;Loṕez-Meza et al., 2011;Huan et al., 2020). The first plant-derived AMP is purothionin, which displays activity against Corynebacterium fascians, Pseudomonas solanacearum, Corynebacterium poinsettia (de Caleya et al., 1972). Plant defensins are cysteinerich AMPs, with four disulphide bridges and a globular structure (Salas et al., 2015); they are basic peptides, composed by 45 to 54 amino acid residues, ubiquitous in the plant kingdom, displaying activities against bacteria and fungi. The PvD1 peptide is a defensin from Phaseolus vulgaris, which inhibits growth of yeasts, such as Candida albicans, Candida tropicalis and Saccharomyces cerevisiae (Mello et al., 2011). Thionins, composed by 45 to 47 amino acids, are basic peptides found in several plant tissues, which are toxic to bacteria and phytopathogenic fungi (Loṕez-Meza et al., 2011). Snakins are small peptides with 12 cysteine residues forming six disulphide bridges, essential for their biological activity (Meneguetti et al., 2017). Snakin-Z from Ziziphus jujuba, composed by 31 amino acids, is more toxic for fungi than bacteria (Meneguetti et al., 2017). Finally, different AMPs have been identified in avocado fruit and in fruits of Capsicum, which for their antimicrobial properties could be used in the treatment of infections caused by S. aureus and E. coli strains (Liu et al., 2006;Guzmań-Rodrıǵuez et al., 2013;Taveira et al., 2014). Considering their efficiency and broad-spectrum activity, plant AMPs may represent a promising alternative to conventional antibiotics for counteracting infections (da Silva and Machado, 2012). Animals as Source of AMPs Animal AMPs are produced at the sites that are constantly exposed to microbes, such as skin and mucosal barriers (Loṕez-Meza et al., 2011). Various AMPs have been isolated from invertebrates and many vertebrate species (including fish, amphibians, and mammals). In invertebrates the innate immune system is extremely efficient since they lack an adaptive immune system, and in this regard, AMPs play a key role in protection against foreign microbial attacks (Jenssen et al., 2006). Invertebrates can produce a wide range of proteins and peptides which are found in phagocytes, in epithelial cells and in hemolymph (plasma and hemocytes) (Jenssen et al., 2006). The b-hairpinlike peptides tachyplesin (Nakamura et al., 1988) and polyphemusin (Miyata et al., 1989) (from horseshoe crab), and melittin (from bee venom) (Raghuraman and Chattopadhyay, 2007) are examples of invertebrate AMPs. A recent study has demonstrated that a pretreatment with Tachyplesin III on mice protects them against P. aeruginosa and Acinetobacter baumannii infection, reduces the production of pro-inflammatory cytokines (IL-1b, IL-6, and TNF-a) and induces the macrophage phagocytosis, fundamental to exert bacterial clearance, in a dose-dependent manner (Qi et al., 2019). All these findings must be confirmed in human clinical trials. More than 200 AMPs have been isolated in insects (Li et al., 2012). The number of these bioactive molecules varies between species. Hermetia illucens and Harmonia axyridis produce up to 50 AMPs, while they are not found in other species, such as Acyrthosiphon pisum (Huan et al., 2020;Moretta et al., 2020). AMPs are produced mainly in the fat body and blood cells (hemocytes) of insects and then are secreted into the hemolymph (Jenssen et al., 2006;Huan et al., 2020). Based on their amino acid sequences and antimicrobial activities, insect AMPs are divided into several groups: cecropins, defensins, proline-rich and glycine-rich peptides (Manniello et al., 2021). Cecropin was the first insect AMP discovered in the hemolymph of the pupae of Hyalophora cecropia (Steiner et al., 1981). Cecropins, which are described only in the order Diptera and Lepidoptera, are linear peptides with a-helix and without cysteines, composed by around 35 amino acid residues and displaying activity against Gram-positive and Gram-negative bacteria (Wu Q. et al., 2018). Insect defensins are inducible peptides which display strong activity against Gram-positive bacteria and less against Gramnegative bacteria. They are composed by 29-34 amino acid residues and have been isolated from several insect orders, such as Coleoptera, Hemiptera Diptera, Trichoptera, Hymenoptera and Odonata (Bulet et al., 1999). Attacins are an example of glycine-rich AMPs, which show activity against Gram-negative bacteria, including E. coli (Carlsson et al., 1991). This group of peptides is heterologous in size, but their common feature is the high content of glycine-residues (10-22%) (Wu Q. et al., 2018), which affect the tertiary structure and consequently their mode of action (Li et al., 2012). Diptericin, Coleoptericin, Sarcotoxin IIA are other glycine-rich AMPs isolated from insects (Ando and Natori, 1988;Dimarcq et al., 1988;Sagisaka et al., 2001). Although insect AMPs could be a good alternative to conventional antibiotics, their clinical use is still limited and most of them are just in vitro tested (Manniello et al., 2021). Among them, the melittin peptide is, currently, in clinical use for its antimicrobial potency. Composed by 26 amino acids, melittin is the principal component of venom from the honeybee Apis mellifera. Melittin has broad spectrum activity, and its ability to protect in vivo against MRSA infections has been demonstrated (Choi et al., 2015). It acts by induction of pore formation following interaction with membrane surfaces (van den Bogaart et al., 2008). Since it also shows anti-inflammatory properties (Lee and Bae, 2016), the Food and Drug Administration (FDA) approved its usage in clinical practice (Dijksteel et al., 2021), for relieving pain associated to tendinitis, arthritis, sclerosis multiple (Park et al., 2004;Son et al., 2007;Yang et al., 2011). Amphibians, especially frogs, are a rich source of AMPs. Most of the amphibian AMPs have been isolated from the frog skin. These biologically active molecules are released from cutaneous glands and excreted towards the skin surface following pathogen stimulations (Patockaa et al., 2018). The prototypic and the most famous AMP from frogs is the a-helical magainin (Zasloff, 1987), which is active against yeasts, fungi, bacteria, and viruses (Borah et al., 2020). Esculentins, nigrocins, brevinins, temporins are some of the best characterized peptides produced by frogs of the genus Rana (Patockaa et al., 2018). The basic esculentin-1 peptide, composed by 46 amino acid residues and a disulphide bridge, exhibits strong activity against several human pathogens, such as C. albicans, P. aeruginosa, E. coli and S. aureus (Patockaa et al., 2018). Esculentin was in vitro tested on human lung epithelium to determine the toxicity, finding a good tolerability in terms of inflammatory effects. Then, it was studied in a mouse model, in which a lung-infection was induced with P. aeruginosa: promising results showed a strong reduction in bacterial load not only in lungs but also in spleen, indicating a decrease in systemic spread of bacteria (Chen C. et al., 2017). Brevinin-2Ta was tested on mice infected with Klebsiella pneumoniae. In this study, it was demonstrated that the peptide decreases the bacterial load, altering the microorganism structures in infection sites and it also showed the ability to faster angiogenesis and granulation tissue maturing process, obtaining comparable results to classical antibiotics. For this reason, this peptide is a good candidate for pre-clinical studies, even if some modifications are needed in order to decrease its hemolytic power (Liu et al., 2017). Liu et al. (2017), hypothesized that amino acid substitutions in the primary structure could be the right strategy to reduce the hemolytic activity, improving, at the same time, the antimicrobial one. Regarding anionic AMPs, the temporin-1Ja, carrying a net charge of -1, has been isolated from the skin secretions of the Japanese frog Rana japonica (Isaacson et al., 2002). This anionic peptide revealed moderate activity against E. coli and S. aureus strains. However, it was found that this peptide synergizes with other temporins, contributing to endotoxin neutralization (Rosenfeld et al., 2006). AMPs can also protect amphibians from ingested pathogens since they are produced in the mucosa of the stomach. The Asian toad peptide buforin and buforin II are the best characterized examples in this regard (Jenssen et al., 2006). Some of these natural AMPs have been used for the production of synthetic peptides, such as the Pexiganan, also known as MSI-78. It is a synthetic 22-aminoacid analogue of magainin-2, which has been tested as a topical cream for treatment of bacterial infections related to diabetic foot ulcers. It showed promising in vitro broad-spectrum activity (Ge et al., 1999), but it was rejected by FDA because there was no advantage compared to conventional antibiotics (Koo and Seo, 2019). Mammalian AMPs have been identified in humans, cattle, sheep and other vertebrates (Huan et al., 2020). Some AMPs from mammalians have a second major function inducing chemoattraction and activation of host cells to engage in innate host defense (Yang et al., 2001). AMPs can be stored in phagocytes and epithelial cells and can be released extracellularly by degranulation in response to different stimuli, becoming available at the site of infection (Yang et al., 2001). For example, cathelicidins are stored within granules of circulating immune cells as inactive propeptides (Jenssen et al., 2006). Cathelicidins and defensins are the main AMPs found in mammalians, such as humans, horses, rabbits, sheep and mice. Cathelicidin family comprises heterogeneous peptides which share the N-terminal pro-region but show a variable antibacterial peptide in the C-terminal region, displaying different structures, including b-hairpin, a-helical, and arginine and proline-rich peptides (Kosćiuczuk et al., 2012). This structural diversity reflets cathelicidin different functions and their diverse spectrum of antimicrobial and immunomodulatory activities (Jenssen et al., 2006). The a-helical BMAP-28 is a bovine AMP of the cathelicidin family which is able to permeabilize the membranes of several bacteria and fungi at a moderate concentration in vitro (Risso et al., 2002;Benincasa et al., 2006). Only one cathelicidin, the hCAP18 (better known as LL-37), is produced in humans and has been isolated from specific granules of neutrophil granulocytes. A second group of mammalian AMPs are the defensins, which require proteolytic processing to acquire their active form (Selsted and Ouellette, 2005). More than 50 defensins have been identified in mammalian species; some of them are stored in granules of macrophages, neutrophils and Paneth cells, while others are produced by mucosal epithelial cells and keratinocytes (Yang et al., 2001). Defensins production can be constitutive, such as for human b-defensin-1 (hBD1), or inducible, such as for hBD2, whose expression is induced by exposure to bacteria or microbial components, as LPS (Jenssen et al., 2006). Maiti et al. (2014) studied mice mortality after the infection with Salmonella typhimurium, demonstrating that the administration of hBD1, hBD2, or a combination of both, lead to an increased mice mortality and a decreased S. typhimurium load in peritoneal fluid, liver and spleen. The anionic peptide Dermcidin, discovered in epithelial and neutrophil granules of humans, is one of the most studied human anionic AMPs. This peptide is proteolytically processed in sweat producing several truncated peptides which display a good spectrum of antimicrobial activity (Schittek et al., 2001). There are several examples of mammalian AMPs proposed for clinical applications. The acid-pepsin digestion of bovine lactoferrin results in the release of the peptide lactoferricin, which shows the strongest antimicrobial activity among mammalian lactoferricins (Vorland et al., 1998) and has potent immunological and antitumor properties (Gifford et al., 2005;Yin et al., 2013;Arias et al., 2017). It exerts its bactericidal activity on Gram-positive and Gram-negative bacteria inducing depolarization of the cell membrane, with fusion of negatively charged liposomes and formation of blebs on the cell surface (Ulvatne et al., 2001;Bruni et al., 2016). The bovine lactoferricin displays useful properties for potential applications in human medicine. It has been successfully utilized for treatment of enterohemorrhagic E. coli infections (Kühnle et al., 2019). Because of its antimicrobial and anti-inflammatory properties, the bovine lactoferricin can be used for treatment of ocular infections, since it potentiates the effect of conventional antibiotics against clinical ocular isolates of P. aeruginosa and S. aureus (Oo et al., 2010). Moreover, it improves diabetic wound healing (Mouritzen et al., 2021) and finds applications in the treatment of osteo-articular diseases (Yan et al., 2013). The saliva of humans and other primates contains various forms of AMPs, among them the histatins, which are small histidine-rich cationic peptides with antifungal properties. Histatin 5, that is the product of histatin 3 proteolytic cleavage, is the most active histatin against several yeasts, such as Cryptococcus neoformans, Candida dubliniensis and Candida albicans (da Costa et al., 2015). Histatins exert their activity by targeting the mitochondria, affecting cell respiration (Kavanagh and Dowd, 2004) and, because of their safety and tolerance, have been successfully tested in topical gels to treat oral fungal infections (Paquette et al., 2002). Several efforts have been made to identify fragments of histatin 5 with pharmaceutical application and have yielded promising results. An example in this regard is the 12-amino acid peptide P113, which was evaluated in phase I and phase II clinical studies as pharmaceutical agent to fight oral candidiasis (Woong et al., 2008;Cheng et al., 2018;Browne et al., 2020). Tables 1 and 2 summarize, respectively, naturally occurring AMPs from different sources and those used in clinical practice. AMPs: INNATE WEAPONS AGAINST DISEASES Given the broad spectrum of action of the AMPs, their diversity in sequences and considering the physico-chemical characteristics related to their several sources, they can find application in different fields. Specifically, below we addressed the suitability of AMPs in the biomedical and pharmacological fields, also taking into account the pharmacokinetic and pharmacodynamic approaches to develop new molecules with antimicrobial activity. The excessive use of antibiotics in clinical treatment has increased pathogens resistance to these compounds (Aminov, 2010). The pharmaceutical industry is trying to solve this problem by looking for new molecules with antibiotic activity or by modifying/improving the existing ones. Nevertheless, pathogens can develop resistance mechanisms that compromise this strategy. Thus, the need to find new active molecules with different mechanisms of action represents one of the most urgent challenges in medicine (Parisien et al., 2008). AMPs are among the most promising alternatives to modern antibiotics and they have already found clinical applications in this field, as previously mentioned, alone or in synergy with existing antibiotics. AMPs are susceptible to proteolysis due to their chemical characteristics and their activity is affected by salts concentration and pH. For this reason, the most promising applications for AMPs in clinical evaluations are those involving topical applications (Hancock and Sahl, 2006). The endogenous production of AMPs is also relevant and worth further studies. For example, sodium butyrate administration has been shown to induce the production of intestinal AMPs, beneficial for the treatment of infectious or inflammatory diseases (Guanı-Guerra et al., 2010). However, AMPs broad spectrum of biological activities suggests other potential clinical benefits such as for the treatment of cancer and viral infections as well as in the immune system modulation (Schweizer, 2009). Involvement of AMPs in Respiratory Diseases Infections in the lower respiratory tract are involved in chronic inflammatory lung disorders such as cystic fibrosis and chronic obstructive pulmonary disease. In cystic fibrosis patients with a P. aeruginosa infection, this organism produces AMPs, such as pyocins, which inhibit the growth of its closest competitors. Thus, the same AMPs could be used as a therapeutic agent to minimize the effects of the infection, besides rooting out other susceptible pathogens. Pyocins derived from P. aeruginosa strains also have toxic effects on Haemophilus, Neisseria and Campylobacter strains and have been successfully used for the treatment of peritonitis in mice (Scholl and Martin, 2008;Waite and Curtis, 2009). It is of interest that neutrophils and airway epithelial cells produce AMPs to prevent infection of the respiratory system by pathogens. In cystic fibrosis patients, P. aeruginosa induces the secretion of sPLA2-IIA by airways epithelial cells via a Krüppellike transcription factor (KLF)-2-dependent pathway, that lead to the selective death of S. aureus (Rahnamaeian, 2011). Moreover, the serum level of the human LL-37 peptide is higher in patients with lower respiratory tract infections than in healthy people (Majewski et al., 2018). Recently, it has been reported that the Esculentin peptide (1−21), active on both P. aeruginosa planktonic and biofilm forms, has the ability to prolong the survival of mouse models with pulmonary infection. The main AMPs detected in lung tissues and secretions of cystic fibrosis patients are sPLA2-IIA, neutrophil a-defensins/HNPs, hBDs and LL-37 (Hiemstra et al., 2016). Similar phenomena have been described in periodontal diseases caused by Porphyromonas gingivalis in which the sPLA2-IIA peptide is produced by oral epithelial cells via activation of the Notch-1 receptor and kills oral bacteria (Balestrieri et al., 2009). AMPs in Wound Healing and Skin Infections Skin and soft tissue infections are one the most common microbial infections in humans and AMPs can be a new therapeutic option thanks to their broad-spectrum of biological activities, since skin pathogens include bacteria but also protozoa, fungi and viruses (Sunderkötter and Becker, 2015). Moreover, AMP preparations have the advantage of high concentration at the target site for topical administration because of their low ability to penetrate into the bloodstream. Moreover, AMPs can promote wound healing by modulating cell migration, angiogenesis, chemotaxis, and cytokine release (Ramos et al., 2011). For example, the hBD2 is induced by the Epidermal Growth Factor Receptor (EGFR) activation and it can increase keratinocyte migration and cytokines production (Sørensen, 2016). Another peptide highly expressed by keratinocytes at wound sites is represented by hBD3 defensin. It promotes cytokine secretion, cell migration and proliferation by phosphorylating EGFR and STAT proteins (Sørensen et al., 2005). It also speeds up the wound closure when topically applied in a porcine model of infected skin wounds (Hirsch et al., 2009). Moreover, it has been demonstrated that hBD3 exhibits anti-inflammatory activity through the inhibition of TLR (Toll-like receptor) signaling pathways in immune cells leading to a transcriptional repression of the proinflammatory genes (Semple et al., 2011). The expression of skin LL-37 peptide is also increased after wounding (Heilborn et al., 2003), and it seems to be involved in the modulation of angiogenesis. Indeed, LL-37 peptide stimulates endothelial cells proliferation and neovascularization by activating the formyl peptide receptor-like 1 (FPR2/ALX) (Koczulla et al., 2003). Psoriasis vulgaris is an inflammatory skin disease characterized by abnormal epidermal proliferation and a cellular infiltrate including neutrophils and T cells (Davidovici et al., 2010). Due to the enhanced proliferation rate of psoriatic keratinocytes associated with a reduction of the cell cycle duration, psoriasis has been thought to be an epidermal disease. However, experiments performed with severe combined immunodeficiency (SCID) mice indicated that psoriatic eruptions are induced by CD4+ cells and T cells are believed to play a key role in the pathogenesis of psoriasis (Ellis et al., 1986;Wrone-Smith and Nickoloff, 1996). The keratinocytes within the epidermis of psoriatic plaques are abnormal and among the abnormalities there is the excessive production of AMPs which, in vertebrates, are believed to modify host inflammatory responses through different mechanisms including regulation of cell proliferation, chemotactic and angiogenic activities (Lai and Gallo, 2009). HNP1, HNP2, HNP3, hBD2 and hBD3 are defensins identified from lesional psoriatic scale extracts and their presence could help to explain why a hyperproliferative and noninfectious skin disease, such as psoriasis, undergoes less cutaneous infections than it would be expected (Harder et al., 2001;Harder and Schröder, 2005). Studies performed on LL-37 peptide demonstrated that it has both pro-inflammatory and anti-inflammatory activity, can promote chemotaxis, angiogenesis and enhance wound repair (Yang et al., 2000;Koczulla et al., 2003;Braff et al., 2005;Tokumaru et al., 2005;Mookherjee et al., 2006). Frohm et al. were the first to report that cathelicidin/LL-37 expression is upregulated in psoriatic epidermis and suggested that this induction increases the antimicrobial defense ability of the disrupted barrier in the lesions (Frohm et al., 1997). Later, it has been hypothesized that LL-37 could drive inflammation in psoriasis by allowing plasmacytoid dendritic cells (pDCs) to recognize self-DNA through TLR9 (Lande et al., 2007). Angiotensin-Converting Enzyme I (ACE) Inhibitory Peptides The angiotensin-converting enzyme I (ACE) is produced by lung or kidney tissue and the luminal membrane of vascular endothelial cells. ACE converts inactive decapeptide angiotensin I (ANG I) into vasoconstrictor octapeptide angiotensin II (ANG II). ANG II is involved in several physiological and pathophysiological cardiovascular conditions such as atherosclerosis and hypertension . ACE inhibitors are used in hypertension treatment, but they may cause serious side effects, such as cough, rush and edema . Hence, it derives the need to identify new and nontoxic ACE inhibitors, whose activity depends on the amount and type of amino acid composition. It has been observed that the binding to ACE is influenced by hydrophobic amino acids at the peptide C-terminus (Salampessy et al., 2017). Moreover, amino acids like alanine, valine, isoleucine, isoleucine and glycinewhich are hydrophobic residues with aliphatic side chainsat the C-terminus have been associated with an increase in the ACE inhibitory activity (Toopcham et al., 2017). SAGGYIW and APATPSFW are two AMPs able to act as ACE inhibitors potentially suitable as antihypertensive peptides. They are produced in wheat gluten hydrolysate by the P. aeruginosa protease and contain tryptophan at the C-terminus (Zhang et al., 2020). This observation led to the idea that the presence of a tryptophan at the C-terminus of a peptide could influence the ACE inhibitory activity by blocking the enzyme active site via weak interactions, such as electrostatic, hydrophobic and Van Der Waals interactions and hydrogen bonds. Another example is the VEGY peptide, which was isolated from the marine Chlorella ellipsoidea and has been demonstrated to exhibit ACE inhibitory activity and to be stable against gastrointestinal enzymes (Ko et al., 2012). This potential use of AMPs certainly represents a fruitful avenue of pursuit and will likely find clinical applications in the future. Pancreatic Lipase Inhibitory Peptides Obesity and fatty acid metabolism disorders are widespread epidemic. One of the pharmacological strategies to counteract these issues is the dietary lipid inhibition. The pancreatic lipase enzyme hydrolyzes 50-70% of food-derived fat in the human organism and its inhibition is exploited by the Orlistat drug used in obesity treatment. However, in long-term treatment, this strategy can cause side effects, such as pancreatic damage and gastrointestinal toxicity (Cheung et al., 2013). For this reason, the search of new compounds able to inhibit pancreatic lipase, without exerting side effects, represents a still alive need to fight these disorders. Several AMPs have been identified so far that are able to show this activity, which depends on the structure and amino acid composition of the peptide (Hüttl et al., 2013). CQPHPGQTC, EITPEKNPQLR and RKQEEDEDEEQQRE are three peptides from purified soybean b-conglycinin that have been demonstrated to inhibit the pancreatic lipase (Lunder et al., 2005;Martinez-Villaluenga et al., 2010), and are under investigation for potential clinical applications (Złotek et al., 2020). Peptides With Antioxidant Activity Oxidative stress, caused by an imbalance between production and removal of reactive oxygen species (ROS) in cells and tissues, can promote diseases like obesity, diabetes, and heart disease (Pizzino et al., 2017). Environmental stressors like pollutants, heavy metals, xenobiotics, high-fat diet and the progression of aging can contribute to an increase in ROS production. Oxidative stress is also involved in several neurological disorders such as Alzheimer's and Parkinson's diseases (Singh et al., 2019). A growing number of antioxidant AMPs have been identified from different sources, including animals, plants and insects (Balti et al., 2010;Villadońiga and Cantera, 2019;. Peptide antioxidant activity is related to their sequence and amino acid composition. Indeed, it has been suggested that isoleucine, leucine and histidine residues could contribute to the antioxidant activity of fermented anchovy fish extracts (Najafian and Babji, 2019). A study carried out by Wu et al. on the QMDDQ peptide, from a shrimp protein hydrolysate, showed that the antioxidant potency could be related to the high number of active hydrogen sites . Peptide antioxidant properties are usually expressed as free radical scavenging, metal ion chelation activity and inhibition of lipid peroxidation . For example, Zhang et al. showed that the VYLPR peptide has a protective effect on H 2 O 2 -induced cell damage (HEK-293 cells) (Zhang et al., 2019). Moreover, Liang et al. investigated antioxidant peptides deriving from a protein hydrolysate of Moringa oleifera seeds and demonstrated their protective effects on Chang liver cells exposed to H 2 O 2 oxidative damage . Jiang et al. identified four peptides AYI(L) and DREI(L) from Jiuzao protein hydrolysates able to decrease ROS production in HepG2 cells . AMPs in Intestine Infection and Inflammation The bacterial microflora is essential for human health and the development of the mucosal immune system. In the small intestine, Paneth cells secrete a-defensins in response to bacterial antigens including LPS and muramyl dipeptide (Ayabe et al., 2000). Petnicki-Ocweija et al. showed that the bactericidal activity of crypt secretions of the terminal ileum was compromised by NOD2 gene deletion (Petnicki-Ocwieja et al., 2009). The human NOD2 protein is a cytoplasmic receptor for bacterial molecules principally expressed in Paneth cells (Lala et al., 2003) and it was identified as a susceptibility gene for Crohn's disease (Hugot et al., 2001). Deficient expression of Paneth cell a-defensins (HD5 and HD6) may contribute to the pathophysiology of Crohn's disease (Bevins, 2006). It has been demonstrated that mice lacking NOD2, fail to express cryptidins, equivalents of human a-defensins (Kobayashi et al., 2005). Moreover, human a-defensin expression is reduced in Crohn's disease patients, particularly in those with NOD2 mutations (Wehkamp et al., 2005). hBD1 was the first defensin identified in the human large intestine and in the not-inflamed colon. It was observed a reduction of hBD1 expression in inflamed mucosa in patients with inflammatory bowel diseases (Wehkamp et al., 2003). hBD1, hBD2, hBD3 and hBD4 expression has been demonstrated to be upregulated in colonic enterocytes in patients with ulcerative colitis (Fahlgren et al., 2004). Moreover, a lot of interest has been given to the role of AMPs in the stomach, which is easily colonized by Helicobacter pylori. Infection by this bacterium leads to the induction of hBD2 (Wehkamp et al., 2003). It has been demonstrated that gastric epithelial cells are induced by Helicobacter pylori to upregulate hBD2 production (Grubman et al., 2010). These observations make defensins very attractive from a pharmacological point of view and can offer a good starting point for future AMP clinical applications. PHARMACOKINETIC AND PHARMACODYNAMIC (PK/PD) APPROACH IN THE EVALUATION OF AMP CLINICAL APPLICATIONS PK/PD Approach to Determine AMP Antibacterial Efficacy PK and PD principles that determine response to antimicrobial AMPs can provide clinicians with useful information on the correct dose regimens. Schmidt and colleagues showed that AMPs (Onc72 and Onc112) reach several organs within 10 min after intravenous and intraperitoneal administration and the PK experiments explain the high in vivo efficacies of AMPs indicating their potential use for the treatment of urinary tract infections (Schmidt et al., 2016). However, these data are not sufficient to predict the exact relationship between dose, exposure, and response and translational PK/PD modeling and simulation are used to identify the most suitable dosing regimen in patients. PK/PD modeling can provide useful clues concerning the multifaceted correlation between the selected kind of AMP, the bacterium characteristics, and the reaction of the host organism. Furthermore, complicating factors can also be incorporated into the in silico approach thus allowing to carefully predict the right balance between bacterial killing, adverse effects, and appearance of resistance. This practice may, therefore, help to identify and to optimize the dose for novel and established antibacterial agents (Rathi et al., 2016). As previously mentioned, AMPs affect growing bacterial populations differently from antibiotics (ampicillin, ciprofloxacin, gentamicin, kanamycin, neomycin, rifabutin, spectinomycin, and tetracycline), particularly from a PD point of view (Yu et al., 2016). Moreover, Yu and colleagues, analyzing the resistance evolution by predictive model, found that differences in PD and in the mutagenic properties between AMPs and antibiotics produce a much lower probability that resistance will evolve against AMPs . More experiments with a variety of AMPs are needed to determine if PK/PD characteristics of AMPs can be generalized and if these characteristics are significantly different from antibiotics. However, all the available data suggest that AMPs are significantly different from antibiotics in terms of PD and mutagenic properties and are good candidates for slowing the evolution of resistance. PK/PD Approach to Determine AMP Efficacy in Non-Bacterial Disease The "right" use of AMPs is imperative, not only in treating bacterial disease but also in other diseases to avoid toxicity and to limit the development of resistance. Few studies have analyzed AMP PK/PD properties in relation to no-bacterial disease. AGPSIVH, FLLPH, and LLCVAV antioxidant peptides were obtained from duck breast protein hydrolysates by Li et al. and beside the nontoxic effects exhibited digestive resistance . Xu and colleagues used in vitro and in vivo models to study the absorption and potential antioxidant activity and the in vivo metabolism, respectively, of WDHHAPQLR derived from rapeseed protein (Xu et al., 2018). Koeninger and colleagues showed that hBD2 displays a good tolerability and rapidly enters the bloodstream in a model of experimental colitis after its subcutaneous administration. Thus, besides being well tolerated in vivo, it might not only act locally but could also have systemic effects (Koeninger et al., 2020). Several other bioactive peptides have been discovered in recent years, but their PK/PD properties are still unknown. It is therefore necessary to increase the studies to determine the PK/PD efficacy of AMPs also in non-bacterial disease. DRUG DEVELOPMENT AND FORMULATION APPROACHES FOR AMP APPLICATIONS Production and Costs -Pilot Study vs. Small Industrial Scale The development of AMPs as APIs (Active pharmaceutical ingredients) has been greatly limited by their high manufacturing costs. Although the chemical synthesis of peptides has high efficiency, it is also complex and expensive. Hence, advanced natural approaches should be considered with the aim to increase the production of alternative molecules. Genetic engineering can be considered one of the most important strategies to obtain higher yields or higher quality of AMPs. To obtain AMPs, biotechnological approaches involving competent bacteria and yeasts, as well as transgenic plants or animals, should be considered (Sinha and Shukla, 2018). Gaglione and co-workers focused on how to optimize the bacterial culturing using a new composition of culture broth. They basically considered inexpensive as well as readily available components containing welldefined amounts of each nutrient. They also substituted IPTG (isopropyl bd-1-thiogalactopyranoside) with cheaper and more harmless sugars, such as lactose. Indeed, IPTG use might result in high-cost accumulation for industrial purposes. Altogether, the optimized bacterial culture strategy can contribute to further development to enhance the manufacturing scalability of AMPs (Gaglione et al., 2019). However, although bacteria can produce some cyclic peptides, they do not produce disulfide-rich peptides, so that recombinant expression of cyclic peptides might be best performed in yeast-or plant-based recombinant expression systems (Thorstholm and Craik, 2012;Moridi et al., 2020). The manufacturing cost of AMPs is estimated to be around $50-400 per gram of amino acid produced by SPPS (Solid Phase Peptide Synthesis), thus biotechnological engineering or fermentation should give cheaper alternatives. Moreover, the identification, characterization and production of new AMPs also with biotechnology improvement is expensive from many points of view, therefore, it could be useful to perform preliminary in vitro screening, to evaluate physio-chemical characteristics, putative modifications in the secondary structure and putative antimicrobial activity . About the peptide drug market in 2018, more than 50 peptide drugs have been commercialized. The annual sales of peptide drugs, including the AMPs, is around 25 billion USD (Koo and Seo, 2019). AMP Dosage Forms Compared to the possible sequence modifications to enhance the molecular stability, the drug delivery platform development has reported a minor attention so far. As described in literature, the dosage forms in ongoing clinical trials encompass topical gel and hydrogel, topical cream, polyvinyl alcohol-based solution for administration in the wound bed, hyaluronic acid-based hydrogel for the administration at the surgical site, oral solutions, and mouth rinse (Mahlapuu et al., 2016). Concerning dermal administration, burn and chronic wounds can exhibit difficult control, especially in the case of upsurges caused by ESKAPE pathogens (Enterococcus faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp). Topical administration of antimicrobials onto the skin provides many advantages since it offers a high local load of the antimicrobial. Moreover, due to the pleiotropic mechanisms of action, AMPs can contribute to fight ESKAPE infections as well as to regulate various mechanisms including the host processes of inflammation and wound healing (Kang et al., 2014;Vassallo et al., 2020). However, AMPs intended to treat chronic skin and soft tissue infections should not (i) be absorbed from the wound or infection site into the systemic circulation; (ii) rouse allergic sensitization. Topical administrations of AMPs have demonstrated to be not free of systemic side effects since the drug transport may also occur via skin layers and through hair follicles. Besides, the stability enhancement against enzymatic degradation needs to be assessed when peptides are developed for clinical purposes. Moreover, the membrane border of the epithelial cells includes several peptidases to be considered (e.g., leukocyte elastase, cathepsins B and D, zinc-dependent endopeptidases, interstitial collagenase), since they are characterized by a broad specificity to degrade exogen peptides (Vlieghe et al., 2010;Lam et al., 2018;Pfalzgraff et al., 2018). Delivery System In the context of Drug Delivery System (DDS), peptides are playing an important role as APIs vehicles, due to the intrinsic biodegradability and biocompatibility (Giri et al., 2021). Novel DDS can also help (i) to reduce adverse side-effects, and (ii) to obtain a controlled release of the AMP (Nordström and Malmsten, 2017;Martin-Serrano et al., 2019). Hydrogels -Overview and Platform Development for the AMP Dermal and Subdermal Delivery Hydrogels (HGs) comprise materials constituted by hydrophilic as well as polymeric vehicles to entangle large amounts of water within their three-dimensional (3D) networks (Liu and Hsu, 2018). As reported in the Eur. Pharm 8 th , gels consist of gelled liquids with suitable gelling agents. Specifically, HGs (i.e., hydrophilic gels) consist of water, glycerol, or propylene glycol-based preparations. These compounds are gelled with starch, cellulose derivatives, poloxamers, carbomers, and magnesium-aluminum silicates (European Pharmacopeia, 2016). HGs exhibit improved bioavailability for applications onto the impaired skin. Moreover, HG-based burn dressings (HBBD) appear appropriate as they provide a suitable wound covering. Thanks to a cooling sensation that occurs via convection and evaporation of the solvent from the wound, HBBD can also contribute to dissipating the heat that occurs from the concomitant inflammation (Fichman and Gazit, 2014;Goodwin et al., 2016). HGs have also been extensively studied since they exhibit different applicability potentials covering the cell culturing (Caliari and Burdick, 2016), the regenerative medicine (Catoira et al., 2019), and DDS developments. After chemical interactions, such as the Michael's addition, the Diels-Alder or Schiff base reactions, chemically-crosslinked HGs form the matrix structure (Overstreet et al., 2012) (Figure 3). To obtain a HG that supports the wound closure, Bian and co-workers used modified chitosan with maleic anhydride and a polyethylene glycol derivative, that was modified with benzaldehyde at both ends. Via a Schiff-base reaction, the obtained HG showed a shear-thinning behavior. Accordingly, it was intended to be injected/applied into/onto wounds, as it was suitable to adopt the contour as well as to seal the defects of the impaired tissue. Afterwards, the in situ HG solidification was promptly realized by using ultraviolet light (Bian et al., 2019). HG can also be prepared by multiple non-covalent interactions, by which the monomeric building blocks can selfassociate in ordered fibrous structures. Also, they are suitable to interact with each other forming the 3D network (Fichman and Gazit, 2014). Moreover, thanks to a self-assembly skill of polymers e.g., via changing pH and temperature, the physical cross-linking method favors the formation of weaker and stimuli-responsive HG. Hence, HG can temporarily modify the structure due to the solicitation of external mechanical forces and the shear-thinning behavior (Yan et al., 2010). Since a substantial change in volume is usually not observed, HGs are also suitable as injectable vehicles (Manna et al., 2019). Moreover, HG can also polymerize in situ becoming a shear-thinning material after injection, allowing, therefore, AMP delivery. The in situ forming HG was demonstrated useful for ophthalmic applications, as well as to support the wound-healing after surgical operations (Travkova et al., 2017). The widely used materials and techniques for surgical closure purposes may contribute to providing some drawbacks. Hence, contaminations by impurities from air or from a fluid leakage can contribute to microbial infection harm (Rajabi et al., 2020). Moreover, medicated HG can release AMPs at the site of action after disruption of the inner matrix by erosion, swelling, or via enzyme interactions (Chen M. H. et al., 2017). Li and co-workers formulated a thermosensitive HG constituted of biodegradable poly (l-lactic acid)-Pluronic L35-poly (l-lactic acid) for cutaneous wound-healing treatment, to investigate whether AMPs encapsulated in this HG formulation demonstrated efficient candidates in wound healing management. They used a type of multifunctional human-derived AMP (i.e., AP-57), with a broadspectrum antimicrobial activity as well as an immune regulation ability. The AP-57 peptide was enclosed first in biocompatible nanoparticles, named AP-57-NPs. Subsequently, to facilitate their application in cutaneous wound repair, the AP-57-NPs were further encapsulated in a HG matrix (AP-57-NPs-H). As reported, the in situ gel-forming system exhibited in vitro a low cytotoxicity and a sustained drug release behavior. After applied to the wound, the formulated peptide achieved additional characteristics, such as a non-flowing gel that consequently become a sustained drug depot. Li and co-workers also demonstrated wound-dressing properties of this formulation. The effect of the formulated AMP was then investigated on full-thickness excision wound using the Sprague-Dawley ® male and albino rat models. At last, the obtained DDS was effective on the wound, and rat models reported a complete wound closure (Li et al., 2015). A different method to obtain HG in the aqueous phase is the mussel-inspired polydopamine chemistry. A study of Khan and colleagues reported the use of catechol, instead of dopamine, as a cross-linker with amine-rich polymers to prepare thin films. Catechol is less expensive than dopamine; hence, it was used with ϵ-poly-L-lysine (EPL), a natural AMP produced by Streptomyces albulus, to fabricate HG with antimicrobial properties. EPLcatechol HG showed in vitro antimicrobial and antibiofilm properties against multidrug-resistant A. baumannii associated with a good biocompatibility with a mouse myoblast cell line and in vivo reduced the bacterial load and improved wound healing when topically applied on the skin of a mouse with a seconddegree burn wound also infected with multidrug-resistant A. baumannii (Khan et al., 2019). Lee and colleagues engineered nanoparticle-HG corneal implants containing the human AMP LL-37: although in vivo studies have not already been carried out, this device could inhibit in vitro HSV-1 attack to ocular cells . An example of insect AMP formulated as HG was recorded from Lucilia sericata, in both wound bandages and cosmetics to hinder dermatological pathogens (Mylonakis et al., 2016). Practically, bicontinuous cubic phases can be obtained by dispersing the amphiphilic lipid system into the aqueous phase using e.g., ultrasonication or homogenization. Subsequently, a dispersed gel is obtained, known as cubosome (CB) (Karami and Hamidi, 2016). As a result of the hydrophobic effect, FIGURE 3 \| Chemical and physical bonds to obtain hydrogels. Hydrogels can also be prepared by a hybrid interaction consisting of physical interactions and/or covalent bond formation, exhibiting at the same time reversible mechanical properties and long-term stability. Moretta et al. AMPs: Biomedical and Pharmacological Applications thermodynamically stable structures with a well-defined disposition of each component (i.e., the cubic liquid crystalline gel) are realized (Figure 3). These nanostructures have demonstrated suitable for loading hydrophilic, hydrophobic, as well as amphiphilic cargos. More importantly, CB can include bioactive compounds, as the structure provides a significantly higher membrane surface area to loading proteins (Barriga et al., 2019). Anatomically, the stratum corneum represents a strong barrier for the transdermal drug delivery of topically applied drugs, due to the presence of the external and highly organized skin layer. The ability of CB to adhere to the stratum corneum makes CB effectively useful in topical drug delivery for mucosal tissues (Gaballa et al., 2020). The structure and properties of CBs provide a promising vehicle for transdermal drug delivery especially for skin infections (Zeng et al., 2012;Meikle et al., 2019). AMPs can be adsorbed onto the CB structure that usually shows a slightly negative charge. For instance, Boge and coworkers demonstrated that the GMO based-CB structure contributes to protecting the AMPs from proteolytic degradation, improving their bioavailability after topical administration. Furthermore, they found that AMPs loaded onto CB are highly released in the milieu whether P. aeruginosa or human neutrophil elastases are present. The authors also reported a study investigating CB interaction with both a bacterial membrane model and E. coli's membrane, to further understand how the interaction between AMPs and the membranes can be accomplished. The authors suggested that the bactericidal effect was due to physical interaction between the product and the bacterial membrane and not solely to the release of the peptide. Moreover, they noted that the presence of LL-37, the chosen AMP, constituted of a secondary structure of a linear a-helix increased the affinity of CB to bacterial membranes (Boge et al., 2017;Boge et al., 2019a;Boge et al., 2019b). Many papers have reported that the composition of GMObased CB generally involves the use of stabilizer molecules. The stabilizer avoids the aggregation of hydrophobic portions with the external aqueous media and consequently helps to reach a thermodynamically stable form (Gaballa et al., 2020). Pluronics, especially poloxamer 407 (F127), represent the most used stabilizing agents. This nonionic copolymer vehicle comprises a central hydrophobic chain of polypropylene oxide with a molecular weight of approximately 12.6 kDa and lateral hydrophilic chains of polyethylene glycol (Barriga et al., 2019). The clinical application of GMO-based CB stabilized by F127 may be limited due to concentration-dependent cytotoxicity. Moreover, F127 may also show hemolytic effect, as well as a poor biodegradability. A novel stabilizer-free antimicrobial nanocarrier was developed by Zabara and co-workers, by dispersing GMO in water using ultrasonication and combining the AMP LL-37 by spontaneous integration in the internal nanostructure. Comparing the new system to the GMObased CBs stabilized with F127, they found that the stabilizer-free nanocarrier showed cytocompatibility and a higher antimicrobial effect, especially against the tested Gram-negative pathogens, among which P. aeruginosa CIP A22 DSMZ 25123 strain (Zabara et al., 2019). Other Drug Delivery Systems Some negative aspects are related to the lipid-based nanocarriers: beside the poor stability, they are also susceptible to aggregation in vitro and to esterase activity. This last aspect might also affect the relationship between the in vitro and the in vivo controlled release of the cargo. Subsequently, materials alternative to lipids have been explored including self-assembled polymeric nanocarriers for preparing both vesicular and bicontinuous systems. Compared to lipids, the block polymeric structures (BPS) can be synthesized from an expansive pool of amphiphilic monomers. Therefore, BPSs, called also polymersomes, have demonstrated high flexibility to functionalization, along with well-defined structures that can be distinguished in both hydrophobic and hydrophilic sections. Hence, the BPS can exhibit substantial rewards involving both mechanical and chemical stability (Allen et al., 2019). Most AMP formulations in ongoing clinical trials belong to semi-solid preparations for external use (Koehbach and Craik, 2019;Koo and Seo, 2019;Sheard et al., 2019). Hence, among the FIGURE 4 \| Cubosomes comprise curved lipid bilayers with a well-defined disposition and divided into two internal aqueous channels that can be exploited by antimicrobial peptides. Figure created with topical formulations, topical gel formulations are often mentioned in several research works to treat e.g., chronic skin and soft tissue infections. Moreover, proteins but also longer peptides ranging between 20 to 30 amino acids can also selfassemble naturally to achieve a-helices or b-sheets motifs. Likewise, two antiparallel b-strands can fold in b-hairpin motif, which contributes to creating higher-ordered fibers and pH-responsive active pharmaceutical ingredient vehicles. Recently, specific functional peptides have been synthesized and utilized as useful nanomaterials. Particularly, important properties have characterized a special group of synthetic peptides called peptide amphiphiles (PAs). They essentially consist of four sequences: (i) a hydrophobic tail (e.g., palmitic acid residue); an internal portion able to form b-sheets, which comprises (ii) an amino acid sequence to promote through hydrogen bond the formation of fibril-like structures; (iii) a spacer containing charged amino acids to allow solubility and cross-linking (Cui et al., 2010); at the opposite end of the structure, (iv) the hydrophilic head can be found that triggers the signaling for the biological response. Due to the molecular organization and the chemical characteristics, PAs can organize spontaneously in a nanostructure using a folding-like behavior to form specific nanostructures, including micelles and microtubes ( Figure 5). Hence, to stabilize the system in a lower energy state, PA molecules can organize the alkyl chains away from the aqueous environment, exposing externally the hydrophilic portion. PAs have attracted special interest as drug carriers due to their (i) advantage of a unique structure of assemblies, (ii) abundant molecular structures, and (iii) ability to give biological functions (Song et al., 2017). Additionally, the self-assembly aptitude of diphenylalanine (Di-Phe) building-blocks can be used to obtain diverse supramolecular nanostructures, such as nanofibrils, or nanowires. These structures have demonstrated large applicability due to their biocompatibility, high loading capacity and simplicity to obtain the self-assembled nanostructures. Furthermore, as reported by Schnaider and co-workers, nano-assemblies formed by Di-Phe exhibited an intrinsic antibacterial activity (Schnaider et al., 2017). Such nanostructures can considerably enhance the active pharmaceutical ingredient stability since they become less sensitive towards enzymatic degradation. Likewise, most AMPs forming a-helices or b-sheets could be inserted into supramolecular nanostructures. This strategy might contribute, therefore, to a suitable delivery of AMPs without using additional vehicles and their molecular stability. Upon contact with the pathogen, the peptide nanostructure is disrupted, especially from peptidases, and releases the AMP. Also inorganic nanomaterials (metal and metal oxide nanoparticles, silica, nanoclays, and carbon-based nanomaterials) have investigated as AMP delivery systems, because they shield the molecules from degradation and avoid peptide aggregation or conformational changes that could inactivate them (Nordström and Malmsten, 2017). Furthermore, they have the ability to control the drug release (thanks to well-defined pore sizes and forms (Vivero-Escoto et al., 2010)) increasing bioavailability and reducing toxicity (Nordström and Malmsten, 2017). In addition, several nanoparticles have been shown to have antimicrobial properties against both Gram-negative and -positive bacteria, suggesting that the complex AMP-nanoparticle may have a synergistic impact (Hajipour et al., 2012). Another synergistic effect could be achieved by a close interaction between AMP and antibiotics, which can be carried together in mesoporous silica nanoparticles with good chemical stability and biocompatibility, even though it is always important to consider the chemical nature of these nanoparticles, the dosage, and the administration route (Nordström and Malmsten, 2017). ADMINISTRATION ROUTES Compared to other routes of administration, the intramuscular, or the subcutaneous routes may not require too much stability of the peptide. Indeed, AMP physicochemical and biological characteristics could be taken into less account in these routes of administration, while size, permeation through gastrointestinal membrane, poor stability to gastric pH and susceptibility to proteolytic enzymes make the oral administration much difficult (Schiffter, 2011). Hence, injection represents the best route of administration for most of the AMPs (Di, 2015). However, the intravenous administration certainly exposes the peptides to the esterase and peptidase activity present in serum (Vlieghe et al., 2010;Fosgerau and Hoffmann, 2015). The oral route remains a patient-friendly option, due to the non-invasive and painless administration. However, considering few exceptions, the oral pharmaceutical technologies have not shown radical improvements regarding the AMP formulation to increase their bioavailability. The principal efforts concern the peptide stability due to the presence of pancreatic peptidases, e.g. a-chymotrypsin, trypsin and pancreatic elastase secreted from the pancreas into the gastrointestinal tract (Vlieghe et al., 2010;Aguirre et al., 2016;Malhaire et al., 2016). Furthermore, high dosage and low systemic exposure allow minimizing systemic side effects when a drug is formulated for the lung administration. Inhaled medications of peptides have demonstrated superior in terms of rapid onset (Larijani et al., 2005). Peptide macrocycles with antimicrobial effect working as protein epitope mimetics can also be formulated for inhalation, due to appropriate chemical stability. The POL6014, a neutrophil elastase inhibitor (i.e., Murepavadin ® ), can be administered via eFlow ® nebulizer system to treat cystic fibrosis lung infections and it is currently in Phases I/II (NCT03748199, 2018). In conclusion, as reported above, topical applications involving AMPs loading in nanoparticles, hydrogels, creams, gels and ointments represent the most used and best developed AMP applications and further studies are needed to exploit new suitable administration routes. AMPs IN ONGOING CLINICAL TRIALS We have described several AMPs approved for clinical applications. However, many others, both natural and synthetic, are still under clinical trials (Table 3). Preliminary results suggested that many AMPs could be useful alone or in synergy with common antibiotics to prevent or treat several diseases, but most of the studies are still ongoing or were stopped because of issues that can be solved, including unfavorable pharmacokinetic profile or unexpected side effects (Browne et al., 2020;Dijksteel et al., 2021). Below we reported some recent clinical trials, focusing the attention on studies still in progress. Bacitracin, a natural cyclic AMP from Bacillus subtilis, is currently reported in several ongoing studies of phase IV to treat Gram-positive bacterial infections (Bacitracin -ClinicalTrials.gov). These studies are evaluating bacitracin (i) in subjects with minor, second-degree burns, for topical use and in combination with a second ointment of collagenase; (ii) as an ointment to treat skin infections in combination with medical-grade honey; (iii) as an ointment for topical antibiotic therapy after eyelid surgery and to evaluate the use of antibiotic prophylaxis in presence of antibiotic side effects and antibiotic allergy; (iv) as topical antibiotic irrigation to reduce surgical site infections and in combination with neomycin and polymyxin (Neomycin ® ) for postoperative urinary tract infections and to extend the antimicrobial effect to Gram-negative bacteria; (viii) to evaluate the efficacy of preoperative oral antibiotic prophylaxis for preventing surgical site infections in elective colorectal surgery (combination of Bacitracin and the antibiotic Neomycin). Another clinical trial was ongoing to evaluate the topical use of bacitracin to reduce surgical site infections in midfacial fracture surgery, but in April 2020 this trial was closed because of bacitracin toxicity. Other phase IV studies involving bacitracin are aimed to the treatment of (v) facial burns, (vi) in combination with topical tranexamic acid (i.e., 5%, and 25%), and (vii) with polymyxin B (Polysporin ® ) to evaluate the use of Biofine ® cream on wounds due to cryotherapy for removing actinic keratosis lesions (Bacitracin -ClinicalTrials.gov). Pexiganan, a linear AMP, is under investigation in four phase III studies for the treatment of diabetic foot ulcers using topical cream formulations (Gottler and Ramamoorthy, 2009). Omiganan, an indolicidin derivative (Sader et al., 2004), has been tested in a total of sixteen studies and thirteen of them have been completed. Looking at the completed ones, three phase III studies have been reported, among which two were aimed to evaluate the efficacy of AMPs as topical gel formulation to treat rosacea. The third phase III study concerned the treatment of catheter colonization, and prevention of bloodstream infections if applied to the skin surrounding the insertion. The innate immunity of mammals comprises also the cathelicidins as a distinct class of proteins. Like defensins, although their structural features clearly distinguish them from defensins, cathelicidins act as precursor molecules that can release an AMP after proteolytic cleavage (Dürr et al., 2006). The human cathelicidin-derived AMP, named LL-37, belongs to the class of a-helical AMPs. Currently it can be found on a Phase II clinical trial by Promore Pharma (Promore Pharma AB, Sweden) evaluating LL-37 safety and tolerability in patients with venous leg ulcers (Grönberg et al., 2014;Sierra et al., 2017;Koo and Seo, 2019). It is also under investigation in patients with diabetic foot ulcers (LL-37 -ClinicalTrials.gov). The synthetic AMP LTX-109 represents a novel class of very short AMPs. It has been described as a synthetic antimicrobial peptidomimetic and has entered the phase II clinical studies (Isaksson et al., 2011) with the aims (i) to assess the clinical and microbiological response of two LTX-109 dosages (i.e., 1%, and 2%) formulated as a topical gel (Lytixar ™ ) for the treatment of nonbullous impetigo; (ii) to evaluate the safety, local tolerability, and efficacy of 1%, 2% and 3% LTX-109 gel formulations for the anterior nare delivery in patients who are carriers of MRSA/MSSA (methicillin-susceptible S. aureus); (iii) defining the magnitude of systemic absorption when LTX -109 is applied to the anterior nares as a topical gel; (iv) to evaluate the safety and tolerability of topical Lytixar ™ formulation onto uncomplicated skin infections, as well as to investigate both the clinical and microbiological effect of Lytixar ™ in patients with uncomplicated skin infection by Gram-positive and to determine the degree of systemic absorption of LTX-109. A further trial in recruiting phase is aimed to demonstrate the safety of a percutaneous application of a 3% gel cream of LTX-109 in Hidradenitis suppurativa, to identify the clinical responses and the influences of specific parameters, including age, disease duration, and body mass index (LTX-109 -ClinicalTrials.gov). Brilacidin is a synthetic AMP, successfully tested in Phase II clinical trials for treatment of acute bacterial skin and skin structure infections. A recent work demonstrated that Brilacidin displays an antiviral activity, inhibiting SARS-CoV2 virus in Vero African green monkey kidney cells and Calu-3 human lung epithelial cells and showing a synergistic inhibitory activity in combination with the antiviral Remdesivir (Bakovic et al., 2021). A Phase II clinical trial is going to start to assess the efficacy and safety of Brilacidin on patients with moderate or severe SARS-CoV-2 infection, hospitalized with respiratory difficulty but not requiring high-level respiratory support (Brilacidin -ClinicalTrials.gov). CONCLUSIONS AMPs can be considered unconventional therapeutic small molecules which have attracted great interest in recent years because of their promising potential, as they can be used as alternative or complement approaches for treatment of microbial infections. Due to their potency, broad-spectrum activity, different sources available in nature, lack of rapid development of resistance, low accumulation in tissue and rapid killing activity, these peptides show several advantages over conventionally used antibiotics. Moreover, AMPs also display immunomodulatory, antioxidant and anti-inflammatory activities and, for this reason, researchers are devoting considerable efforts to implement the use of AMPs as commercially available drugs. This review examined the features of AMPs, their mechanisms of action and their sources, highlighting their antimicrobial activity against several pathogens involved in human infections. Thus, the efficacy and potentially applicability of AMPs in human diseases has been analyzed. Particularly, we examined the beneficial role of several AMPs in the treatment of skin infections, but we also reviewed their potential use in respiratory diseases and oxidative-stress disorders, such as obesity, diabetes and chronic inflammatory intestinal disorders. Indeed, AMPs display several potential applications in medicine, since they can regulate proinflammatory reactions, stimulate cell proliferation, promote wound healing by modulating the cell migration, angiogenesis, chemotaxis and cytokine release. On these bases, pharmaceutical companies are performing great efforts to develop AMPs as therapeutic agents, improving their chemical and metabolic stability, setting up smart and novel formulation strategies, with the aim to improve AMP delivery and, consequently, their activity.	2021-06-15T13:16:51.821Z	2021-06-14T00:00:00.000	{ "year": 2021, "sha1": "993b7b0a3fda762185362fcb1da130e8fce7262f", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fcimb.2021.668632/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "993b7b0a3fda762185362fcb1da130e8fce7262f", "s2fieldsofstudy": [ "Medicine", "Biology", "Chemistry" ], "extfieldsofstudy": [ "Medicine" ] }
154856361	pes2o/s2orc	v3-fos-license	Recent HR practices of selected banks in India Introduction The banking sector has been an instrument for the economic development of any nation and its role in a developing nation like ours is a vital importance. Globally, the banking activities worldwide are undergoing rapid diversification. Technological changes have become the very essence of the banking sector and the Indian banking sector it also prone to these changes. In order to maintain their status in the present competitive environment, banks have to concentrate to utilize the human resources efficiently for the better development. It should faster cohesive team work and create commitment to improve the efficiency of its human capital. Thus, the primary apprehension of the bank should be to bring in proper integration of human resource management strategies with the business strategies. In any organization, the quality and the amount of productivity mainly depends on the skills and interest of its employees. Therefore, every business organization should take the lead for upgrading the skills and knowledge of its employees for the mutual benefits and progress. In this, direction Human Resource (HR) is an essential process for every organization in order to optimally utilize its human resource and in turn to attain its designed objectives. Like any other industrial organizations, banking sector is also highly dependent on the quality of HR practices for the motivation of its employees. An effective implementation of HR activities would results in excellent organizational climate, for the people to be competent and productive. Nevertheless, it is not lonely the implementation of the HR practices but also their review from time to time helps the banking institutions maintained their status in the competitive environment. Many literature surveys reveal that a paradigm shift is taking place in the role of the HR function in these organizations. There is a real danger of the function itself being outsourced in most organization. The danger comes through the following shifts: The line manger has become increasingly competent even to handle the matters. The government is seriously thinking of privatization and this leads to a government-protected system, to an environment where it has to be contended with market forces and large corporations with significant brand equity and also follow vastly different HR strategies & practices. Technology is eliminating routine paper activities. Virtual organizations are coming up. Human Resource HR contributes to organizational performance in different ways: Introduction The banking sector has been an instrument for the economic development of any nation and its role in a developing nation like ours is a vital importance. Globally, the banking activities worldwide are undergoing rapid diversification. Technological changes have become the very essence of the banking sector and the Indian banking sector it also prone to these changes. In order to maintain their status in the present competitive environment, banks have to concentrate to utilize the human resources efficiently for the better development. It should faster cohesive team work and create commitment to improve the efficiency of its human capital. Thus, the primary apprehension of the bank should be to bring in proper integration of human resource management strategies with the business strategies. In any organization, the quality and the amount of productivity mainly depends on the skills and interest of its employees. Therefore, every business organization should take the lead for upgrading the skills and knowledge of its employees for the mutual benefits and progress. In this, direction Human Resource (HR) is an essential process for every organization in order to optimally utilize its human resource and in turn to attain its designed objectives. Like any other industrial organizations, banking sector is also highly dependent on the quality of HR practices for the motivation of its employees. An effective implementation of HR activities would results in excellent organizational climate, for the people to be competent and productive. Nevertheless, it is not lonely the implementation of the HR practices but also their review from time to time helps the banking institutions maintained their status in the competitive environment. Many literature surveys reveal that a paradigm shift is taking place in the role of the HR function in these organizations. There is a real danger of the function itself being outsourced in most organization. The danger comes through the following shifts: The line manger has become increasingly competent even to handle the matters. The government is seriously thinking of privatization and this leads to a government-protected system, to an environment where it has to be contended with market forces and large corporations with significant brand equity and also follow vastly different HR strategies & practices. Technology is eliminating routine paper activities. Virtual organizations are coming up. Human Resource HR contributes to organizational performance in different ways: The banking sector has been an instrument for the economic development of any nation and its role in a developing nation like ours is a vital importance. Globally, the banking activities are undergoing rapid diversification. In order to maintain their status in the present competitive environment, banks have to concentrate to utilize the human resources efficiently for the better development. It should faster cohesive team work and create commitment to improve the efficiency of its human capital. Thus, the primary apprehension of the bank should be to bring in proper integration of human resource management strategies with the business strategies. The long-term vision for India's banking system is to transform itself from being a domestic one to the global level may sound far-fetched at present. To take up this industry to the heights of international excellence requires combination of new technologies, better processes of credit and risk appraisal, treasury management, product diversification, internal control, external regulations and human resources at the most. The most important need in this service industry is naturally the HR. During the early phase of banking development in India after independence, opportunities for employment of the educated man-power were relatively limited. This sector was the preferred employer for the educated persons in the country in addition to civil services. In recent years, this position has changed dramatically. Certain rigidities have also developed in HR within the banking system as this system is public sector. Its hierarchical structure gives preference to seniority over performance, and it is not the best environment for attracting the best talent among the young people. How well Challenges are met will be mainly depend on the extent to which the banks leverage their primary assets i.e., HR in the context of the changing economic & business environment. In this paper a research survey is conducted to elicit the information about Recent HR practices of selected banks in India. This paper is an attempt to review the HR practices in the Indian banking sector taking place in the aftermath period of the new competition emerged with the arrival of new private banks and foreign banks through the process of liberalization. i) through sound functional basics; ii) through effective realignment when the external environment changes; iii) by building an organizational context to that the organization can cope with the dualistic forces (2002). Emergence of HR Practices To make the Indian Banking System stronger, efficient and low-cost, the creation of fundamentals must include in the bank's operations, strategies and processes: strengthening the prudential norms and market discipline; adoption of international benchmarks; management of organizational change and consolidation within the financial system; upgrading the technological infrastructure of the financial system; and human resource development as the catalyst of the transformation (2002). The Human Resource field in the Banking Industry is considered as one of the process of discovery and transformation. The field of Human Resource can be described as emergent and dynamic within the cultural business aspect in a Banking Industry. The success of today's banking business will sparsely depends on the human resources of the organization, in which plays a crucial role in providing the services needed. The primary strength of the industry is the human resource that is why the efforts to develop the skills and management are the main subject placed before the human resource. A major challenge for many banks will be to develop the special competencies and skills for credit appraisal and risk management. Putting the information technology is a key contributed in human resource development. Therefore, the HR model of the future will require professionals to be both driving and anticipating change, understanding the complexities of the new business environment and forces shaping it (2002). Human Resource in Banking The core function of HR in banking industry is to facilitate the performance improvement among its people. Factors such as skills, attitudes and knowledge of personnel, play a critical role in determining the competitiveness within the organization or the industry (2002). The quality of human resources indicates the ability of banks to deliver the value to clients or customers. Indian banking industry has been an important driving force behind the nation's economic development. The emerging environment poses both opportunities and threats, particularly to the public sector banks, as well as the human resource in changing economic and business environment. The primary emphasis needs to be on integrating human resource strategies with the business strategy. Above the aspects of recruitment, placement, performance management, rewards and employee relations -a radical transformation of the existing personnel structure in public sector banks like the seniority over performance is not the best environment for attracting the best talent from the young competitive environment. However, recruitment practices as well as on-the-job-training and redeployment are considered as one of those many improvements of HR in Indian Banks (2002). HR Practices in Banking Industry: A. Staff Meetings • Staff Meeting aims at group synergy, team building, open culture, family feeling and talent recognition which individually and cumulatively benefit the organizations. • Goals/Targets set for the unit/Bank is discussed in the monthly Staff Meetings conducted at all branches/units and action plan is drawn in achieving them. • The forum is being effectively utilized for harmonious functioning of all the branches and administrative units through greater involvement and collective contribution of all staff members. Brain Storming Sessions • This is a technique for generating ideas and suggestions on topics of relevance and also to provide alternate solutions to problems by simulative thinking and imaginative power of cross section of employees. • Corporate Topics are selected for each quarter and brain storming sessions are conducted in administrative offices/ branches on the topic during every quarter. • Worthy implement able suggestions emanated are circulated for necessary action. Study Circle • Concept of Study Circle aims at self development of employees by kindling the desire to acquire/update knowledge, information and experience. • Guest lectures, Power Point Presentation, Group Discussions, etc are arranged on topics of general interest by inviting experts in the field. • Study Circle Meeting are conducted once in two months in administrative offices and once in a quarter in branches Quality Circles • It is a time tested tool of Total Quality Management (TQM) which promotes team spirit, cohesive quality work culture, commitment and involvement of employees. Best Employers in Banking Industry The US$ 28 billion Indian financial sector has grown at around 15 per cent and has displayed stability for the last several years. The total asset size of the Indian banking sector is US$ 270 billion while the total deposits amount to US$ 220 billion with a branch network exceeding 66,000 branches and 17,000 ATMs across the country. Currently, India has 88 scheduled commercial banks -28 public sector banks, 29 private banks and 31 foreign banks. According to a report by ICRA Limited, a rating agency, the public sector banks hold over 75 percent of total assets of the banking industry, with the private and foreign banks holding 18.2% and 6.5% respectively. In the year (2006-07) was marked by surplus liquidity, slowly rising interest rates, good credit growth, good returns, mergers and status quo on reforms. Indian banks broadly are classified as nationalized banks, private banks and cooperative banks and foreign banks. Reserve bank of India is the central banks and supreme monetary authority. It is being predicted that India could become the third largest banking hub in the world by 2040. The total assets of all scheduled commercial banks by the end of March 2010 is estimated at Rs 40,90,000crore, which will form about 65 per cent of GDP at current market prices as compared to 67 per cent in 2002-03. Banks assets are expected to grow at an annual composite rate of growth of 13.4 per cent in the coming years. The Indian Banking Sector is witnessing a dearth of qualified professionals. And to fix this issue, many organizations are joining hands with premier institutes. To quote some examples, Standard Chartered has tied up with SP Jain Institute and BSE with Birla Institute of Management Technology (BIMTECH). There has also been a need to groom and sharpen the skills required in dealing with banking sector. This is one of the greatest challenges that concern the sector. Specialized skills are required in retail banking, investment banking, risk management, foreign exchange, development banking, etc. As a consequence of this, banks and financial companies are required to streamline their human resource and IT processes. As far as compensation is concerned, salaries in banking sector have raised to 17 per cent hike in financial year 2008. The annual entry level salary ranges from Rs.2.62 lakh to Rs.4.46 lakh throughout India. Moreover MNC banks like ABN AMRO, Citibank and others are paying higher compensation through employee stock option plans, bonuses and increments. HR Practices and Policies followed by various Banks Axis Bank Axis Bank (earlier popular as UTI Bank) was the first of the new private banks to begin its operations in 1994. The bank has been promoted jointly by the Unit Trust of India, Life Insurance Corporation of India, General Insurance Corporation Ltd. and its associates, viz., National Insurance Company Ltd., The New India Assurance Company, The Oriental Insurance Corporation and United Insurance Company Ltd. Presently, the Bank operates through a network of more than 608 branch offices and Extension Counters. The bank, currently, has a total headcount of 13,389. It works on strong ethical practices and believes in achieving customer satisfaction by providing quality service effectively and efficiently, maximizing stakeholder value and adding more customers to its base. The Bank has adopted a human resource policy that is not only robust and flexible, but also aims to create and nurture a committed, motivated and knowledgeable pool of talent. The bank hires employees through lateral recruitments as well. With a view to promote rural talents, it recruits aspirants from Tier-II business schools as well. Continuous training, the opportunity to work on challenging tasks, and job rotation are part of the Bank's talent retention strategy. Training is an area of continuing focus for the Bank in order to ensure that its professionals are equipped to maintain high standards of customer service and are also aware of the latest developments in their specializations. The training system in the Bank focuses on upgrading the professional skills of each individual employee through classroom sessions, outbound training, and in-house and external domain skills programmes. The bank has a wellstructured performance-linked scheme of variable pay and employee stock options for all employees across grades and functions. Bank of India Bank of India is a premier public sector bank which was founded on 7th September, 1906. It has a purposeful existence of 100 years. The Bank has 2725 branches in India spread in all states and union territories including 120 extension counters. These branches are controlled through 48 Zonal Offices. There are 25 branches/ offices (including three representative offices) abroad. It has an employee base of over 41,511 employees. The bank has a joint venture abroad by the name of Indo Zambia Bank Limited. The bank has a strict policy of prudence and caution and conducts business on basis of its values and ethics. Realizing the importance of human resource, the bank focuses on continuous empowerment of its employees. Appropriate performance recognition and motivation schemes are already in place a proper training is provided to the staff wherever needed. The bank initiates serious efforts to redeploy redundant staff if any. Citibank Citibank is a part of the Citigroup, a premier global financial services organization that caters to almost 130 million customers in more than 100 countries. Citibank India is the largest MNC bank in India with a network of 16 branches across the country. Its presence is in India since 1902. Citibank offers host of value added services such as retail banking, investment, insurance, loans, wealth management, NRI banking etc. While dealing with customers, Citibank India values truthfulness, superior quality of products and services, transparency in credit decisions, safeguarding public funds. It provides a professional work environment that attracts and retains outstanding individuals who are committed to excellence. Valuing integrity, teamwork and partnership, it looks for creative and dynamic people who take enormous pride in their work. Citibank India's human resources department has moved its regular HR functions online so as to concentrate on the other, more important issues such as training processes and staffing requirements. Being among the most valuable brands in the banking industry, Citibank has managed to attract and retain talented employees. The bank focuses on its people, and believes in hiring experienced bankers and thought leaders and providing additional career development for existing teams. Corporation Bank Corporation Bank is regarded as one of the well-run banks among the public sector banks in the Country. The bank was established in 1906 and went public in 1997. It is one of the technologically advanced banks with 100 per cent computerized branch network. The bank has a capital market subsidiary, namely CorpBank Securities and a home loan subsidiary named CorpBank Homes Ltd. The bank has also sponsored Regional Rural Bank (RRB) named Chikmagalur Kodagu Grameena Bank (CHIKO Bank). Corporation Bank is regarded as one of the well-run banks among the public sector banks in the Country. The bank was established in 1906 and went public in 1997. It is one of the technologically advanced banks with 100 per cent computerized branch network. The bank has a capital market subsidiary, namely CorpBank Securities and a home loan subsidiary named CorpBank Homes Ltd. The bank has also sponsored Regional Rural Bank (RRB) named Chikmagalur Kodagu Grameena Bank (CHIKO Bank). The Bank has a relatively young, dynamic and efficient manpower of 11,880 employees which is the key factor for bank's success. The bank actively continues its endeavors in the development of human capital so as to provide unmatched services to its clientele. The bank's firmly believes in serving the society. Its good customer service, adherence to prudential accounting norms, consistent profitability and adoption of modern technology for betterment of customer service has earned it a place of pride in the Banking Community. Excellence in performance and uniqueness in customer service form the central core of Bank's organizational culture. As the industry is witnessing attrition even at the lower levels (clerk cadre), the bank has initiated steps to minimize the attrition at bottom levels by recruiting pre-university candidates. The bank believes that such candidates are expected to stay for long with the company. HDFC Bank HDFC Bank is one of India's premier banks providing a wide range of financial products and services to its customers. It was promoted in 1995 by Housing Development Finance Corporation (HDFC) and currently has a nationwide network of 2000 Branches and 5,998 ATMs in 996 Indian towns and cities. Total number of employees working with the bank is 52,687 as of March 31, 2009. The Bank focuses on training its employees on a continuous basis, both on the job and through training programs conducted by internal and external faculty. The Bank has consistently believed that broader employee ownership of its shares has a positive impact on its performance and employee motivation. The Bank's employee stock option scheme therefore extends to all levels and so far covers around 59% of the employees. The bank's sole focus is on product quality and service excellence. It values integrity, commitment, teamwork and excellence in customer service. HSBC India HSBC India is a part of HSBC Group which is one of the largest banking and financial services organizations in the world. The Group has around 10,000 offices in 82 countries and territories in Europe, the Asia-Pacific region, the Americas, the Middle East and Africa and serves over 110 million customers. HSBC India came into being after HSBC Limited acquired Mercantile Bank in 1959. The bank offers personal financial services including NRI banking, Wealth management, Gold and Classic credit cards, etc. it has over 50,000 customers in India. In commercial banking, it has its presence in 47 branches covering 26 cities. The bank values its employees and considers them the backbone of its business. It tends to create an environment that stimulates the challenges and rewards each employee, taking care of their personal and professional development. ICICI Bank The ICICI Bank was promoted by Industrial Credit and Investment Corporation of India in 1994 as a wholly owned subsidiary. The Bank has a network of about 950 branches and 3,300 ATMs in India and has presence in 17 countries. ICICI Bank is one of the largest recruiters in India's private sector and inducts as 15,000 people each year. With a headcount of 33,321, it believes that employees are the greatest driver of its growth and development. The bank has started a unique initiative of industry-academia partnership. It has collaborated with NIIT to set up the Institute of Finance, Banking & Insurance to provide skilled resources to the financial services industry at the entry level. ICICI Bank's performance and aspirations are underpinned by a strong organizational culture of dynamism, meritocracy, excellence in execution and high standards of professional integrity that have helped us become an industry leader. The bank runs a leadership development program which aims to build leadership talent within the organization. The program attempts to tap into the potential of employees and develop them into global leaders. It has also extended its role beyond economic growth concerns to directly participate in the pursuit of human development. IDBI Bank Industrial Development Bank of India Ltd (IDBI) is one of the largest commercial banks in India. It came into existence in October 2004 with the merger of erstwhile IDBI Bank with its parent company, IDBI Ltd. The bank operates through a network of 453 branches and 536 ATMs spread across 256 centers. The Bank has a highly competent and dedicated workforce and state-of-art technology to deliver personalized banking services. On March 31 2007, IDBI had a combined employee base of 7500, comprising 4277 officers, 1936 Clerical (Class III) and 1269 Sub-staff (Class IV) employees. The workforce includes professionals from the fields of accountancy, management, engineering, law, computer technology, banking and economics. The Bank trains its employees through various training programmes either in-house or through training institutes like Jawaharlal Nehru Institute for Development Banking (JNIDB), Hyderabad and Training Centre at Belapur. Oriental Bank of Commerce Oriental Bank of Commerce is a leading public sector bank in India with over eight million customers and 1,121 branches. The bank works towards achieving customer satisfaction and instills the same philosophy in all its employees. OBC has a network of 530 branches spread throughout India out of which 490 branches offer centralized banking solutions. It also has a network of 505 ATMs. The Bank's business continues to grow at an impressive rate of 27.57 percent. As on 31 st March 2007, the Bank had total staff strength of 14730. This includes 6809 Officers, 5002 non-subordinate and 2919 Subordinate staff. Due to the changing face of banking sector, there is a need to streamline the HR policies so as to attract and retain new talent. Towards this endeavor, the bank hires Specialist Officers under various streams such as Financial Analyst, Forex, SSI, Agriculture, IT, Law, Company Secretary, HRD etc. Besides, the Bank has also initiated the process for recruiting clerks with computer literacy which further strengthens & improves the service delivery channels and improves the age profile of employees and also leads to optimum utilization of Human Resources. The bank actively participates in social development by taking over projects with a goal of eliminating poverty, promoting self-employment, encouraging women empowerment, amending the lives of rural people and nurturing cottage industries. These programs cover 15 villages in total -10 in Punjab, 4 in Haryana and 1 in Rajasthan. The Bank has an effective grievance redressal mechanism. The grievances of the employees, if any, are promptly resolved through mutual and bilateral discussions over the regularly held industrial relations meetings. Punjab National Bank Punjab National Bank (PNB) is a public sector commercial bank which was established in 1895 at Lahore. It was nationalized in 1969. The bank has a rich customer base of over 3.5 crore customers. It operates through 4525 offices. It offers a wide range of products and services including corporate and personal banking, industrial finance, agricultural finance, financing of trade and international banking. The Bank's operations are classified into two segments: treasury operations and banking operations. Some of the core values of bank include customer satisfaction, working on ethical practices, motivated teams, and social economic development. The bank is committed to create a progressive work culture and encourages employees to deliver better services everyday. The bank was ranked 21 st amid top 500 companies by The Economic Times. It also stood on 9 th place in a study conducted by Economic Times on India's Most Trusted Top 50 service brands. State Bank of India The State Bank of India is India's largest commercial bank and is ranked one of the top five banks worldwide. The Bank is actively involved since 1973 in non-profit activity called Community Services Banking. It serves 90 million customers through a network of 9,000 branches. It offers a wide range of banking services including Personal Banking, Gold Banking, NRI Banking, International Banking, Corporate Banking, Small Business Finance, Rural Banking and Home Loans. SBI operates through 52 Foreign Offices with presence in 34 countries. SBI India serves the international needs of its foreign customers, in addition to conducting retail operations. The bank has eight banking subsidiaries in Bikaner and Jaipur, Hyderabad, Indore, Mysore, Patiala, Saurashtra and Travancore. The bank works towards improvement group dynamics and work culture through team building. The bank also has a dedicated facility for training and development of its employees. Syndicate Bank Syndicate Bank was established in 1925 in Udupi. Earlier it was known as Canara Industrial and Banking Syndicate Limited. The bank was nationalized in 1969. Today, the Bank has a network of 1801 branches spread all over India. Syndicate Bank has won many awards over the years for its excellent performance. In 1999, the Bank won FICCI AWARD for institutional initiative in the field of "Rural Development" and in 2003; the Bank was awarded Banking Technology Award awarded by IDRBT, Hyderabad. The bank has a positive, harmonious and productive work environment which emphasizes integrity and transparency in its methods of working. The bank encourages an open and informal culture that values integrity, commitment, teamwork and excellence in customer service. Challenges faced by the banks: The real challenge of this sector is how to transform the operations into global corporations without losing the positive values and culture that they have developed. As most part of the jobs in this industry is monotonous/repetitive and routine, the HR Department has to empower, engage and energize the employees to create effectiveness & efficiency through motivation organizational structures, systems & procedures are facilitators of these, and there is a need to focus greater attention on these aspects by the industry. Certain rigidities have also developed in HR Department within this banking system itself because this industry is largely in the public sector. Suggestions: Following suggestions are very essential to adhere for effective Banking Administration: HR functions to be linked to corporate goals. Team work is another important skill that is necessary in this industry. The HR Professionals have to introduce & improve the adaptability of their structure that will be able to absorb, draw and retain the best. HR practices are to be regularly reviewed against business outcomes as part of strategic and operational planning. To overcome the public sector's hierarchical structure, which gives preference to seniority over performance, is not the best environment to attract the best talent from among the young. Recommendations There is a need to adopt global best practices in financial sector regulation and supervision and adapt them to the domestic environment. This largely depends on the functioning and policies of public institutions, such as the RBI as it is increasingly subject to public discussion and debate. This calls for greater transparency more effective communication, and a high degree of professionalism in the bank's staff, are the need of the hour. Incentive structures need to be conceived, supported by appropriate training and motivations, which aligns the employee's goals and orientations with the core competencies and strategic advantages of the institution This service sector has to explore the feasibility of entering into collaborative arrangements with universities and other institutions in India and abroad to identify and provide specialized training in the financial services industry with an ongoing flow of emerging training packages. Information technology is an area where human resources development is critical fortunately, Indian professionals are world leaders in this area and spirit of co -operation and partnership between them in banking industries will result in a strong and modern financial system comparable to the best in the world. This committee recommended a system of recruitment from open market, including lateral induction of experts. Conclusion: In a nutshell, it is felt that, the changing environment, the forces of globalization and liberalization and the advances in information and communication technology have major HR implications for the RBI as well. Continuous up gradations of human resources management strategies with a view to enhancing the level of knowledge, sharpening skills are important and the necessary work culture must be installing to develop work practices which encourage efficiency in this banking sector.	2019-05-16T13:06:58.984Z	2011-10-01T00:00:00.000	{ "year": 2011, "sha1": "c2d95ae26a65b80c34d8e0562cfc8be757e05a1b", "oa_license": "CCBY", "oa_url": "https://doi.org/10.15373/2249555x/nov2011/24", "oa_status": "HYBRID", "pdf_src": "MergedPDFExtraction", "pdf_hash": "67ef64907f17e75178d3cca3fbedf160f9c9e295", "s2fieldsofstudy": [ "Economics", "Business" ], "extfieldsofstudy": [ "Economics" ] }
236966855	pes2o/s2orc	v3-fos-license	The Impact of COVID-19 Pandemic on Emergency Department Visits at a Canadian Academic Tertiary Care Center Introduction Public health response to the coronavirus 2019 (COVID-19) pandemic has emphasized social distancing and stay-at-home policies. Reports of decreased emergency department (ED) visits in non-epicenters of the outbreak have raised concerns that patients with non-COVID-19 emergencies are delaying or avoiding seeking care. We evaluated the impact of the pandemic on ED visits at an academic tertiary care center. Methods We conducted an observational health records review between January 1–April 22, 2020, comparing characteristics of all ED visits between pre- and post-pandemic declaration by the World Health Organization. Measures included triage acuity, presenting complaints, final diagnoses, disposition, and mortality. We further examined three time-sensitive final diagnoses: stroke; sepsis; and acute coronary syndrome (ACS). Results In this analysis, we included 44,497 ED visits. Average daily ED visits declined from 458.1 to 289.0 patients/day (−36.9%). For the highest acuity triaged patients there was a drop of 1.1 patients/day (−24.9%). Daily ED visits related to respiratory complaints increased post-pandemic (+14.1%) while ED visits for many other complaints decreased, with the greatest decline in musculoskeletal (−52.5%) and trauma (−53.6%). On average there was a drop of 1.0 patient/day diagnosed with stroke (−17.6%); a drop of 1.6 patients/day diagnosed with ACS (−49.9%); and no change in patients diagnosed with sepsis (pre = 2.8 patients/day; post = 2.9 patients/day). Conclusion Significant decline in ED visits was observed immediately following formal declaration of the COVID-19 pandemic, with potential for delayed/missed presentations of time-sensitive emergencies. Future research is needed to better examine long-term clinical outcomes of the decline in ED visits during pandemics. INTRODUCTION On March 22, 2020, the World Health Organization (WHO) designated the outbreak of a novel coronavirus (SARS-CoV-2) first reported in January 2020 as an international pandemic causing coronavirus disease 2019 (COVID-19). 1-3 COVID-19 was thought to spread from person-to-person by respiratory droplets and contaminated surfaces or fomites, with asymptomatic transmission suspected. [4][5][6] In an effort to "flatten the curve" public health response to COVID-19 encouraged social distancing, self- Impact of COVID-19 Pandemic on ED Visits Kwok et al. Population Health Research Capsule What do we already know about this issue? Responses to the coronavirus disease 2019 pandemic have emphasized social distancing and stay-at-home policies with subsequent reports of decreased emergency department (ED) visits. What was the research question? We evaluated the impact of the pandemic on ED visits at a center not overwhelmed with COVID-19 admissions. What was the major finding of the study? Decline in ED visits including time-sensitive emergencies was observed after declaration of a pandemic. How does this improve population health? Public health responses to pandemics affect ED visit behaviors. Further research is needed to examine long-term clinical outcomes of the decline in ED visits. year. At our center, confirmed COVID-19 admissions were limited (as of April 22, 2020, Ottawa had eight COVID-19 patients in intensive care, and 22 COVID-19 patients on inpatient wards 8 ) and had not overwhelmed acute hospital capacity. The sudden drop in ED visits caused concern that patients with non-COVID-19 emergencies were delaying or avoiding seeking appropriate ED care during this pandemic. We sought to rapidly review the immediate impact of the COVID-19 pandemic on ED visits at a tertiary care hospital not overwhelmed with COVID-19 admissions. We aimed to characterize and compare trends of pre-vs post-COVID-19 ED populations in terms of the Canadian Triage Acuity Score (CTAS) level, presenting complaints, discharge/admission diagnoses, and patient flow metrics. In addition, we sought to examine the effect of the pandemic on ED visits and mortality rates of three time-sensitive diagnoses: stroke; sepsis; and acute coronary syndrome (ACS). Design We conducted a retrospective observational electronic health records (EHR) review. Setting The Ottawa Hospital (TOH) is a 1202-bed academic tertiary care hospital with the ED receiving >174,000 visits per year. It is the main regional referral center for specialized services including trauma, stroke, neurosurgical, thoracic, oncological, and vascular emergencies. Adjacent to TOH is the regional cardiac center, the Ottawa Heart Institute, which receives prehospital Code STEMI (ST-elevation myocardial infarct) cases bypassing TOH EDs. It was not included in this study. Patient Population and Time Period We included all patients presenting to TOH ED between January 1, 2019-April 22, 2020. We excluded all patients who were "direct-to-service," which included patients already assessed at another hospital/outpatient clinic being transferred directly for admission to a specialized service at TOH. We used the date March 11, 2020, when the WHO declared COVID-19 to be an official pandemic, to define pre-and postpandemic periods. Measures We collected ED visit characteristics including patient demographics, presenting complaints, final diagnoses, and disposition. Mortality rates were observed for the entirety of patients' ED or in-patient stays. We also collected data on patients' CTAS, which is a triage tool used internationally to allow EDs and their staffs to prioritize patient care requirements upon arrival to the ED. Levels of CTAS range from 1 (most acute) to 5 (least acute). 9 For presenting complaints and final diagnoses, two authors independently reviewed all primary chief complaints listed for each ED visit, as well as final discharge/admission diagnoses, and assigned them into the most appropriate categories based on symptom-or specialty-related headings. Any discrepancies were resolved with discussion between the reviewers, with arbitration by the third author if necessary. We used a similar process to critically review all discharge/ admission diagnoses for three time-sensitive emergencies: stroke; sepsis; and ACS. Data Collection The Ottawa Hospital transitioned to Epic EHR (Epic Systems Corporation, Verona, WI) in June 2019. A quality improvement coordinator with Epic-reporting expertise pulled the required data elements from the EHR using integrated reporting functionalities and entered the data into a Microsoft Excel database (Microsoft Corporation, Redmond, WA) for further analysis. We retrieved historical patient volume data from TOH's previous performancemeasurement data warehouse. Data Analysis We present patient demographics, CTAS acuity, presenting complaints, final diagnoses, process measures, time metrics, and mortality using descriptive statistics. For comparison between pre-and post-pandemic periods, we examined the total number of ED visits within each Kwok et al. Impact of COVID-19 Pandemic on ED Visits time period, as well as the number of ED visits per day. We plotted relevant results temporally to provide visual trends over time, with annotation to provide context around specific milestones. We assumed normal distributions and performed statistical analysis using Student's two-sided t-test to compare pre-vs post-pandemic periods, and chi-squared test for comparison of proportions, with P-value of <0.05 considered to be significant. Ethical Considerations We obtained research ethics approval for this project by the Ottawa Hospital Research Institute Research Ethics Board, dated Apr 24, 2020, protocol ID# 20200262-01H. RESULTS A total of 44,497 ED visits met our inclusion/exclusion criteria during the study period (32,068 in pre-pandemic; 12,429 in post-pandemic) ( Table 1). The mean age was 49.9 years old with 46.5% being male patients. Overall, average daily ED visits declined from 458.1 patients/day in the prepandemic period, to 289.0 patients/day in the post-pandemic period (-36.9%). There was a significant decrease in the proportion of patients with incomplete ED visits (ie, leaving without being seen, etc), from 8.6% in the pre-pandemic period to only 3.5% in the post-pandemic period. Relative CTAS levels distribution remained stable throughout the study period, with the exception of an increase in the proportion of CTAS 5 patients (pre: 4.5%, post: 5.0%). For the most severe and critical CTAS 1 acuity patients, on average there was a significant drop of 1.1 patients/ day (-24.9%) in the post-pandemic period. For the second most critical CTAS 2 acuity patients, on average there was a significant drop of 45.9 patients/day (-37.7%). There was a sharp drop in overall ED visits immediately following the WHO declaration of a pandemic, followed by a second acute sustained drop in ED visits immediately after the city's local announcement of social distancing policies (Figure 1). The distribution of chief complaints presenting to the ED remained similar between the pre-/post-pandemic periods except for a number of categories ( Table 1). The only categories that increased in proportion relative to all presenting complaints were respiratory (pre: 10 There was a volume decline in all presenting complaint categories except for respiratory complaints, which rose acutely following the WHO declaration of the COVID-19 pandemic (Figure 2). At its peak on March 12, 2020, there were 131 ED The distribution of final diagnoses also changed following the WHO pandemic declaration. Diagnoses related to respiratory complaints increased from 4.2% to 6.1% of all diagnoses; infectious increased from 13.2% to 20.0%; and mental health increased from 4.6% to 5.5%. The top five final diagnosis categories with the greatest absolute numbers Patients diagnosed with infection-related issues spiked immediately after WHO's declaration of the COVID-19 pandemic, peaking at 168 ED visits (35.4%) on March 12, 2020 ( Figure 3). The number of patients diagnosed with mental health and respiratory-related issues appeared to be stable over time. Diagnoses related to musculoskeletal, abdominal/GI, and neurological issues had sustained declines in the post-pandemic study period. There was a significant increase in overall mortality rate for all ED visits in the post-pandemic period (pre: 1.1%, post: 1.6%), but no difference in mortality within the three subgroups of stroke, ACS, and sepsis ( Table 2). There was a significant drop in average daily ED visits for stroke (5.8 patients/day in pre-pandemic; 4.8 patients/day in post-pandemic) and ACS (3.3 patients/day in pre-pandemic; 1.7 patients/day in postpandemic), but no significant change in average daily number of ED patient diagnoses with sepsis (2.8 patients/day in prepandemic; 2.9 patients/day in post-pandemic). Patient flow metrics significantly improved in the postpandemic period. Physician initial assessment, defined as time from patient arrival to the ED to the time when first seen by a physician, improved by one hour (hr) and 50 minutes (min) (pre: 3hr 00 min, post: 1hr 10 min). Average ED length of stay for both discharged and admitted patients also significantly improved by 2 hr 12 min, and 11 hr 35 min, respectively. Finally, average total hospital length of stay for admitted patients decreased by 21 hr 39 min (pre: 214 hr 35 min, post: 192 hr 54 min). DISCUSSION Following WHO's declaration of COVID-19 as an official pandemic, we found a significant drop in overall visits to our ED. Patients presenting to the ED with respiratory and infectious issues sharply increased, while visits related to many other complaints decreased. Musculoskeletal-and trauma-related complaints appear to be the most impacted; this may in part have been due to social distancing and stayat-home public health messaging resulting in fewer outdoor activities and vehicles on the road. It is important to note the drop in absolute numbers of patients who presented to the ED with potentially life-threatening CTAS 1 and 2 acuities (-47 patients/day; a 37.3% decline), strokes (-1.0 patient/ day; a 17.6% decline), and myocardial infarction (MI) (-1.6 patients/day; a 49.9% decline). This a concerning proportion of patients with time-sensitive emergencies who were not presenting to the ED immediately following the pandemic declaration, given that there are no known physiological reasons for the prevalence of these conditions to be lower. Interestingly, the number of patients diagnosed with sepsis appears to have remained stable, which may reflect the fact that septic patients often present to the ED via prehospital emergency Table 2. Overall mortality rates and average mortality per day between pre-and post-COVID-19 pandemic status for patients diagnosed with stroke, acute coronary syndrome, and sepsis. *"in ED," mortalities within the emergency department; "in Hospital," after admission into hospital. ACS, acute coronary syndrome. medical services (EMS), and thus may be less affected by an individual's fear of coming to the ED. 10,11 Among patient groups whose volume of ED visits did not appear to be affected by the pandemic were those presenting with mental health-related issues. Anecdotally, physicians in our group reported seeing escalating cases of anxiety-related cases due to the COVID-19 pandemic; this may have been further augmented by closure of regular mental health community supports. Finally, we noticed significant improvements in all ED crowding and flow metrics. This is likely a result of the drop in hospital occupancy and improved internal operations after non-essential healthcare services were ceased during the pandemic period. A decline in the number of non-COVID-19 patients presenting for emergency care has been anecdotally observed elsewhere, with numerous news media articles citing concerns of unintended consequences in North America. 12,13 A regional hospital in Germany reported total ED visits to their center dropped by 23% within four weeks of admitting their first COVD-19 patient. 14 Although the article did not report details on acuity levels, presenting complaints, or clinical outcomes, it did note a respective 53% and 30% decline in the hospital's cardiology-and neurology-related ED populations. The authors postulated that these unintended consequences may have been a result of individuals' extreme reactions to dread risks, defined as "low-probability events in which many people are killed at the same time," such as the COVID-19 pandemic. Wong et al described a similar drop in overall ED visits in a community hospital in California, and interviews with patients confirmed fear as the overarching theme affecting decisions to avoid ED visits. 15 There are few other studies examining the ED population as a whole, although more reports are being published with respect to how the COVID-19 pandemic may be affecting specific diagnoses such as acute MIs and strokes. 16,17 Our findings also support the risk-avoidance behavior of ED patients with non-COVID-19 related issues in the setting of this pandemic. However, we did not power the study to robustly examine mortality rates for all subgroups of patients (due to limited time frame), and it is difficult to fully understand meaningful clinical impact. We did note an increased overall mortality rate in our study population, but this may simply be a reflection of the drop in non-emergent ED visits in the post-pandemic period rather than a true increase in severity of disease. Of note, our national statistics agency StatsCan found no increase in "excess deaths" between January 1-March 31, 2020 when compared to the same time period in the previous year. 18 It is very difficult to accurately attribute any potential delayed/avoided ED visit directly to patients' fears and behaviors in response to the pandemic. Future studies are needed to help identify this subgroup of patients who delayed ED presentation as a result of the pandemic, and to further examine relevant clinical consequences. LIMITATIONS There are a number of important limitations to our study. Firstly, this was a single-center study in North America and may not reflect nuances around ED visit behaviors of patients in other healthcare systems. Although our center is the regional referral center for specialized emergencies including stroke code bypass, STEMI cases identified in the field by EMS are redirected to a separate cardiac center and thus were not included in this study. As a result, our findings may underestimate the potential impact on ED visits related to cardiac and ACS presentations noted in our findings. Secondly, our findings reflect a center with relatively low COVID-19 burden in terms of admissions and critical care resources, and thus should be interpreted in relation to similar centers that were not epicenters of the COVID-19 pandemic. Thirdly, the pre-/post-design was limited by our institution's recent switch from paper charts to full Epic EHR; thus, we were unable to directly compare data from the same time period from previous year(s) without extensive manual chart review. However, we do not believe there are any seasonal variation factors between January-March vs March-April that would significantly invalidate our data. Finally, given the nature of a timely rapid review our study period was limited to just over a month past the WHO declaration of pandemic status. Future research with more detailed individual chart reviews are needed to assess delayed findings and clinical significance. CONCLUSION Significant decline in ED visits was observed immediately following declaration of global pandemic status, with potential for delayed/missed presentations of time-sensitive emergencies. We believe it is important for public health communication strategies to take our findings into account, as messaging regarding staying at home may have created potential extreme reactions to dread risks. Future research is needed to examine long-term and impactful clinical outcomes related to significant decline in ED visits during pandemics.	2021-07-31T13:09:39.381Z	2021-07-01T00:00:00.000	{ "year": 2021, "sha1": "84dd831782002ff18b969a49bd2de2c9bb9c975f", "oa_license": "CCBY", "oa_url": "https://escholarship.org/content/qt5rt4474k/qt5rt4474k.pdf?t=qy3u9e", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "75825301da5425a4b811ccfe5118c852f4957228", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
266077096	pes2o/s2orc	v3-fos-license	Implementation of Cement-based nano composite Energy Absorption Damper to improve the damping properties of concrete and monitoring applications . The energy from the moving seismic waves through a building structure is dispersed by means of dampers. Dampers work by converting the kinetic energy into heat energy, dissipating it into the hydraulic fluid. Damper systems are designed and manufactured to protect structural integrity, reduce structural damage, and prevent injury to people by absorbing energy from earthquakes and minimizing structural deformations. The most effective way to achieve good vibration damping is by tailoring the construction materials such as cement with nanomaterials like Silica, Alumina, Graphene, CNTs, etc. This paper focuses on developing a vibration damper, prepared by cement nanocomposite containing MWCNTs and Carbon fibers. The tests, such as the Impact, Flexural, and Compressive strength tests, are conducted to investigate their energy-absorbing capacity, strength, and durability. The microstructural analysis SEM is performed to know the morphology of concrete mix with MWCNTs and Carbon fibers on damping mechanism. Impact test results indicate that the beams without MWCNTs and CFs exhibited an average energy absorption of 248 J, while those with MWCNTs and CFs absorbed an average energy of 262 J which shows almost 15% more energy absorption. Adding nanomaterials in a cement matrix improves concrete's frictional damping energy consumption ability and increases structures' energy-absorbing properties, flexural strength, and compressive strength. Introduction Typhoons and earthquakes are the main natural disasters that can destroy reinforced concrete structures, causing significant societal costs for adapting the structures.Although concrete is the most used structural material, its inadequate damping qualities cause catastrophic damage.For people's safety and comfort, better concrete damping characteristics are now necessary.To date, vibration control has been used in structures to increase their lifespan and save expenditures.Vibration control attempts to mitigate the harm that dynamic vibration responds to due to civil infrastructure.The three categories that best describe it are passive energy dissipation, smart control, active, and semiactive and vibration isolation of the substructure [1].To modify the structural material itself is the best way to apply these techniques to enhance vibration capacity such that endures high strength while contributing to ideal damping properties.The material which is exposed to cyclic stress undergoes damping.It will be more practicable to add the mixture to the cement matrix rather than outfit structures with damping devices to boost the damping property.To date, it has been standard practice to increase the damping qualities of cement matrix by adding admixtures, such as polymers, steel, and carbon fiber [2][3][4].The promising carbon material that has astonishing properties is the carbon nanotube (CNT).The compressive strength of road composites doped with multi-walled carbon nanotubes has dramatically risen, increasing by 46.8% MPa year over year, according to experimental data.The density of the composites is more or less going to rise as the particle size grows under the same carbon nano content.[5].One benefit of employing carbon nanotubes to enhance the qualities of concrete is the prevention of cracking, which has led to a substantial increase in their use in the sector.[6].A single CNT fiber has a tensile strength of 100 GPa, Young's modulus of 1 TPa, can sustain a 15% fracture strain, and has a specific surface area of up to 1000 m2/g.[7][8].Due to its bonding, bridging, and mesh-filling properties, CNT may be utilized as a reinforcing element in cement matrixes [9][10][11], which would stop microcracks propagation and improve damping qualities.CNT has been used in several studies to increase the ability of polymer composites to reduce vibration.For instance, according to Dai and Liao's [12] investigation into the damping properties of epoxy resin, adding CNT can improve the composite's damping characteristics without subjecting it to strain.Tehrani et al. [13] observed that as compared to the reference sample, the performance of the hybrid CNT-carbon fiber-reinforced epoxy composite had a better loss tangent of 56%.However, there haven't been many studies done on how to increase the cement matrix's damping capacity.Additionally, the effects of the CNT on the cement composite's damping capacity and its operational mechanism have not yet been described. Numerous studies have identified the potential of carbon fiber-reinforced cement composites (CFRCCs) in terms of bending strength, impact strength, electrical conductivity, and behavior that senses strain [14][15][16].Due to the hydrophobic surface and low weight, it is difficult for carbon fibers to disperse uniformly in cement paste [17].And the higher flexural strength, conductivity, and toughness of the CFRCCs depend on the good dispersion of carbon fibers in the cement matrix [18][19][20].By comparing it was concluded that the pre-mixing method which involved the addition of 2-5 mm carbon fibers before the addition of cement achieved better dispersion than the post-mixing method.According to SEM findings, carbon fiber's capacity to prevent the development of microcracks and absorb energy by overcoming it pulling out is what accounts for the improved qualities of cement mortar filled with carbon fiber.[21].The interfacial modulation may greatly improve the damping ratio, loss factor, and energy dissipation ratio of UHPC, with the maximum damping ratio being provided by chemical modification of steel fibers.[22]. In the proposed work, we are developing a novel cement-based nano composite damper system to improve the damping properties and evaluate its efficiency, and mechanical and microstructural properties.Nanomaterials like MWCNTs and Carbon fibers have been incorporated into Cement to improve its energy-absorbing capacity by conducting various trial experiments like Impact Strength tests, Flexural strength tests, Compressive testing, etc. for the first time.The samples were cast as per the Mix design given in Table 3.The specifications of MWCNT's and Carbon fiber is presented in Tables 1 & 2. Three samples S1, S2, and S3 are the normal cement beams and are cast without any nanomaterials.D1, D2, and D3 are cast with the addition of nanomaterials viz MWCNTs which is taken as 0.5 wt% of the volume of cement, and Carbon fibers which is taken as 2 wt% of the total volume of cement as per the literature review as shown in Figure 1 properly in water and then put in an Ultra Sonicator for proper and uniform mixing.Then the required mold is properly cleaned and oiled and then the concrete mix is prepared by mixing the cement, sand, coarse aggregates, MWCNTs, Carbon Fibres, and water and then poured into the mold and kept for curing for 28 days in water in a tray at room temperature.One set of samples consists of the Dampers with nanomaterials and the other set is controlled specimens without nanomaterials.After 28 days of curing, the samples are tested for Damping and Energy Absorption Properties using the Impact, compression, and Flexural Beam Tests.The complete methodology and experimental procedure are shown in Fig 2. Izod Impact Test A conventional notched specimen's energy absorption while breaking under an impact force is measured by the Izod impact test.As demonstrated in Fig. 3, this test is still employed as a low-cost quality control procedure to evaluate the notch sensitivity and impact toughness of engineering materials such as metals, composites, ceramics, and polymers. To strike a piece of material or sample with a notched edge, a pendulum with a hammer of known mass and length as shown in Fig 3 .is released from a certain height as part of the equipment.The energy absorbed is calculated by comparing the difference in the height of the hammer before and after the fracture Compressive Strength Test The concrete compressive strength gives an understanding of all the properties of concrete.By this test one can infer whether the concrete pouring was done correctly or not.Concrete compressive strength ranges from 15 MPa (2200 psi) to 30 MPa (4400 psi) for normal construction.The commercial and industrial structures having greater compressive strengths.For a compressive strength test either a cube or a cylinder is used as they are advised as the standard specimen for the test by several standard codes.Hence the compressive strength test was carried out to know the compressive strength of the samples. Calculations of Compressive Strength Size of the dampers =5 cm x 5 cm x 2.5 cm.The average compressive strength involves using the formula for stress, which is defined as the force applied divided by the cross-sectional area of the specimen SEM Analysis (Scanning Electron Microscopy) SEM analysis of the composite is necessary to evaluate the various properties of composites like bonding, fracture behavior, interfacial adhesion, and other properties.The SEM analysis of the samples was carried out at Karnataka University Dharwad, India.Figure 9 proves that the fiber pullout zones are hardly visible, as it is just a Cement composite without nanomaterials whereas fiber breakage is more visible in Fig 10.This is indicative that the composite has good fiber-matrix adhesion which leads to a higher interlaminar shear strength between the layers.And good adhesion and bonding lead to good damping and better Energy Absorption Capacities of the composite.FTIR spectroscopy is a controlling technique used to characterize the characteristics of vibrational bands/modes within the material and the presence of various functional groups associated with the bonds in the compound.Fourier Transform Infrared Spectroscopy (ASTMD5477-18, 2018) was carried out at KLE Pharmaceuticals Hubli and was adopted to determine the FTIR spectra of the cement-based nanocomposite damper doped with MWCNTs and Carbon Fibers.FTIR spectra, between 400 and 4500 cm -1 with a wavelength accuracy of 1 cm -1 , were gathered.FTIR spectra can be employed to quantify the constructive and destructive bonds to evaluate the strength.It is clear that the MWCNTs have their characteristic peaks band at 2171.51 cm−1 and 2021.54cm−1 corresponding to their octahedral and tetrahedral site vibrational modes as seen from Fig 12, it can also be stated Fibres clattered due to improper mixing Uniformly clustered fibres at some locations that IR spectra for oxygen display a strong and broad peak around 2981cm−1, which corresponds to the stretching mode of the O-H group. Figure 8 Figure8shows a graph of the difference of compressive strengths test results of the specimens with and without nanomaterials.The compressive strength is highest for the specimens with Nanomaterials and it is 81.24N/mm 2 whereas the specimens without nanomaterials are having less compressive strength i.e., 77.18 N/mm 2 .The highest compressive strength is attributed due to the good bonding of Cement with MWCNTs and Carbon Fibers. Figure 8 . Figure 8. Compression test results of both the specimens with & without nanomaterials Figure 7 . Figure 7.The specimen under Compression Testing Figure 9 .Figure 10 . Figure 9. SEM analysis of the Cement Composite without nanomaterials 5 Conclusions Experimental investigations provide more insight into the influence of MWCNT and Carbon fibres on the damping, flexural, and Compressive strength properties of the composite.The energy absorption capacity of the composite was observed to increase with the content of MWCNTs and CFs in the range of 0.5% & 1 to 2wt.% in all samples.Samples loaded with MWCNTs and CFs exhibit an increase in Energy absorption capacity in a range of 15% more than the normal Cement Composite.Experimental results also an increase in flexural strength.Samples supplemented with MWCNTs and CFs display an increase in compressive strength of up to 5%.The addition of nanomaterials in a cement matrix improves concrete's frictional damping energy consumption ability and increases structures' energy-absorbing properties, flexural strength, and compressive strength.mechanical and electrically conductive properties of cement-based materials", Construction and Building Materials Volume 125, 30 October 2016.22. Huinan Wei, Tiejun Liu, Ao Zhou, Dujian Zou, Ye Li.Toughening static and dynamic damping characteristics of ultra-high performance concrete via interfacial modulation approaches, Cement and Concrete Composites, Volume 136, February 2023, 10487 4. Multi-walled carbon Nanotubes (MWCNTs)-These are 2.1 Materials1.Cement-ULTRATECH brand, OPC 43 grade cement was used.Ordinary Portland cement confirming to IS8112-1989 has been used, it is used for all types of buildings.2. Aggregates-The coarse aggregate gives the completed product the volume, stability, wear, erosion resistance, and other necessary physical attributes.5mm aggregates are obtained from Sieve analysis and are mostly angular in shape.The fine aggregate used is a granular material composed of finely divided mineral particles.Sand has various compositions but is defined by its grain size.It was procured locally from Tungabhadra River Mundargi.3. Carbon Fibres-Carbon fibers are about 5 to 10 micrometers in diameter and are composed mostly of carbon atoms.Table 1 lists the characteristics of the carbon fibers.They were procured from Intelligent Materials Pvt Ltd, Village Sunderban, Derabassi-140507, Punjab India.a special type of carbon nanotubes in which the single-walled carbon nanotubes are layered one inside the other.The specifications of the MWCNTs are presented in Table 2.They were procured from Intelligent Materials Pvt Ltd, Village Suderban, Derabassi-140507, Punjab India. Table 1 . Specifications of Carbon Fibers. Table 3 . Mix design for Concrete Mix 2.2 Preparation of Samples-Experimental Procedure	2023-12-08T16:05:47.861Z	2023-01-01T00:00:00.000	{ "year": 2023, "sha1": "762f9544f158817e19a526f9a122bda2f7aba531", "oa_license": "CCBY", "oa_url": "https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/92/e3sconf_icgest2023_03019.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "4f0bdd1b4c091a8efc4b822d8cafc5c0b6fff78b", "s2fieldsofstudy": [ "Engineering", "Materials Science" ], "extfieldsofstudy": [] }
221321232	pes2o/s2orc	v3-fos-license	Therapeutic Use of Silver Nanoparticles in the Prevention and Arrest of Dental Caries Dental caries is one of the major diseases of the oral cavity affecting humans worldwide. Different alternatives have been used for its control, but its incidence and prevalence are still high. On the other hand, silver has been used for centuries due to its antimicrobial properties. With advances in nanotechnology, the use and research in nanomaterials has increased, recently, and silver nanoparticles have become an essential part of the dental practice, giving materials physical and chemical improvements in their properties, used for their antibacterial capacity preventing and arresting dental caries. The objective of this review was to examine the use of silver nanoparticles, in the treatment of dental caries in the remineralization of teeth hard tissues, as well as the antimicrobial potential, cytotoxicity, and long-term effectiveness. Introduction Dental caries is a disease caused by a specific biofilm that forms acid [1], which begins with demineralization on the enamel surface showing what is called white spot lesion, this stage is represented by white and opaque colour [2,3], and the main pathogens responsible for dental caries Streptococcus mutans and Lactobacillus [3][4][5][6][7]. Although, in recent decades, there has been an advance in dental care, dental caries is a global health problem [2,8] and costly, compromising the health and quality of life of children and adults [1]. The most successful treatments used for the prevention and control of early-stage dental caries are based on the use of fluorides [9][10][11][12]. In dentistry, silver has been used for over a century as an antimicrobial agent due to its broad spectrum, low toxicity, and absence of cross-spectrum bacterial resistance [13][14][15][16][17][18]. Silver nitrate was used for reducing the incidence of caries in the deciduous dentition, as a caries preventive agent for permanent molars, a cavity sterilizing agent, and as a dentine desensitizer [9,19]. Subsequently, in the 1960s, it was suggested to combine silver with fluoride (silver diamine fluoride) as an anticaries agent for a possible combined effect [20,21]. However, its clinical application has been limited due to the black pigmentation produced by its application [9,[22][23][24][25][26]. The use of nanotechnology in dentistry has attracted significant attention in the recent years [1], showing novel methods in the prevention and treatment of caries, controlling plaque-related biofilms, and remineralization of primary dental caries [27], among these methods are the formulations of silver nanoparticles (AgNPs) with antimicrobial properties against a wide variety of microorganisms [1,9,[28][29][30][31][32][33][34]. Silver nanoparticles are considered potent antimicrobial agents and have been proven to be effective in vitro against cariogenic bacteria such as Streptococcus mutans [35], having an antibacterial activity 25 times greater than chlorhexidine, as well as an antiviral and antifungal activity [33,36], so multiple studies have proposed their use in various preparations, showing good results in the treatment of early dental caries [4,37,38]. For these reasons, the objective of this report is to present a review of the literature on the possible therapeutic use of silver nanoparticles in dentistry, in the remineralization of teeth hard tissues, their mode of action, and biocompatibility. AgNPs Formulations in Caries Prevention and Arrest Dental caries is one of the most widespread diseases in the world, affecting 95% of the population [39,40] at different stages of their lives [41,42]. Several studies have designed formulations containing silver nanoparticles against cariogenic pathogens mainly against Streptococcus mutans, evaluating their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and in the control of biofilm formation; showing that they are good oral antimicrobial agents [28][29][30]43], especially at dimensions between 80100 nm, since cytotoxicity increased to dimensions lower than 20 nm [44] has been demonstrated ( Table 1). New formulations have been evaluated against cariogenic pathogens and their cytotoxic activity through haemolytic activity in different concentrations provisionally called silver nanofluoride (NSF). MIC and MBC were, respectively, around 33.54 µg/ml and 50.32 µg/ml. Silver diamine fluoride (SDF) showed similar values. NFS proved to be an antimicrobial agent similar to silver diamine fluoride, but less cytotoxic; however, the effect was not tested or if it stained the enamel surface [37]. Another important aspect to evaluate is the microhardness of the enamel after remineralization with formulations containing AgNPs and evaluating the cariostatic effects using microhardness and microbiological tests as outcome variables, using Saforide ® , Cariestop ® , Ancarie ® , and Ag-Nano (experimental cariostatic agent). Naturally exfoliated deciduous teeth were submitted to an initial test (after a pH cycle to obtain an initial like-caries lesion) and a final microhardness test (after application of the cariostatic agents), with an antimicrobial effect evaluated with Streptococcus mutans, Escherichia coli, and Enterococcus faecalis strains. All the cariostatic showed an improvement in the remineralization of the enamel, in which Saforide ® had a higher percentage of microhardness 28.55 ± 11.75 than that of Ag-Nano 14.63 ± 13.38. Also, in the agar diffusion tests, a greater inhibition of S. mutans, Enterococcus faecalis, and Escherichia coli by Saforide ® was observed than by Ancarie ® and Ag-Nano. However, the MIC Ag-Nano inhibited the growth of microorganisms at a concentration lower than the other agents, remineralizing also the enamel of deciduous teeth with initial like-caries lesion [45]. In another study were prepared dentin discs from previously demineralized premolars. SDF (38%), nano-silver fluoride (NSF) (3.16%, 3.66%, and 4.16%), and propolis fluoride (PPF) (3%, 6%, and 10%) were applied and subjected to pH cycles using a demineralization solution (pH 4.4) for 30 minutes and a remineralization solution (pH 7) for 10 minutes, and this cycle was performed 6 times a day for 8 days. The amount of calcium, phosphate and fluoride ions on the dentine discs surface was compared by energy dispersive X-ray spectroscopy. The results showed that the levels of calcium ions, phosphate, and fluoride in the NSF and PPF groups increased significantly compared to SDF [48]. According to the previous results and with the same procedures, fluorapatite crystals were found on the dentine surface in the SDF and NSF, as well as greater hardness and increased intensity and quality of apatite crystal compounds [49]; on the other hand, the use of optical coherence tomography images showed a greater remineralization effect of NSF compared to sodium fluoride (NaF) in deciduous teeth [50]. Clinical Evidence Although most studies on cariogenic bacteria are still in vitro, in recent years, controlled clinical trials have been conducted to measure the efficacy of silver nanoparticles as anticariogenic agents in remineralization. A controlled clinical trial was performed on decayed teeth in vivo. A formulation of nanoparticle silver, fluoride, and chitosan (NSF) was used as the experimental group and water as the control group. The caries was diagnosed clinically, and only one application was made, with an evaluation of seven days, five and twelve months. On the seventh day, 81% of the teeth in the treatment group had arrested cavities and 0% in the control group. After five months, the treatment group had 72.7% with arrested caries, and the control group had 27.4%. At 12 months, 66.7% of the caries lesions treated with NSF were still arrested, while the control group had 34.7%. No dark stains were observed on the teeth [4]. In a similar study, the effect of AgNPs added to a commercial fluoride varnish on remineralization of deciduous teeth was evaluated. The study was conducted in children with white spot lesions in anterior deciduous teeth diagnosed with the DIAGNOdent ® laser cavity detection pen to measure tooth demineralization. A mixture of silver nanoparticles (powder) and fluoride varnish (Fluor Protector Varnish ® , Vivadent, Schaan, Liechtenstein) was prepared at 0.1% wt. The teeth were assigned, one tooth of each hemi arc to the experimental treatment which was fluoride varnish adding 0.1% AgNPs and the other for the control treatment, which was commercial fluoride varnish (Fluor Protector Varnish ® ). Each tooth was submitted to these treatments once a week for 3 weeks, and measurements were taken again with the DIAGNOdent ® at three months to evaluate changes in remineralization, being higher in treatment with AgNPs with a p � 0.001 [46]. In another research, the efficacy of silver nanoparticles added to a pit and fissure sealant was evaluated in permanent molars to determine the demineralization of this mixture against a control group. They observed that the conventional sealant presented an average microleakage of 30.6%, and the sealant added with silver nanoparticles showed 33.6% (P � NS). A three times greater reduction in fluorescence was found in the AgNPs group compared to the conventional group (p < 0.05). No associations were found based on sex or age, concluding that the sealant with silver nanoparticles significantly reduced dental demineralization and probably increased remineralization compared to the conventional sealant [47]. Finally, a study was recently conducted using nanosilver 5% incorporated sodium fluoride (NSSF) dental varnish and 38% SDF. No significant differences were observed between the NSSF and SDF groups during their 12-month follow-up. NSSF did not cause dark staining of dentinal tissue [51]. Discussion and Perspectives SDF has been used as a gold standard in several investigations [23][24][25][52][53][54][55][56][57][58][59] in different concentrations in deciduous teeth with a high risk of active caries [20,59,60], effectively preventing and controlling them [23,24,57,[61][62][63]. However, it has shown adverse effects, such as causing ulcerations and staining in oral mucosa [64] that are painful due to accidental contact with the solution [20,65], which may disappear within 48 hours [21,65] and the black staining of carious tissue [65][66][67] due to the oxidation process of the ionic silver contained in its formulation [4,68]; however, as previously demonstrated, the use of a cariostatic agent with silver nanoparticles does not cause darkening in demineralized teeth [46][47][48]51]. Besides, practically all studies have demonstrated the anticariogenic and remineralizing effect of silver nanoparticles alone [45] and in combination with various components, being superior to conventional treatments [48][49][50][51] such as silver diamine fluoride when applied once a year, with the advantage of not staining black dental tissue, the reason being they do not form oxides when in contact with oxygen in the environment, no metallic taste, and lower cost compared to SDF [4,51]. Although the cariostatic effect on dental tissues has not been clarified yet [4], the effectiveness in arresting cavities can be explained by the synergism of the components of their formulation (nanoparticles of silver, chitosan, and fluoride) [69]. Chitosan is a biocompatible, biodegradable, and nontoxic biopolymer widely known for its activity against a wide range of microorganisms [69,70], including Annual application of NSSF is better than or equal to SDF in preventing the progression of dentinal caries in deciduous molars, and it does not cause dark staining of dentinal tissue AgNO 3 : silver nitrate, AgNPs: silver nanoparticles, TEM: transmission electron microscopy, OCT: optical coherence tomography, NaF: sodium fluoride, MIC: minimum inhibitory concentration, NSF: silver nitrate, chitosan, and fluoride, NH 4 F: ammonium fluoride, ICDAS II: International Caries Detection and Assessment System, EDX: energy-dispersive X-ray spectroscopy, NFS * : silver nitrate: gelatin, glucose, and ammonium fluoride, NSSF: nanosilver 5% and sodium fluoride, ADT: agar diffusion test, EDX: energy-dispersive X-ray spectroscopy, SEM: scanning electron micrograph, XRD: X-ray diffraction. Streptococcus mutans [69] and Streptococcus sanguinis [71], and by adding the antibacterial mechanism of silver nanoparticles, the silver ions can adhere to the cell wall and cytoplasmic membrane due to electrostatic attraction and affinity to sulfur proteins leading to disruption of the bacterial envelope. Once the ions enter the cells, the respiratory enzymes are deactivated, generating reactive oxygen species. As phosphorus and sulphur are components of DNA, the interaction of silver ions with these elements can cause problems in DNA replication and cell reproduction. Furthermore, silver ions can inhibit protein synthesis causing structural changes and cell death [72]. Besides, the reduction in the size of silver nanoparticles implies an increase in the contact surface, which is an important condition for the antimicrobial effects of silver and which could prevent black stains on teeth [36], as it occurs after the application of SDF [20] and reduces toxicity [36]. Therefore, the activity and stability of silver nanoparticles are believed to be influenced by the nature of their stabilizing agent (chitosan) within their formulation, having a low level of constant interaction between silver nanoparticles and bacteria [35,73]. Although the use of silver nanoparticles in the medical and dental field is of great importance, knowledge about the effect of human exposure and the possible toxicity of these products is limited [74]. However, haemolytic activity tests were performed to evaluate the cytotoxicity of NSF compared to SDF and found not to cause damage to the human erythrocyte membrane, regardless of the blood type. When evaluating absolute cytotoxicity values, NSF is less toxic than SDF (p � 0.05) [37]. The present review showed that these formulations can prevent and arrest demineralization of deciduous tooth enamel, so it is important to take into account that these have a thickness of their enamel layer thinner than the permanent teeth [75][76][77], being more prone to the caries progression in comparison with the permanent tooth [76][77][78]. Despite these disadvantages, the deciduous tooth is more sensitive to fluoride treatments, because its enamel has a higher permeability which is approximately 150 times greater than that of a permanent tooth [35,75]. Since this enamel is more sensitive to acid action, the bactericidal potential of silver will be able to increase the effect of fluoride while preserving the remineralization potential [50]. Currently, the use of these formulations with silver nanoparticles and fluoride in different concentrations display a positive effect on dental hard tissue for enamel remineralization in deciduous and permanent teeth, increasing the remineralizing, and they may enhance the performance of fluoride with antimicrobial action of silver added to these formulations, preventing and arresting the carious lesion without causing staining. However, no publication has reported the long-term stability and antibacterial effect of silver nanoparticles and fluoride. Conclusions The investigations conducted in laboratory and clinical trials have shown the superiority of silver nanoparticle compounds in the prevention and arrest of dental caries, without the adverse effect of dental pigmentation. This therapy is easy to use and of a lower cost than SDF. The benefit of the combination of AgNPs with other components must be examined to know its interaction with dental tissues, as well as to determine ideal concentrations, the optimal size of AgNPs, delivery vehicles, doses related to cellular toxicity, and adverse effects. These studies can guide future clinical application protocol for permanent and deciduous dentition whose structure is different, and the behaviour of the formulations added with silver nanoparticles could show different results. Data Availability The data that support the findings of this study are available on request from the corresponding author. Conflicts of Interest The authors declare that they have no conflicts of interest.	2020-08-20T10:06:55.656Z	2020-08-12T00:00:00.000	{ "year": 2020, "sha1": "2b10ce6966ed262671d0591502bc9ee639bcc1e3", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/bca/2020/8882930.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "b262b5c2610a36570e6e7bf66b9d877a94973a23", "s2fieldsofstudy": [ "Medicine", "Materials Science" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
226248817	pes2o/s2orc	v3-fos-license	Atogepant Is Not Associated With Clinically Meaningful Alanine Aminotransferase Elevations in Healthy Adults Atogepant is a potent, selective, oral calcitonin gene–related peptide (CGRP) receptor antagonist in development for migraine prevention. The chemical structure of atogepant is distinct from previous CGRP receptor antagonists, which were associated with elevated serum alanine aminotransferase (ALT) in clinical trials. Here, we report the safety, tolerability, and pharmacokinetics (PKs) of a once‐daily supratherapeutic dose (170 mg) of atogepant for 28 days from a randomized, double‐blind, placebo‐controlled phase I trial in healthy participants. Overall safety, hepatic safety, and plasma PK parameters were evaluated. Thirty‐four participants aged 23–55 years enrolled; 28 (82.4%) completed the study in accordance with the protocol. Multiple doses of 170 mg atogepant for 28 consecutive days were generally well‐tolerated. All adverse events (AEs; reported in 87.0% of the atogepant group; 72.7%, placebo) were mild in severity except one serious AE of subarachnoid hemorrhage due to a bicycle accident and not considered related to treatment. There were two discontinuations due to AEs, both with atogepant, one considered possibly related to treatment. Over 28 days of treatment, no participant receiving atogepant had an ALT elevation above 1.5 × upper limit of normal. Change from baseline in serum ALT levels was not different between atogepant and placebo. Atogepant is rapidly absorbed (median time to maximum plasma concentration, ~ 2 hours) with an apparent terminal half‐life of ~ 11 hours, and no evidence of accumulation after once‐daily dosing. Overall, atogepant at a high oral dose is safe and well‐tolerated in healthy participants with no clinically meaningful elevations in ALT. Migraine is a highly prevalent and burdensome chronic neurological disease. 1,2 It is the second largest cause of disability worldwide, 3 and the leading cause of disability during the most productive ages of 15-49 years. 4 The disabling effects of migraine can also exert negative changes on many aspects of life, including work productivity, quality of life, and finances. [5][6][7] Effective and safe preventive treatment options are needed to help reduce the burden of migraine. Inhibition of calcitonin gene−related peptide (CGRP), a potent vasodilatory protein strongly implicated in the pathophysiology of migraine, has emerged as a targeted approach for migraine prevention and treatment. 8 Small-molecule oral CGRP receptor antagonists (called gepants), such as ubrogepant 9 and rimegepant, 10 have demonstrated efficacy in the acute treatment of migraine attacks and were recently approved by the US Food and Drug Administration, [11][12][13][14][15] and rimegepant is being evaluated for prevention in adult patients with migraine (NCT03732638). Monoclonal antibodies that target CGRP or the CGRP receptor are currently available for adults in the United States and Europe. 8 Monoclonal antibodies require parenteral administration (subcutaneous or intravenous injection) and have a long elimination half-life. 8 Therefore, the development of orally administered CGRP WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ✔ Once-daily supratherapeutic doses of atogepant for 28 days were found to be safe and well-tolerated in healthy participants with no clinically meaningful elevations in ALT. HOW MIGHT THIS CHANGE CLINICAL PHARMACOL-OGY OR TRANSLATIONAL SCIENCE? ✔ These findings will provide clinicians with the knowledge that atogepant, an oral CGRP receptor antagonist, is unlikely to be associated with drug-induced liver injury. Hepatic Safety of Atogepant Min et al. receptor antagonists may provide an alternative for people who prefer an oral route of administration over an injection. Atogepant is a potent, selective, small-molecule antagonist of the CGRP receptor that is currently in development for the prevention of migraine, with a half-life of ~ 11 hours. Atogepant is chemically distinct from prior oral CGRP receptor antagonists, notably telcagepant and MK-3207, which were discontinued because of drug-induced liver injury (DILI). 16 The efficacy and safety of atogepant in migraine prevention was demonstrated in a phase IIb/III clinical trial conducted subsequent to this trial in which treatment with atogepant, compared with placebo, significantly decreased monthly migraine days over 12 weeks. 17 Atogepant is in phase III development for migraine prevention (ClinicalTrials. gov NCT03700320, NCT03777059, NCT03855137, and NCT03939312). This study evaluated the safety, tolerability, and pharmacokinetics (PKs) of multiple oral 170 mg doses of atogepant in healthy adult participants. The 170 mg dose is substantially higher than doses being tested in phase III clinical trials (once daily 10, 30, and 60 mg, and twice daily 30 and 60 mg). The primary objectives were to evaluate the safety and tolerability, and the mean fold change from baseline of alanine aminotransferase (ALT) after 28 days of once-daily atogepant dosing in healthy participants. The secondary objective was to obtain preliminary plasma PK data following multiple-dose administration of atogepant. The primary safety end point was mean fold change from baseline in serum ALT. Trial design This was a randomized, double-blind, placebo-controlled, single-site, phase I trial (MSD Protocol MK-8031 PD004). After screening, participants were randomly assigned in a 2:1 ratio to oral atogepant 170 mg or matching placebo administered once daily for 28 days. For this study, a dose of 170 mg in an oral compressed tablet formulation administered once daily was chosen because it was expected to provide a 3-fold to 5-fold margin over the expected highest clinical dose. Follow-up visits to evaluate safety (including liver function tests) were conducted at 14, 30, and 60 days after the final dose of study medication. This trial was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. All participants provided written informed consent prior to initiation of any trial-specific procedures. The trial was approved by the Thomas Jefferson University Institutional Review Board. Participants Eligible participants were adults (18-55 years of age at screening) judged to be in good health, with body mass index between 18 and 32 kg/m 2 , nonsmokers, and had a fasting glucose value below the upper limit of normal (ULN). Key exclusion criteria included history of clinically relevant medical conditions; estimated creatinine clearance ≤ 80 mL/min based on the Cockcroft-Gault equation; or major surgery or blood donation or loss within 4 weeks before screening. Participants could not use cytochrome P450 enzyme inhibitors or inducers, P-glycoprotein inhibitors, or prescription medications (particularly substrates of transporters, such as organic-anion-transporting polypeptide or breast cancer resistance protein). Assessments Pharmacokinetics. Blood samples for PK assessments were collected before dosing and at 20 and 40 minutes, and at 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, and 36 hours postdose on days 1 and 28 (full PK sampling days), and before dosing on days 7, 14, and 21. The concentration of atogepant was assessed in plasma samples: liquid-liquid extraction was used to isolate analytes, which were detected and quantified using reversed-phase chromatographic-tandem mass spectrometry. The lower limit of quantitation was 1.657 nM with a linear calibration range of 1.657-1657 nM for atogepant. Overall safety. Safety assessments included reports of treatment-emergent adverse events (AEs) and serious AEs (SAEs) throughout the trial, and physical examination, vital sign determination, electrocardiogram, and laboratory assessments performed at prespecified times during the trial. Hepatic safety. Liver function tests, including ALT and aspartate aminotransferase (AST), were conducted on blood samples collected on day -1, day 1 predose, at 24 hours after dosing on days 1, 7, 14, 21, and 28, and at the 3 safety follow-up visits at ~ 14, 30, and 60 days after the last dose. Statistical analysis As an estimate of expected precision, assuming a true total SD for log-transformed fold-change from baseline ALT of 0.299 with 20 participants receiving atogepant and 10 receiving placebo, the estimated half-width of the 95% confidence interval (CI) for the true mean difference (atogepant -placebo) is 0.237 for log-transformed fold-change from baseline ALT. For comparison of adverse experience rates, if an adverse experience occurs at a rate of 10%, then the chance of observing such an adverse event among 20 participants receiving atogepant would be 88%. If no AE of a given type is observed in any of the 20 participants receiving atogepant, then with 80% confidence, the true incidence of the adverse experience at that dose is at most 8% (11% with 90% confidence). AUC 0-24hr and C max values were natural log-transformed and analyzed using linear fixed-effects models with fixed effects for day and sex, day-by-sex interaction, and a random effect for participant. Geometric means (GMs) for PK parameters are reported with 95% CIs on day 1 and day 28 and accumulation of atogepant was assessed by construction of a 90% CI for the GM ratio (day 28 value/day 1 value) for PK parameters. Natural log-transformed fold-change from baseline values for serum ALT were analyzed in a linear mixed-effects model with fixed effects for treatment, time and sex, treatment-by-sex interaction, treatment-by-time interaction, and a random effect for participant within treatment. Least squares mean and 90% CIs for mean treatment differences (atogepant -placebo) in natural log-transformed change from baseline at each timepoint were calculated from the same model. Treatment differences and CIs were exponentiated to obtain GM treatment fold differences (atogepant/placebo) in fold-change from baseline at each timepoint. The proportion of participants exceeding 3 × ULN for serum ALT was reported. Pharmacokinetics Oral atogepant was rapidly absorbed (median T max of ~ 2 hours) with an apparent t 1/2 of ~ 11 hours ( Table 2). Consistent with the t 1/2 , there was little to no accumulation of atogepant following 28 days of once-daily dosing, and the ratio of day 28 to day 1 AUC 0-24hr was ~ 1. Mean atogepant plasma concentrations over time following once-daily dosing at 170 mg/d are shown in Figure 2. Overall safety Most participants (atogepant 87%, placebo 73%) reported at least one AE during the trial ( Table 3). The sole SAE (subarachnoid hemorrhage from a bicycle accident on day 12 after dosing) was not considered to be related to treatment. In addition to the participant with the SAE, 1 other participant discontinued the study because of an event of tachyphrenia (racing thoughts) on day 6 before dosing, which was evaluated by a psychiatrist and considered possibly related to treatment. Tachyphenia was not reported as an AE in the phase IIb/III trial. 17 The most commonly reported AEs were fatigue, headache, and dizziness ( Table 3). Sixteen participants in the atogepant group reported AEs that were considered to be treatment related, most frequently fatigue (n = 9), decreased appetite (n = 5), dizziness (n = 5), and headache (n = 4). Six participants in the placebo group also had treatment-related AEs, most commonly fatigue (n = 4) and headache (n = 3). All AEs were rated as mild except for the SAE (subarachnoid hemorrhage). No clinically significant abnormalities related to treatment were observed in routine serum chemistry, hematology, urinalysis, electrocardiogram, or vital signs. Hepatic safety Mean serum ALT levels were below the ULN for both atogepant and placebo groups throughout the trial (Figure 3). The mean serum ALT levels for participants in the atogepant group were lower during the dosing period than at baseline. Point estimates for the model-based mean foldchange from baseline in treatment differences were < 1.00 at all times tested during the dosing period ( Table 4). No participant had an ALT value ≥ 3 × ULN at any timepoint. None of the transaminase elevations were considered to be clinically significant by the investigator. DISCUSSION Atogepant 170 mg, a dose 2.8-fold greater than the highest dose being evaluated in phase III clinical trial participants, administered once daily over a 28-day period was safe and generally well-tolerated in healthy adult participants. The mean fold-treatment differences for change from baseline in serum ALT were less than one throughout the study, suggesting no apparent effect of atogepant on ALT level. Consistent with its known PK profile, atogepant was rapidly absorbed following oral administration, with a median T max of ~ 2 hours and mean apparent terminal t 1/2 of ~ 11 hours. Notably, atogepant did not appear to exhibit any features of DILI, namely, increases from baseline in mean ALT or events of markedly elevated ALT as had been seen with the two first-generation gepants, as described below. Importantly, this trial was conducted to explore the possible occurrence of signals of liver injury prior to conducting the previously reported phase IIb/III trial. 17 Telcagepant was the first gepant to be clinically evaluated, and demonstrated efficacy for the acute treatment of migraine attacks across several clinical trials. [18][19][20][21][22] However, a retrospective analysis of data from phase I trials showed that some participants experienced elevated ALT levels and the group mean levels of ALT were elevated following repeated dosing for 14 days or longer (either once daily for 7 days or twice daily for 12 weeks), and these concerns regarding DILI ultimately led to discontinuation of telcagepant development. 21,22 MK-3207, another gepant, was associated with delayed liver test abnormalities in phase I trials (ALT elevations following the discontinuation of MK-3207 administration), which also led to discontinuation of its clinical development. [23][24][25] Integrated mechanistic data suggested that DILI associated with telcagepant and MK-3207 was at least partly attributable to reactive metabolites. 26 In addition, it is believed that the formation of covalent adducts between MK-3207 and endogenous liver proteins, which does not occur with atogepant, caused the delayed DILI. Atogepant is chemically distinct from telcagepant and MK-3207, and has characteristics hypothesized to be important for reducing the potential for DILI, such as greater potency and higher target engagement and, therefore, a lower dosing needed for clinical efficacy, and a reduced potential to form reactive metabolites. 16 In a phase IIb/III trial, all tested doses of atogepant (10 mg q.d., 30 mg q.d., 60 mg q.d., 30 mg b.i.d., and 60 mg b.i.d.) were superior to placebo in reducing mean monthly migraine days across 12 weeks of treatment. 17 Nausea was the only treatment-related treatment-emergent AE occurring in at least 5% of participants in multiple dose atogepant groups. During the trial, no treatment-related SAEs were reported with atogepant. After daily dosing for 12 weeks, the overall percentage of participants who had ALT/AST elevations at least 3 × ULN ranged from 0.6-2.2% with atogepant compared with 1.1% for placebo. There were no cases of Hy's Law of concurrent ALT/AST elevations at least 3 × ULN or bilirubin elevation at least 2 × ULN. The phase IIb/III trial results, combined with results from the present study and other ongoing and completed 27 atogepant phase I studies, all of which have not observed incidences of DILI, support the notion that the toxicity observed with telcagepant and MK-3207 was due to the chemical structures of those particular molecules and is not inherent to all gepants. 16 Nonetheless, the potential for DILI has been instrumental in the design and implementation of the clinical development program for atogepant, and the safety profile of atogepant continues to be closely monitored across the phase III clinical trials for migraine prevention (ClinicalTrials.gov NCT03700320, NCT03777059, NCT03855137, and NCT03939312). The present trial has several limitations. Comparisons in ALT levels between groups were not adjusted for multiplicity for the numerous timepoints tested. The trial had a relatively small sample size, although this is a limitation of phase I studies in general and only healthy participants were included. Additionally, most of the participants were men and, because migraine is more prevalent in women, these data may not be generalizable to the overall population of people with migraine. Participants received atogepant for only 28 days, which is shorter than the proposed long-term, once-daily dosing regimen for migraine prevention. Data from larger randomized clinical trials are needed to more fully evaluate the overall safety and risk of DILI associated with atogepant in the target therapeutic population, people with migraine. Strengths of the trial include the use of a supratherapeutic dose of atogepant: a 170 mg dose is ~ 3-fold higher than the highest once-daily dose administered in the dose-ranging phase IIb/III study, 17 which was safe and well-tolerated when used once daily for 28 days. These results are supported by the lack of serious DILI found in the phase IIb/III clinical trial, further adding to the evidence supporting a lack of atogepant-associated DILI. In conclusion, atogepant was rapidly absorbed (median T max of 2 hours), had a t 1/2 of ~ 11 hours, and exhibited little to no accumulation following 28 days of once-daily dosing. Multiple doses of atogepant 170 mg administered once daily had no clinically meaningful effect on ALT levels in healthy adults. Data Availability Statement. This clinical trial data can be requested by any qualified researchers who engage in rigorous, independent scientific research, and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). Data requests can be submitted at any time and the data will be accessible for 12 months, with possible extensions considered. For more information on the process, or to submit a request, visit the following link: https://www.abbvie.com/our-scien ce/ clini cal-trial s/clini cal-trial s-data-and-infor matio n-shari ng/data-and-infor matio n-shari ng-with-quali fied-resea rchers.html.	2020-11-05T09:08:59.384Z	2020-11-03T00:00:00.000	{ "year": 2020, "sha1": "a3a8f35560e240f2c1eab4d896d9919f110b0970", "oa_license": "CCBYNC", "oa_url": "https://ascpt.onlinelibrary.wiley.com/doi/pdfdirect/10.1111/cts.12917", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "9d7d66765d2a550683317f81b3e6dc230df08d68", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
244120454	pes2o/s2orc	v3-fos-license	Microsatellite and RAS/RAF Mutational Status as Prognostic Factors in Colorectal Peritoneal Metastases Treated with Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (HIPEC) Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) leads to prolonged survival for selected patients with colorectal (CRC) peritoneal metastases (PM). This study aimed to analyze the prognostic role of micro-satellite (MS) status and RAS/RAF mutations for patients treated with CRS. Data were collected from 13 Italian centers with PM expertise within a collaborative group of the Italian Society of Surgical Oncology. Clinical and pathologic variables and KRAS/NRAS/BRAF mutational and MS status were correlated with overall survival (OS) and disease-free survival (DFS). The study enrolled 437 patients treated with CRS-HIPEC. The median OS was 42.3 months [95% confidence interval (CI), 33.4–51.2 months], and the median DFS was 13.6 months (95% CI, 12.3–14.9 months). The local (peritoneal) DFS was 20.5 months (95% CI, 16.4–24.6 months). In addition to the known clinical factors, KRAS mutations (p = 0.005), BRAF mutations (p = 0.01), and MS status (p = 0.04) were related to survival. The KRAS- and BRAF-mutated patients had a shorter survival than the wild-type (WT) patients (5-year OS, 29.4% and 26.8% vs 51.5%, respectively). The patients with micro-satellite instability (MSI) had a longer survival than the patients with micro-satellite stability (MSS) (5-year OS, 58.3% vs 36.7%). The MSI/WT patients had the best prognosis. The MSS/WT and MSI/mutated patients had similar survivals, whereas the MSS/mutated patients showed the worst prognosis (5-year OS, 70.6%, 48.1%, 23.4%; p = 0.0001). In the multivariable analysis, OS was related to the Peritoneal Cancer Index [hazard ratio (HR), 1.05 per point], completeness of cytoreduction (CC) score (HR, 2.8), N status (HR, 1.6), signet-ring (HR, 2.4), MSI/WT (HR, 0.5), and MSS/WT-MSI/mutation (HR, 0.4). Similar results were obtained for DFS. For patients affected by CRC-PM who are eligible for CRS, clinical and pathologic criteria need to be integrated with molecular features (KRAS/BRAF mutation). Micro-satellite status should be strongly considered because MSI confers a survival advantage over MSS, even for mutated patients. The local (peritoneal) DFS was 20.5 months (95% CI, 16.4-24.6 months). In addition to the known clinical factors, KRAS mutations (p = 0.005), BRAF mutations (p = 0.01), and MS status (p = 0.04) were related to survival. The KRAS-and BRAF-mutated patients had a shorter survival than the wild-type (WT) patients (5-year OS, 29.4% and 26.8% vs 51.5%, respectively). The patients with micro-satellite instability (MSI) had a longer survival than the patients with micro-satellite stability (MSS) (5-year OS, 58.3% vs 36.7%). The MSI/WT patients had the best prognosis. The MSS/WT and MSI/mutated patients had similar survivals, whereas the MSS/mutated patients showed the worst prognosis (5- Conclusion. For patients affected by CRC-PM who are eligible for CRS, clinical and pathologic criteria need to be integrated with molecular features (KRAS/BRAF mutation). Micro-satellite status should be strongly considered because MSI confers a survival advantage over MSS, even for mutated patients. Colorectal cancer (CRC) represents the third most common neoplasm and the third leading cause of death among the population of developed countries. 1 For untreated CRC metastatic patients, the median survival is shorter than 9 months, whereas with systemic chemotherapy it can be as long as 24 months. 2,3 Peritoneal metastases (PM) from CRC are estimated to develop in about 19% of patients after radical surgery and are the cause of death in more than half of CRC patients. [3][4][5] Patients affected by CRC PM and treated with standard systemic chemotherapy show a shorter median survival, estimated to be about 16.3 months in isolated PM, compared with CRC patients affected at other metastatic sites (lung, liver, lymph nodes). 4 The introduction of cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) leads to prolonged survival (up to 45 months) for a selected subgroup of patients. [6][7][8][9] Selection of the patients who might benefit from CRS-HIPEC procedure has always been considered crucial. Besides the extent of PM [measured as the Peritoneal Cancer Index (PCI)], and residual disease after surgery [completeness of cytoreduction (CC)], lymph node status, tumor differentiation, and signet ring histology are recognized as important risk factors in the selection process for CRS-HIPEC. 10 Mutations in RAS and RAF kinase genes, present in up to 50% (KRAS) and up to 10% (BRAF) of CRC, 11,12 are related to impaired prognosis for liver and lung metastatic patients. 13 Recent studies have identified mutations of prognostic value in RAS and RAF genes, making their determination crucial in the selection process for surgery. [14][15][16][17] In parallel, a defective mismatch repair system (dMMR) and micro-satellite instability (MSI), found in 10% to 15% of CRC patients, are gaining an emerging role in the selection of patients who may potentially benefit from immunotherapy with checkpoint inhibitors. 18,19 However, available evidence for the prognostic role of micro-satellite (MS) status in an advanced CRC stage, especially for patients with PM, remains scant and discordant. [20][21][22][23][24] This study aimed mainly to analyze the prognostic impact of MS status and RAS/RAF gene mutations in CRC patients with PM treated according to a standardized protocol of CRS. Data Collection and Patients Data were retrospectively collected from 13 Italian centers with peritoneal malignancies expertise by members of a collaborative group (Peritoneal Surface Malignancies Oncoteam) in the Italian Society of Surgical Oncology (SICO). All the participating centers are referral centers certified by SICO for the surgical treatment of patients with peritoneal metastases. The study was approved by the ethics committee of the lead center (Veneto Institute of Oncology IOV Padua, nr. 194/2019). All the patients, treated according to the national guidelines for metastatic CRC, were discussed and selected for CRS-HIPEC at a multidisciplinary board meeting. All the selected patients underwent a preoperative thoracoabdominal CT scan, and when necessary, 18-fluorodeoxyglucose-positron emission tomography ( 18 FDG-PET). Cytoreductive surgery was performed according to a standardized operative procedure with the aim of eradicating all visible tumor nodules, performing en bloc resection of the affected organ or organs, and stripping the parietal peritoneum if involved. 25 Residual disease after CRS was classified according to the grade of cytoreduction [completeness of cytoreduction (CC) grading system]. 26 Tumor extent was scored at the time of laparotomy using the PCI (range, 1-39 in 13 abdominal regions). 26 Only patients with residual disease less than 2.5 mm in size (CC0 or CC1) who underwent HIPEC were considered for analysis. At the end of CRS, HIPEC was performed by a circuit connected to a pump, supplied with a heat exchanger, using mitomycin C, oxaliplatin, or cisplatin according to center-specific protocols. Clinical and pathologic data [including patient demographics, perioperative systemic treatments, tumornode-metastasis (TNM) staging, histology, grading, RAS/ RAS and MS status, follow-up status, site and date of recurrence, date of death] were retrieved from referring hospital records. RAS/RAF and Microsatellite Status Analysis Analysis was performed at each center according to internal protocols for clinical purposes. In general, mutational analysis was performed on extracted tumoral DNA (in the majority of cases from primary tumor considering the high rate of concordance with PM) 27,28 through forward and reverse sequencing of amplified tumor DNA. 29 Cases analyzed before 2010 were determined predominantly by the Sanger technique, 30,31 and in the period between 2010 and 2015, by the pyrosequencing technique, 32,33 whereas in more recent cases, reverse transcription-polymerase chain reaction (RT-PCR) was the most frequently used method. 34,35 All KRAS, NRAS, and BRAF mutations were classified as binomial [mutated vs wild-type (WT)] or categorical variables (codon site and type of mutation) according to reported results. Analysis of MS status was performed with direct DNA testing on a specific gene panel for older cases (before 2015), 36,37 whereas in more recent cases, immunohistochemistry (IHC) assay of four proteins (MLH1, MSH2, MSH6 and PMS2) 38,39 and PCR analysis of mononucleotide repeat microsatellite markers were used for confirmation in doubtful cases. 40 Statistical Analysis In general, continuous variables were described using median and interquartile range (IQR). Categorical variables were summarized using frequency counts and percentages. Proportions were calculated on the number of patients with available data. The median follow-up time was based on the reverse Kaplan-Meier estimator. Due to the exploratory nature of the study, there was no formal hypothesis or power sample size calculation. The number of subjects was determined by the number of eligible patients from the participant clinical centers. Overall survival (OS) was defined as the time from HIPEC to the date of death due to any cause, and diseasefree survival (DFS) was defined as the time from HIPEC to the date of local or distant relapse or death. Patients without a documented event were censored at the last known date. Survival curves were estimated with the nonparametric Kaplan-Meier method, and comparisons among strata were performed using the log-rank test. Survival rates at 5 years and the corresponding 95% confidence intervals (CIs) were estimated from the Kaplan-Meier analysis. The 95% CI for the median survival was calculated according to Brookmeyer and Crowley. The independent role of each covariate in predicting survival was verified in a multivariable Cox proportional hazard model with Efron's method of tie-handling, considering all characteristics significantly associated with the outcome in the univariate analyses. No deviation from the proportional hazards assumption was found by the numeric methods of Lin et al. 41 The hazard ratios (HRs) and their 95% CIs from the Cox model were reported. No missing data imputation was performed or reported in tables. All statistical tests were two-sided, and p values lower than 0.05 were considered statistically significant. In the multivariable analysis, p values up to 0.08 were reported. Statistical analyses were performed using the RStudio (RStudio: Integrated Development for R, RStudio Inc., Boston, MA, USA). The patients had limited peritoneal disease, with a median PCI of 9 (IQR, 5-14). The PCI was lower than 15 in 76.3% of the cases and lower than 20 in 91.3% of the cases. Surgery was without residual disease (CC0) in 84% of the cases, whereas residual disease smaller than 2.5 mm (CC1) was present in 16% of the cases. The majority of the patients (71.8%) had metachronous PM, and the median time from the primary tumor resection to the CRS procedure was 20.6 months (IQR, 13.3-32.0 months). In 85.7% of the cases, the primary tumor was located in the colon (equally distributed among left and right), and in 13.8% of the cases, it was located in the rectum. Less than 1% of the cases had multiple neoplasms. The TNM staging of the primary tumor showed that 97.4% of the patients had T3-T4 tumors, and one third had no nodal involvement (30.4% were N0). Regarding pathologic characteristics, only 6.7% of the tumors were well-differentiated (G1), whereas one third were mucinous (30.8%), and 2.5% showed signet-ring cell histology. Patient Characteristics The majority of the patients (70%) received systemic chemotherapy before CRS-HIPEC (oxaliplatin-based for 45.6%, irinotecan-based for 32.4%, combination of oxaliplatin and irinotecan for 11.5%). Adjuvant systemic chemotherapy after surgery was administered to 52.2% of the patients. Only two patients with MSI (4.5%) received checkpoint inhibitors (both before and after CRS). Detailed data are presented in Table 1. Prognostic Factors In the univariate analysis, the prognostic factors for survival were PCI (considered as a continuous variable with cutoff levels of 15 and 20 points; all p values, 0.0001), Table 3; Fig. 2). Analogous results were observed for DFS. Actually, the MSI/ all-WT patients had a 5-year DFS of 62.5% compared with 3.6% for the of MSS/mutated patients (p = 0.00001, logrank; Table 3; Fig. 2). DISCUSSION Despite remarkable and constant progress in systemic treatments, patients who have isolated PM of colorectal origin treated with cytotoxic/targeted agents show a significantly worse survival (16,3 months) than patients with isolated non-peritoneal sites (liver, lung, lymph nodes). 2,4 For selected patients treated in high-volume referral centers, CRS-HIPEC provides a long-term survival of up to 43 months. Even as the real value of HIPEC over CRS alone still is debated, surgery for PM is widely adopted worldwide, especially in the presence of limited disease (PCI \15/20) and when CC can be obtained. 9,[42][43][44][45] Our study showed that CRS-HIPEC is a valid option for a selected group of patients affected by isolated PM of colorectal origin, achieving a median survival of 42.3 months, quite identical to recent high-level evidence-reported data. 9,16 Moreover, our results, obtained in highvolume centers with shared selection and treatment protocols, are in line with already established clinical and pathologic prognostic factors for PM patients treated with CRS-HIPEC. 10 The current study focused on KRAS/BRAF mutational and MS status as prognostic factors for patients with CRC peritoneal metastases treated with radical surgery. The study confirmed that KRAS and BRAF mutations have a negative prognostic impact affecting both OS and DSF. 16,17,23 In addition, we observed for the first time that MS instability is a relevant prognostic factor that can mitigate the detrimental effect of KRAS/BRAF mutation. Therefore, MS status should be considered as a new factor for risk stratification of patients eligible for CRS-HIPEC. In the vast field of research on metastatic CRC, few data exist on the role of systemic chemotherapy for patients affected by isolated PM compared with other metastatic sites such as liver, lung, and lymph nodes. A study analyzing a large sample of previously untreated patients enrolled in 14 randomized trials demonstrated that patients with peritoneal metastases have a worse prognosis than other stage 4 patients with a single metastatic site. 2,4 Several possible explanations have been advocated for this difference. Compared with other patients, PM patients could have a higher tumor burden because radiologic detection of small peritoneal nodules is more difficult than radiologic detection of liver or nodal metastases. 46 The severity of PM symptoms (from early onset of cachexia due to malnutrition to bowel occlusion) may lead to reduced therapy adherence or administration, although this seems to be refuted by a retrospective analysis of two CAIRO randomized trials, in which worst prognosis could have been due to relative resistance of peritoneal metastases, even if adequately treated. 47 Finally, the so-called ''sanctuary effect'' could be responsible for the 10% to 20% response rate reduction of peritoneal metastases compared with liver metastases. 48 In the last two decades, the role of peritoneal surgery has progressively but steadily gained in importance, achieving results similar to those for surgical treatment of liver and lung metastases. [49][50][51][52] Currently, CRS combined in a multimodal treatment strategy of perioperative systemic chemotherapy is considered the best therapeutic option and the only potentially curative treatment for PM patients with limited disease. [42][43][44][45] Our study confirmed that surgery provides a survival advantage for patients treated in referral centers according to a standardized protocol and in a setting of multimodal treatment with systemic chemotherapy. The median survival obtained for our patients (42.3 months) was quite identical to that reported in other studies, 9,16 confirming the pivotal role of surgery performed for PM. Addition of HIPEC to CRS has been questioned during the last few years, after results of randomized controlled trials in a proactive/prophylactic (patients at risk for the development of PM) 53,54 or curative (''adjuvant'' treatment after surgery) setting. 9 Of relevance, reported results of a still unpublished PRODIGE 7 randomized trial showed a notable median OS survival in both arms, but failed to demonstrate a survival advantage of CRS?HIPEC with oxaliplatin over CRS alone, reporting a higher rate of complication in the HIPEC arm. 9 Although publication of the full study is needed for any final conclusion to be drawn, no substantial evidence for advantage of oxaliplatin-based HIPEC after CRS (except for patients with 173 147 96 64 52 36 26 15 10 8 8 188 148 84 43 24 13 6 4 2 2 2 27 20 8 7 2 1 0 0 0 0 0 173 107 52 35 31 23 18 13 10 8 8 188 82 29 13 8 5 3 2 1 1 1 27 10 1 0 0 0 0 0 0 0 0 288 239 146 79 51 32 21 12 8 7 7 44 37 25 19 13 9 6 6 5 3 3 288 157 61 30 23 18 15 11 7 7 7 44 27 12 11 9 6 4 4 limited-extent disease in a subgroup analysis) has been found. However, the role of HIPEC after CRS in colorectal cancer remains an open question, considering that a recent randomized controlled trial showed a survival advantage of 11.8 months in the HIPEC arm compared with CRS alone for ovarian PM. 55 Also, the role of perioperative systemic chemotherapy for patients selected to undergo surgery remains debated. Despite the lack of high-quality evidence, systemic chemotherapy currently is administered before or after CRS. A survival benefit of neoadjuvant (and perioperative) therapy may be suggested. 56,57 Our data reflect this clinical attitude, with 70% of patients receiving systemic chemotherapy before CRS versus 52.2% of patients treated with adjuvant therapy. The regimens used among centers do not differ, but we observed that a combination of neoadjuvant oxaliplatin and irinotecan was administered preferentially in most recent cases (2017 was the median administration year for FOLFOXIRI vs 2015 for the other regimens; p = 0.04), possibly reflecting a treatment shift after publication of the TRIBE trial subgroup analysis. 58 A constant effort is being made to identify prognostic factors to drive the multidisciplinary decision of this dismal-prognosis subset of patients. Historically, the PCI (a tumor burden surrogate) and completeness of cytoreduction (a score of surgical radicality) have been used as selection and prognostic criteria. 7,[46][47][48][49][50][51][52][53][54][55][56][57][58] Our results clearly showed the independent role of PCI (HR 1.03 per increasing point) and completeness of surgery (CC score) (HR, 2.8 for complete vs suboptimal cytoreduction) in predicting survival and disease relapse. Indeed, patients with a PCI lower than 15 have a median survival twice as long as patients with a higher PCI (55.0 vs 25.1 months). The main limitation of PCI and the CC score is the difficulty of having a reliable radiologic PCI and predictive criteria for an optimal cytoreduction before surgery. 59,60 Among the pathologic factors, nodal involvement of the primary tumor (N status), grading, and the presence of signet-ring cells (SRC) are related to survival. In the multivariate analysis, only N status (HR, 1.6) and signetring histology (HR, 2.4) remained related to OS (Fig. 3). These results are consistent with previously reported data showing an increased risk of disease-related death in the case of lymph-node involvement 16 and signet-ring histology. The latter represents a contraindication to CRS in some referral centers. [61][62][63] Currently, RAS/RAF mutational status is part of the standard clinical evaluation since demonstration that constitutive activation of the RAS pathway leads to an impaired response to anti-epidermal growth factor receptor (EGFR) targeted therapy, which is an important therapeutic option for CRC metastatic patients. 64,65 It is widely reported that RAS and RAF mutations have a negative effect on the survival of stage 4 CRC patients treated with chemotherapy 66,67 and that they also represent a negative prognostic factor after surgery for liver or lung metastases resection. 13,15,68,69 Currently, strong evidence exists that RAS/RAF mutations also act as negative prognostic factors for PM patients treated with surgery. 16,17 According to our results, the rates of RAS (46.2%) and RAF (6.6%) mutations are comparable with already reported data on PM patients. 16 16,17 To date, the prognostic role of MS status in CRC has not been clearly defined. In some studies, MSI is related to an improved prognosis in American Joint Committee on Cancer (AJCC) stages 2 and 3 patients. [70][71][72] Conversely, stage 4 MSI patients show a reduced OS. 21,22 In addition, neither incidence (estimated to be \15%) nor prognostic relevance of MS for patients with peritoneal metastasis has been defined to date because the vast majority of data derived from studies are focused on liver or lung stage 4 patients. [73][74][75][76][77] According to the few data on MS status for patients with PM, the MSI detection rate is similar to our results (13.2%). 18,78,79 The univariate analysis showed that MSI is related to a remarkably improved survival (median OS, 95 months vs 41 MSS, p = 0.04). This result is consistent with reported data on stages 1 to 3 CRC 70-72,77 and stage 4 for peritoneal malignancies, 23 but seems to be in contradiction to results obtained in other series of stage 4 patients. 21,22 The multivariable analysis failed to demonstrate a direct correlation between MS status and survival, possibly because of the relatively small sample of MSI patients compared with MSS patients. In addition, only 4.5% of the MSI patients (2 cases) had received immune checkpoint inhibitors, whereas 95.5% had been treated with 5-fluorouracil (5-FU) and cytotoxic drugs, which have a postulated detrimental effect on survival. 80,81 Considering these factors, our results could possibly have underestimated the survival of MSI patients, reflecting a reduced power in the multivariable analysis. Although KRAS and BRAF mutations play a major role in determining the prognosis for the whole PM population, MSI seems to have protective effects for mutated patients. In our series, the KRAS-or BRAF-mutated MSI patients had a significantly better prognosis than the MSS-mutated patients (median OS, 43.7 vs 34.4 months; p = 0.002). Even if for a different subset of patients, similar results had been reported for a large group of MSI patients receiving nivolumab plus ipilimumab (CheckMate 142 trial), in which objective response rates (ORR) were similar independently from KRAS and BRAF status. 19,82 The multivariable analysis confirmed the MSI survival advantage by using a combination variable of mutations and MS status. The prognosis of MSI (mutated or not) and MSS/ WT was significantly better than the prognosis of the MSSmutated cases [HR, 0.4 (p = 0.08) and 0.5 (p = 0.0001), respectively]. The main limitation of our study was its retrospective nature and lack of centralized specimen analysis for mutational and MS status, which were unavoidably related to a certain degree of missing data in the series. However, the study results demonstrate the same mutational/MS rates and survival outcomes, showing prognostic stratification factors identical to those of previous studies, indirectly confirming the homogeneity of the study population. This study represents the largest series analyzing MS status in a homogeneous peritoneal-only stage 4 population with similar disease extension (91.3% of cases had a PCI\ 20) treated with radical surgery accordingly with a shared protocol. These results will be useful for improving patient selection, but further large, prospective studies are required to consolidate the role of MS as a prognostic factor in colorectal peritoneal metastases. In the near future, it may be possible to expand surgical eligibility to MSI patients with negative prognostic factors or contraindications such as high tumor burden (PCI [ 20) or pathologic features (SRC). CONCLUSIONS The role of clinical and pathologic criteria in the selection pathway for the surgery of patients affected by CRC PM needs to be integrated constantly with tumor molecular features (KRAS and BRAF mutations). Based on our results, MS status also should be strongly considered in the selection process for patients potentially eligible for CRS because MSI confers a significant survival advantage over the survival of stable patients, even in the group with KRAS/BRAF mutation. ACKNOWLEDGMENT We thank Christine Ann Drace for linguistic editing and grammar revision, and Santiago Gonzalez-Moreno (MD Anderson Cancer Center, Madrid, Spain) for advice and suggestions. DISCLOSURES There are no conflict of interest.	2021-11-16T14:45:21.530Z	2021-11-16T00:00:00.000	{ "year": 2021, "sha1": "c773f805dbcf2e0efb04dccc78a0b324a99d60ae", "oa_license": "CCBY", "oa_url": "https://iris.unimore.it/bitstream/11380/1270270/1/Tonello2021_Article_MicrosatelliteAndRASRAFMutatio.pdf", "oa_status": "GREEN", "pdf_src": "Springer", "pdf_hash": "c773f805dbcf2e0efb04dccc78a0b324a99d60ae", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
382199	pes2o/s2orc	v3-fos-license	Effects of a Single Bout of Interval Hypoxia on Cardiorespiratory Control and Blood Glucose in Patients With Type 2 Diabetes OBJECTIVE Hypoxia may cause functional autonomic imbalance in diabetes. Intermittent hypoxia (IH), a technique improving the adaptation to hypoxia, might improve cardiorespiratory reflexes and, ultimately, blood glucose concentrations in patients with type 2 diabetes. We tested whether a single bout of IH could initiate a long-lasting response potentially leading to better adaptation to hypoxia. RESEARCH DESIGN AND METHODS In 14 patients with type 2 diabetes without autonomic complications, we measured blood pressure, heart rate, oxygen saturation, chemoreflex (hypoxic and hypercapnic ventilatory responses, ventilatory recruitment threshold), and baroreflex sensitivity before, immediately after, and 3 and 6 h after a 1-h single bout of IH (6-min breathing of 13% oxygen mixture 5 times each separated by 6-min recovery). The measurements were repeated on a placebo day (at least 1 week apart, in random sequence) when subjects were only breathing room air (single-blind protocol). RESULTS IH significantly increased hypercapnic ventilatory responses and reduced ventilatory recruitment threshold, and increased oxygen saturation and blood pressures, whereas increases in heart rate variability and baroreflex sensitivity were not significant. Blood glucose significantly decreased after IH. No such changes were observed during the placebo day, except an increase in oxygen saturation. Some of the effects lasted 3 h after IH, and some even persisted until 6 h after IH. CONCLUSIONS A single bout of IH induced an initial adaptation to hypoxia, with improvement in cardiorespiratory reflexes and reduction in blood glucose. Patients with type 2 diabetes could potentially benefit from the application of a full (>2 weeks) IH intervention. I n diabetes, abnormalities of the autonomic nervous system (ANS) represent one important complication of the disease (1) because it can predispose to severe cardiovascular events (2,3). ANS dysfunction is not exclusively induced by anatomic lesions but has an important functional component (4). Low oxygen content (hypoxia), described in most organs and tissues of diabetic patients (5)(6)(7)(8)(9), recently has been suggested as one cause of ANS abnormalities (10,11). As a consequence, improvement of existing hypoxia might improve autonomic abnormalities that, in turn, also might have consequences on glucose metabolism. One possible strategy to improve hypoxia could be the application of intermittent hypoxia (IH). IH improves exercise capacity in athletes, improves the acclimatization to high altitude in climbers (12,13), and improves ANS in various patients (14,15). The technique consists of intermittent exposures to hypoxic stimuli (3-5 times per day, lasting at least 5-6 min, and spaced at least by 5-6 min) repeated over 2-3 weeks. The principle of the method is like any other type of training: a given stress (here, hypoxia), if appropriately administered and spaced in time, creates a counter-regulatory response that lasts longer and, when repeated a sufficient number of times, leads to a sustained "training" effect (16). IH could increase resting oxygen saturation by increasing the ventilation and the chemoreflexes and, consequently, could reduce the sympathetic activation associated with hypoxia, as previously shown in patients with chronic bronchitis (17). However, until now the effects of IH in patients with type 2 diabetes are unknown, even though respiratory and cardiovascular reflexes (18)(19)(20)(21)(22)(23)(24) and molecular responses to hypoxia (25) have been found to be generally impaired. Therefore, performing a short course of IH might initiate a chain of events that may eventually lead to an "acclimatization" process (when prolonging IH to .1 day). The consequence of relieving hypoxia should be restoration and correction of the cardiorespiratory reflexes. If positive results could be found from this initial study performed in type 2 diabetic subjects without complications, then performing a full training period of IH could be justified in diabetes to test whether this intervention is able to prevent the development of diabetes complications. Participants In this single-blind, placebo-controlled study, we tested 14 type 2 diabetic subjects (3 female, 11 male) without clinical evidence of respiratory dysfunction or autonomic complications. Patients were recruited through general practitioners in and around Innsbruck, Austria. Exclusion criteria were the presence of exercise-limiting pulmonary or musculoskeletal diseases, unstable diabetes, previous or acute myocardial infarction, proliferative retinopathy, cardiovascular complications, ventricular arrhythmias and atrial fibrillation, severe hypertension ($180/110 mmHg), unstable or stable angina, smoking, insulin treatment, and treatment with b-blockers. Participants were advised to maintain their habits concerning medications, nutrition, and extent of physical activity. The conditions mentioned in the exclusion criteria were assessed using a medical interview. In addition, the participants underwent clinical examination, including blood pressure measurements, determination of red and white blood cell counts, and lung function testing. A stress test (i.e., spiroergometry including electrocardiogram monitoring) was performed to determine exercise capacity (VO 2peak ) and to reveal ischemic heart disease. The study was accomplished in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Medical University of Innsbruck. Written informed consent was obtained before the study from each subject. Characteristics of the study groups are shown in Table 1. Protocol To test this hypothesis, we exposed patients with type 2 diabetes to 1 h of IH and followed the response over the course of 6 h thereafter in a single-blind, placebocontrolled protocol. All subjects were examined on 2 different days with a minimum time interval of 3-5 days to avoid possible carryover effects. Baseline measurements on each day were performed during the morning at least 2 h after breakfast and subjects were advised to abstain from caffeinated beverages for 12 h and from alcohol for 36 h before the examinations. The intermittent hypoxic protocol was applied on one day (IH day), whereas on the other day the same participants underwent an identical protocol of solely breathing room air (placebo day). Thus, subjects served as their own controls (crossover design). The sequence of hypoxia day or placebo day was randomized to avoid possible confounding factors attributable to the "learning" effect. After the baseline measurements, the hypoxic or placebo sessions were performed during 1 h in the morning under standardized conditions ( Supplementary Fig. 1). Each hypoxia session had five hypoxic periods (13% O 2 inspired fraction of oxygen) each lasting 6 min, interspersed with five normoxic exposures of the same duration. The procedure for the normoxia day was the same, but the participants inhaled normoxic air. Patients were breathing hypoxic or normoxic air in a supine position using a facial mask. Breathing periods were regulated and controlled by a technician under the supervision of a medical doctor, and without being noticed by the patient. After the hypoxic or placebo exposure, three further structured measurement sessions were performed immediately after, after 3 h, and after 6 h. During the exposure, blood pressure (Portapres; FMS Medical Systems, Amsterdam, the Netherlands), heart rate, and arterial oxygen saturation (COSMOplus; Novametrix) were continuously monitored. The appearance of symptoms or a decline of oxygen saturation ,80% was set as criteria for interrupting the administration of hypoxia until saturation levels recovered to $80%. After measurements immediately after IH, a meal was given to the patients, based on their specific diet requirement. The same meal was administered to each patient on both the hypoxia and the placebo days. Cardiovascular and respiratory testing To examine the control of the respiratory systems, measurements of hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR) were performed four times per day (before, immediately after, and 3 and 6 h after a 1-h single bout of IH) during IH and placebo day. All participants were evaluated in a lying position in a silent and well-tempered room. At first, 4-min recordings at rest were obtained during spontaneous breathing for the measurement of baroreflex sensitivity (BRS). Then, using a mouthpiece and an antibacterial filter, they were connected to a rebreathing circuit, as previously described and validated (17,(26)(27)(28). During the testing, we continuously measured end-tidal CO 2 (CO 2 -et) applying a capnograph connected to the mouthpiece (COSMOplus) and oxygen saturation (SaO 2 ). Continuous airway flow measurements were arranged through a heated Fleish pneumotachograph (Metabo, Epalinges, Switzerland) that was connected to a differential pressure transducer (RS part N395-257; Corby) connected in series to the expiratory component of the rebreathing system. The electrocardiogram was recorded via chest leads, whereas the continuous blood pressure was recorded using a digital plethysmograph (Portapres). Ventilation is stimulated when inhaled oxygen is progressively reduced or carbon dioxide concentration increases. To test the response to hypoxia, a variable part of the exhausted air was passed into a scrubbing circuit filled with soda lime, subsequently returning it into the rebreathing bag. This enabled a permanent adjustment of the CO 2 -et values to maintain the values at a constant level. When the percentage of arterial SaO 2 reached 80%, the examination of HVR was terminated. The response to hypercapnia was evaluated by continuously supplying oxygen at very low flow to maintain the SaO 2 percentage at constant levels (.98%). At the same time, bypassing the reservoir with soda lime, the exhaled air directly arrived in the rebreathing bag, resulting in CO 2 -et values progressively increasing. The test was completed when CO 2 -et increased 13 mmHg above resting levels. Resting data were collected before each rebreathing test during 4 min of spontaneous breathing of room air. Data acquisition and analysis All signals were continuously acquired on a personal computer (Apple Macintosh, Coupertino, CA) at 600 samples per channel. An automatic and interactive program, written in BASIC by one of our group members (L.B.), was implemented to identify each single breath and to obtain breath-by-breath minute ventilation, tidal volume by integration of the flow signal, and, in addition to breathing rate, CO 2 -et and SaO 2 percentage. Measurement of chemoreflex sensitivity The slope of the linear regression line of minute ventilation versus SaO 2 or CO 2 -et indicates the chemoreflex sensitivity to hypoxia or hypercapnia, respectively. The change in ventilation attributable to hypercapnia is interpreted as a main indicator for the central chemoreflex sensitivity, whereas the change in ventilation attributable to hypoxia is interpreted as a main indicator for peripheral chemoreflex sensitivity. The point at which the ventilation started to increase during the progressive HCVR (called ventilatory recruitment threshold [VRT-CO 2 ]; Supplementary Fig. 2) was identified by interpolating the ventilation/CO 2 -et plot using a fourth-order polynomial function. Assessment of baroreflex sensitivity The BRS was measured during recordings of spontaneous breathing at each measurement session. Previous studies have shown a poor correlation between different indices of BRS without better performance of one over the others (29). Accordingly, BRS was calculated as the average of the following seven different methods (30): positive and negative sequence methods (31); the a coefficient in the low-and high-frequency bands and its average (32); the transfer function technique (33); and the ratio of SDs of R-R interval and systolic blood pressure variability. In addition to the BRS, a global index of heart rate variability was assessed using the SD of the R-R interval (SDNN), because this variable has a more normal distribution as compared with other indices of variability (e.g., variance). Blood analyses Venous blood samples were taken to analyze total serum cholesterol, HDL, and hemoglobin. To determine fasting plasma glucose, HbA 1c , creatinine, and triglycerides, capillary blood sampling was performed after a 10-h overnight fasting using heparinized glass capillaries. Blood analyses were accomplished by standard local devices (Reflotron sprint; Boehringer Mannheim; Miniphotometer LP 20, Dr. Lange). Glomerular filtration rate was calculated by the Cockroft-Gault formula. Nonfasting plasma glucose levels before and after the hypoxic or normoxic exposures were estimated taking capillary blood samples (Multicare In-Vitro Diagnostic System; Biochemical System). Urine samples First morning urine samples were collected for determination of microalbuminuria (Micral-Test; Roche Diagnostics GmbH) and macroalbuminuria (Combur-Test; Roche Diagnostics GmbH). Statistical analysis Data are presented as means 6 SEM. Data analysis was performed applying the SPSS statistical software package 18. P # 0.05 (two-tailed) was considered as statistically significant. Comparisons were performed between the same time points of the hypoxia and placebo days and, within each day, by comparing the differences from the first measurement (baseline) of each day by paired t test. In addition, the differences with respect to baseline in the hypoxia and placebo days were compared by paired t test to assess whether the intermittent hypoxia induced different trends during the 2 days. RESULTSdThe hypoxic exposure and the evaluation of respiratory function were well-tolerated by each participant and adverse events did not appear. Resting respiratory data and the complete results of the cardiovascular, respiratory, and metabolic variables are described in Table 2. Baseline respiratory and cardiovascular data There were no significant differences in any of the measured variables at baseline on the placebo day or the IH day. In general, respiratory reflexes and BRS appeared slightly reduced as compared with our reference database (17). Effects of interval hypoxia Effects of IH on respiratory data. HCVR significantly increased after IH (Fig. 1) and tended to remain elevated (3 h after IH: P = 0.096; 6 h after IH: P = 0.105) thereafter. In contrast, an opposite trend could be observed immediately after the placebo exposure, which, however, was no more apparent at 3 h or 6 h after exposure. Additionally, the VRT-CO 2 was reduced immediately after the hypoxia exposure, whereas on the placebo day VRT-CO 2 did not decrease (Fig. 1). As a consequence, the differences from baseline were significant not only immediately after the intervention but also 3 h later. The HVR care.diabetesjournals.org DIABETES CARE, VOLUME 36, AUGUST 2013 showed no significant changes after IH (Fig. 1). After both placebo and IH, CO 2 -et increased with respect to baseline. Slight increases in tidal volume and minute ventilation were observed 6 h after IH, whereas respiration rate did not change. Oxygen saturation increased significantly after both IH and placebo; however, the extent of the increase was not significantly higher after IH than after placebo. Effects of IH on cardiovascular data. The R-R interval significantly increased immediately after the exposure during both testing days. Thereafter, R-R interval decreased more on the placebo day than after IH. Heart rate variability (SDNN) increased, although not significantly (P = 0.059). Systolic and diastolic blood pressures significantly increased subsequent to the hypoxia exposure, but decreased again at 3 h and 6 h from baseline. Changes in baroreflex sensitivity were not significantly different between the IH day and the placebo day. Changes in blood glucose Baseline blood glucose concentrations before the chemoreflex testing did not differ between the intervention day and the placebo day (P = 0.12). A significant decrease in plasma glucose (P , 0.01) occurred after the hypoxic exposure, whereas no significant change was apparent on the placebo day ( Fig. 2A). During the second evaluation after the intervention, the glucose concentrations increased during both days because of the effect of the meal; however, when evaluated in terms of differences from the baseline values a clear trend emerged, showing a progressive decrease in blood glucose after the IH as compared with the corresponding times of the placebo day (Fig. 2B). To our knowledge, this is the first study specifically evaluating the cardiovascular and respiratory effects of IH in patients with type 2 diabetes without autonomic complications. The main findings of the present investigation, limited to a single bout of IH, are of potential practical interest. Exposure to IH rebalanced the main cardiorespiratory reflexes, accompanied by a sustained reduction in blood glucose after IH. Effects of IH on respiratory control in type 2 diabetes A decreased ventilatory response to hypercapnia (20)(21)(22) or hypoxia (18)(19)(20) previously has been described in diabetes, and this impaired ventilatory response may increase the risk of diabetes complications. Likewise, in our study, baseline respiratory reflexes appeared slightly decreased as compared with our reference values (17). In line with these findings, VRT-CO 2 was shifted to the right at baseline, indicating that these patients need to reach a higher level of CO 2 before starting ventilation. After IH, we observed a significant increase in HCVR and a shift to the left in VRT-CO 2 . No change was observed in HVR. IH caused a slight increase in tidal volume and in minute ventilation. The evident increase in the HCVR and the lack of change in the HVR seems, at first sight, paradoxical. However, the first effect of an increase in ventilation is a reduction in the CO 2 -et. This limits or even stops the hyperventilation, because the carbon dioxide is a potent stimulus of ventilation. Accordingly, the only possibility to allow a persistent hyperventilation is through resetting of the VRT-CO 2 at lower levels. The changes in HCVR and VRT-CO 2 thus are compatible with the initial chain of events leading to a training effect on the cardiorespiratory reflexes. The oxygen saturation increased significantly after the IH, but also during the placebo day. Because these changes did not seem to result from an increase in ventilation, alternative explanations could be an improvement in gas exchange (8) or a favorable change in the oxygen transport of hemoglobin (34). The increase in oxygen saturation during placebo might suggest that the exposure to hypoxia during the measurement of the HVR could have modified some variables also during the placebo day. Effects of IH on cardiovascular control Abnormalities of the cardiovascular control, evidenced by decreased BRS and heart rate variability, have been observed in patients with type 2 diabetes (23,24). Impairment of cardiovascular control can lead to severe multiorgan dysfunction and, finally, to adverse cardiovascular events in people with diabetes (2,3). Immediately after IH, a significant increase in the R-R interval was found, together with a nonsignificant increase in BRS and SDNN, suggesting an increase in parasympathetic activity. Increases in systolic and diastolic blood pressures also were evident after IH, and they could be explained by the increased oxygenation of the hypoxic patients. In a previous article (10), we showed that patients with type 1 diabetes had increased blood pressure in response to hyperoxia. This might have stimulated the increase in parasympathetic activity that prolonged the R-R interval (i.e., induced a relative bradycardia) and increased the SDNN and the BRS (similar to what found in the current study). However, Haider et al. (17) showed that the long-term effect of IH (1 h per day for 3 weeks) in chronic obstructive pulmonary disease patients was even a slight decrease in systolic blood pressure. Thus, one may suggest that the increase in systolic blood pressure observed in our study may be an initial response that is followed by adaptation. Changes in blood glucose Surprisingly, a significant decrease in blood glucose appeared. The link between hypoxia, cardiorespiratory reflexes, and blood glucose might be highly complex and will certainly require further studies. As speculation, this first observation that IH improves both cardiorespiratory reflexes and glucose concentrations could be the consequence of improving hypoxia through improved tissue oxygenation and reduced sympathetic activity. This may lead to better glucose utilization. Preexisting hypoxia in type 2 diabetic patients Hypoxia is a new, yet common, finding in diabetes and may result from various factors. Diabetic patients show decreased skin oxygenation (reduced transcutaneous partial pressure of oxygen) and increased venous oxygen partial pressure (5). Glycosylation of hemoglobin decreases oxygen transport (6), whereas in the lungs the glycosylation of basal membranes leads to impaired diffusing capacity for carbon monoxide (7). Obstructive sleep apnea is another cause of hypoxia, particularly in patients with type 2 diabetes (9). In addition, low SaO 2 at rest has been observed in diabetic subjects (11). In contrast to healthy subjects, responses to experimental (in isolated cell and tissues) and clinical hypoxia in diabetes are impaired (25,35), possibly leading to diabetes complications. Limitations In this investigation, the control day included a minor exposure to IH because each HVR test (which was repeated four times on IH days and placebo days) exposes the subject to a transitory hypoxia. In addition, some of the effects observed in this study might be mediated by hyperoxia (a small amount of oxygen had to be administered to perform the HCVR test). For safety reasons, because of the novelty of this approach, the subjects in this first study were a selected group of patients without evidence of autonomic complications. Therefore, the present findings may not be generalized or transferred to patients with diabetes with evidence of ANS abnormality. CONCLUSIONSdExposure to a single bout of IH improved hypoxia, rebalanced cardiorespiratory reflexes, and reduced glucose levels in patients with type 2 diabetes without autonomic complications. These preliminary findings support future studies applying IH for a longer period of time to test whether a full IH protocol may potentiate or prolong these initial positive effects, which potentially can have an important clinical impact. study, contributed to data collection, interpretation, and analysis, and edited the manuscript. L.B. developed the study concept and design, contributed to interpretation and analysis, edited the manuscript, and revised the manuscript. T.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.	2016-04-30T01:05:23.138Z	2013-03-27T00:00:00.000	{ "year": 2013, "sha1": "c6971244b8ca9ad356cd40204cd76a4961528ce9", "oa_license": "CCBYNCND", "oa_url": "https://care.diabetesjournals.org/content/diacare/36/8/2183.full.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "ca2a290e72a21ebcc876eee9386476319ab2c2aa", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
58900100	pes2o/s2orc	v3-fos-license	PRELIMINARY ANTIPROLIFERATIVE EVALUATION OF NATURAL , SYNTHETIC BENZALDEHYDES AND BENZYL ALCOHOLS Vanillin, o-vanillin, natural and synthetic benzaldehydes and benzyl alcohols were assessed for antiproliferative effects using different human cell lines. Benzyl alcohols were synthesized from benzaldehydes reduced with NaBH4 in methanol solution. A new method for deprotection of ether compounds with TiCl4 solution was achieved with better performance, than previously reported. Twenty four compounds were tested. The in vitro growth inhibition assay was based on sulphorhodamine dye to quantify cell viability. Catechol 9 derived from piperonal as well as compounds 4 and 12 showed higher cytotoxicity on breast and prostate cancer cell lines (MDA-MB-231 and PC-3 respectively). o-Vanillin 5 has the highest cytotoxicity for all cell lines. IC50 values of 35.40 ± 4.2 μM Breast MDA-MB231; 47.10 ± 3.8 μM Prostate PC-3; 72.50 + 5.4 μM Prostate DU-145; 85.10 + 6.5 μM and Colon HT-29, were obtained without toxicity towards dermal human fibroblast (DHF cells). INTRODUCTION Methoxybenzaldehydes effect over cancer cells has been reported, vanillin (4-hydroxy-3 methoxybenzaldehyde) exhibits a potent anti-proliferative effect on a broad spectrum of cancer cell lines.In 1986, Ohta et al. first tested the anti-mutagenic effect of vanillin on bacteria and found that vanillin could reduce 4-nitroquinoline-1-oxide 1 .Another compound from this family is anisaldehyde, which exhibits a concentration dependent cytotoxicity against murine B16 melanoma cells 2 .Another in vitro study has demonstrated inhibitor effect on hemoglobin S polymerization, of isovanillin, o-vainillin, m-hydroxybenzaldehide and the p-hydroxybenzaldehyde 3 . It was reported by King et al., that vanillin is effective on the repair of mutations in colon cancer cells line HCT-116, this essay suggests colon cancer cells to be suitable for studying vanillin anti-mutagenic effect and its cytotoxicity relationship 4 .On the other hand, in 2002 da Silva et al. have reported that vanillin inhibits spontaneous mutation in bacteria 5 .Studies in Drosophila showed that vanillin inhibited mitomycin C-induced mutations and dramatically increases recombinations 6 , demonstrating that vanillin is a modifying factor that blocks the mutagenic pathway, and consequently directs the mitomycin-induced lesions into a recombinational repair.Other experimental tests suggested that post-treatment with vanillin protects against point mutations and chromosome aberrations induced by chemical and physical agents. 7he purpose of the present work is to examine the effect of a series of 24 compounds related with benzaldehydes and benzyl alcohols.These compounds were tested on one human tumor breast cancer cell line (MDA-MB-231), one human colorectal cancer cell line (HT-29), two human prostate cancer cell lines (PC-3, DU-145), and one dermal human fibroblast cell line (DHF). General Unless otherwise stated, all chemical reagents purchased (Merck or Aldrich) were of the highest commercially available purity and were used without previous purification.IR spectra were recorded as thin films in a FT-IR Nicolet 6700 spectrometer and frequencies are reported in cm -1 .Low resolution mass spectra were recorded on an Agilent 5973 spectrometer at 70eV ionising voltage in a DB-5 m, 30 m x 0.25 mm x 0.25 um column, and dates are given as m/z (% rel.int.). 1 H, 13 C, 13 C DEPT-135, sel.gs1D 1 H NOESY, gs2D HSQC and gs2D HMBC spectra were recorded in CDCl 3 solutions and are referenced to the residual peaks of CHCl 3 at δ = 7.26 ppm and δ = 77.0ppm for 1 H and 13 C, respectively, on a Bruker Avance 400 Digital NMR spectrometer, operating at 400.1 MHz for 1 H and 100.6 MHz for 13 C. Chemicals The natural and synthetic benzaldehydes were purchased from Sigma-Aldrich (St. Louis, MO, USA).All other chemicals (reagents and solvents) were obtained from Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA). General procedure for the reduction of benzaldehydes All benzaldehydes were reduced using the method developed by Brown 8 was slightly modified in this synthesis.A solution of piperonal 7 (300.0mg, 2.0 mmol) in methanol (50 mL), sodium borohydride (176.0 mg, 4.65 mmol) was added in small portions and carefully.The reaction mixture was stirred at -10 ºC for 2 h.After work-up as in the reduction of alcohols, the resulting residue was recrystallized from hexane to give a tan solid identified as compound 22 (188.8mg, 68.4 %); m.p.: 52. 3 General procedure for acetylation and methylation of benzaldehydes and benzyl alcohols. Acetylation and methylation of benzaldehydes and benzyl alcohols of this work were used typical protocols in synthesis organic 9 . Procedure for the cleavage with TiCl 4 solution The new method of synthesis consisted of a TiCl 4 solution (0.9 mL, 8.20 mmol) cooled to −20 °C, was slowly added to a solution of piperonal 7 (300 mg, 2 mmol) in CH 2 Cl 2 (20 mL) at −10 °C under an atmosphere of N 2 with gentle stirring.The reaction was continued for 4 h at −20 °C.After this, the mixture was taken up in water and then extracted with ethyl acetate (3 × 50 mL).The watery layer was discarded and the organic layer was washed to neutrality with a saturated solution of NaHCO 3 .The organic layer was dried over MgSO 4 , filtered and evaporated.Then it was absorbed on silica, chromatographed by CC eluting with mixtures of petroleum ether/EtOAc increasing polarity (19.0:1.0→13.0:7.0) to obtain a white solid identified as compound 9 (188. Cell Lines The experimental cell cultures were obtained from American Type Culture Collection (Rockville, MD, USA).All cancer cell lines were grown in DMEM-F12 medium containing 10% FCS, 100 U/mL penicillin, 100 μg/ mL streptomycin and 1 mM glutamine.Cells are seeded into 96 well microtiter plates in 100 μL at plating density of 3 × 10 3 cells/well.After 24 h incubation at 37 °C (under a 5% humidified carbon dioxide to allow cell attachment) the cells were treated with different concentrations of drugs (aldehydes and derivatives) and incubated for 72 h under the same conditions.Stock solution of compounds was prepared in ethanol and the final concentration of this solvent was kept constant at 0.1%.Control cultures received only 0.1% ethanol. In vitro Growth Inhibition Assay The sulforhodamine B assay according to the method of Skehan et al. 10 was used with some modifications 11 .Briefly, the cells were set up as 3 × 10 3 cells per well of a 96-well, flat-bottomed 200 μL microplate.Cells were incubated at 37 °C in a 5% humidified CO 2 plus 95% air mixture and treated with the compounds at different concentrations for 72 hours.At the end of drug exposure, cells were fixed with 50% trichloroacetic acid at 4 °C (TCA final concentration 10%).After washing with destilled water, cells were stained with 0.1% sulforhodamine B (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 1% acetic acid (50 μL/well) for 30 min, and subsequently washed with 1% acetic acid to remove unbound stain.Protein-bound stain was solubilized with 100 μL of 10 mM unbuffered Tris base.The cell density was determined using a fluorescence plate reader (wavelength 540 nm).Values shown are the mean + SD three independent experiments in triplícate.Untreated cells were used as a negative control while, cells treated with doxorubicin were used as a positive control. Chemistry All benzaldehydes were reduced using NaBH 4 in methanol solution obtaining 8 the corresponding benzyl alcohols with almost 100 percent yield, the high reduction performance was achieved by the only in those compounds which showed no free hydroxyl as case of piperonal (Scheme 1), whereas in those molecules with a free hydroxyl as vanillin yield decreased to a half, and for molecules bearing two or more free hydroxyl groups yield decreased in about 20%.Based on the low reduction yields, other methods such as LiAlH 4 or zinc in acid media were tested to increase efficiency, for aldehydes with two or more hydroxyl groups, but with both methods the yields remained low; to solve this problem, molecules were subjected to reactions of methylation and acetylation, to block the free hydroxyl, and thus increase the benzyl alcohol yield close to 100%, (Scheme 1).All the compounds were characterized by NMR, IR and MS spectral data and their structures were confirmed by comparison with spectral data in literature. Biological Results These natural compounds used in this study were chosen because they are predominant flavor components of some plants used in the food industry, for this reason, foods fortified with these substances (natural or synthetic derivatives) have a potential role preventing diseases. The in vitro cytotoxicity evaluation of natural and synthetic compounds 1-24 (see Figure 1 and 2) natural and synthesized indicated that cell viability expressed as % vs. control vehicle (ethanol 0.1%) was dose-dependent (μM).Doxorubicin was used as positive control in this study (IC 50 value < 10 μM).Among IC 50 values for compounds 4, 5, 10 and 12 were the most potent, are summarized in Table 1 known as the micromolar concentration that produces 50% cell growth inhibition after 72 hours of drug exposure.We are showing only the biologically active compounds with IC 50 lower than 100 μM, while cytotoxicity exhibited by benzyl alcohols in tested cancer cell lines were higher than 100 μM.In this preliminary study, the differences in biological activity attributable to the position of the substituents on the aromatic ring are observed for in the different molecules tested. Scheme 1. General scheme of synthesis of Benzaldehydes derivatives The cytotoxic effects of vanillin are well known 12 .However, o-vanillin was the most active compound in all the cell lines, especially in breast cancer cells MDA-MB231.The structures difference is in the position of the hydroxyl group, therefore o-vanillin may generate an intramolecular hydrogen bond, decreasing in polarity and increasing its ability to pass through cell membranes 13 . On the other hand, the cytotoxic activity of these molecules could be explained by the formation of Schiff bases derived from benzylic aldehydes.Amino groups play an important role in the tertiary structure of cellular enzymes such as tyrosinase, involving the amino group hydrogen bonding which is essential to maintain the tertiary structure of the enzymes 14 .The low conformational stabilities of native proteins make them easily susceptible to denaturation by altering the balance of weak non-covalent bonds that maintain the native conformation.Native proteins form a sort of intramolecular micelle in which the non-polar Schiff base portion is largely out of contact with the water-based environment and the aromatic ring provides stability to the system 15 .This would be enhanced in our active compounds due to the presence of electron donor groups (OH and OCH 3 ).Compound 9 has two hydroxyl substituents in position p and m and compound 12 possesses three methoxy substituents: one in p position and two in o position, which should stabilize the Schiff base and increase cytotoxicity.Thus, the higher activity of compounds 4 and 5 in comparision to the compounds 9 and 12 is directly related to the existence of intramolecular hydrogen bonding and to proton transfer in the equilibrium 16 .This tautomeric equilibrium has been confirmed, at room temperature by NMR spectroscopic studies 17,18 .Compound 4 can form an intramolecular hydrogen bond which would decrease the effect attributed to the tautomerism, therefore o-vanillin (compound 5) is the molecule with the highest non-specific cytotoxicity. Cytotoxicity assays performed by Ho et al. on colon cancer cells HT-29 were made in a high concentrations range of 0-10mM (to 6.58 mM) for the tested compounds 10 .Unlike Ho's assays, we used a concentration range between 0 and 100 µM for all the tested compounds.We obtained an IC 50 for vanillin >100 µM on cancer cells HT-29 and an IC 50 = 85.10 ± 6.5 µM for o-vanillin in the same cell line.Therefore the cytotoxic effect reported by Ho can be explained because the range of concentrations used is some thousand times higher than the concentration we used for this cell line test. We are reporting the toxicity of o-vanillin on prostate cancer cell lines PC-3 and DU-145, calculated IC 50 are 47.10 + 3.8 and 72.50 + 5.4 µM respectively.However, the highest toxicity was observed against breast cancer cell line MDA-MB231, presenting an IC 50 of 35.40 ± 4.2 µM, lower than the other tested compounds (for example compound 4 has an IC 50 of 59.90 ± 3.9 µM).Compounds 9 and 12 have lower cytotoxic effect in breast cancer cell line, being their IC 50 were 82.70 ± 6.7 and 78.71 ± 8.3 µM respectively, lower than those presented by compounds 4 and 5 (See Table 1). CONCLUSIONS In chemical synthetic terms, the development of a new method for deprotection of ether compounds with TiCl 4 solution was achieved with better performance than that previously reported by our group 19 . We can conclude that benzyl alcohols showed no significant cytotoxicity in the cancer cell lines tested.Aldehydes 4 and 5 showed higher cytotoxic activity than 9 and 12 compounds against the selected cancer cell lines.These results indicate that compound 5 exerts this cytotoxic and selective effect on all cancer cells lines over a wide concentration range without effects in nontumoral cell line (DHF). In order to continue with this preliminary work, we will study mechanisms of cell death such as caspase activity and cytochrome c release, triggered by these four compounds 4, 5, 9 and 12. Table 1 . Cytotoxic activity (IC 50; µM) of natural and synthetic Benzaldehydes and Benzyl Alcohol derivatives against various human cancer cell lines.	2018-12-17T20:32:57.331Z	2013-09-01T00:00:00.000	{ "year": 2013, "sha1": "28acfd7f9674f61cdc2c94f0fb080261efefade9", "oa_license": "CCBYNC", "oa_url": "http://www.scielo.cl/pdf/jcchems/v58n3/art03.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "28acfd7f9674f61cdc2c94f0fb080261efefade9", "s2fieldsofstudy": [ "Chemistry", "Medicine" ], "extfieldsofstudy": [ "Chemistry" ] }
9149566	pes2o/s2orc	v3-fos-license	Visualizing Spoken Discourse: Prosodic Form and Discourse Functions of Interruptions In this paper we show that interruptions are important elements in the interactive character of discourse and in the resolution of issues of cognitive uncertainty and planning. By representing discourse graphically, we also show that interruptions are part of the local and global coherence that is brought about through the systematic phrase-to-phrase prosodic patterns of discourse. The specific pitch height of the interruption varies with the expression of emotion, signals of attention-getting, and signals of competitiveness. These prosodic forms are potentially usable in spoken dialogue systems to provide intelligent responding systems that are responsive to human motivations in dialogues. In this paper we show that interruptions are important elements in the interactive character of discourse and in the resolution of issues of cognitive uncertainty and planning. By representing discourse graphically, we also show that interruptions are part of the local and global coherence that is brought about through the systematic phrase-to-phrase prosodic patterns of discourse. The specific pitch height of the interruption varies with the expression of emotion, signals of attention-getting, and signals of competitiveness. These prosodic forms are potentially usable in spoken dialogue systems to provide intelligent responding systems that are responsive to human motivations in dialogues. Introduction: Interruptions and Dialogue One characteristic of human conversation is that it's highly interactive, spontaneous and mutual information building, and the demands of the ongoing mutual negotiation process often cause imbalances in informational adequacy and desired topic direction. Interruptions play a key role in signaling and resolving these imbalances and in bringing about a mutually satisfactory accommodation of the interests and knowledge states of participants. Because interruptions act to mediate the content and redirection of a conversational exchange, and are informationally packed with respect to these communicative pivot points, it is important to understand how interruptions are used in human communication, and determine which elements of interruptions can be accommodated in building a more flexible, more efficient spoken dialogue system. Research Goals and Procedures In this study, our goal is to look at the distribution of interruptive occurrences in natural speech, and investigate their respective functions and characteristics. Several questions that we address are the following: What are the different types of interruptions present in dialogues, and to what extent are prosodicacoustic features significant in distinguishing between these different types of interruptions? What are some of the underlying factors that cause interruptions to occur, and how can such information on the prosodic features be utilized in spoken language systems in detecting interruptions and in constructing appropriate response strategies in human-computer interactions? Our data for this research consists of fifteen dialogue segments extracted from a corpus of 2 hours of spontaneous conversation. The speech data were digitized and annotated for discourse relations, topic structure, interruptions, and speaker turns. The acoustic measurements of f0, amplitude and duration were correlated with the specific characteristics of the interruptions in the data. In this paper we concentrate mostly on pitch, but make reference to amplitude and duration where appropriate. In our analysis we take a multi-level approach. In order to capture the different domains at which prosodic patterns are manifested, we analyzed the data at the within-phrase as well as the inter-phrase level. An additional level of our analysis focuses on how discourse evolves over extended stretches of conversation. As a way of representing the prosodic structures in spoken dialogue, we plotted the highest pitch points of 600 continuous utterances, about 20 minutes conversation, for each speaker and color-coded the interruptions by speaker and type. This What constitutes an interruption? Interruptions can be seen as situations in which one person intends to continue speaking, but is forced by the other person to stop speaking, at least temporarily, or the continuity or regularity of that person's speech is disrupted. This can happen when the interruption causes the main speaker to become hesitant in speech, even while continuing on the intended path or when the speaker continues speaking, but the interruption causes the speaker's topic direction to be modified. Interruptions, therefore, can be seen as consisting of three essential ingredients: intention of the main speaker to continue, entrance of the other person into the conversation, and disruption or stopping of the main speaker, at least temporarily. In general, interruptions can be of two types: competitive vs. cooperative. Competitive interruptions occur when one speaker attempts to take the floor by making his or her own remarks a higher priority over the main speaker's speech when the main speaker intends to continue. This competitiveness can be on two different levels. Speakers can compete for speech space and they can also compete for topic or idea. In either of these competitive cases, interruption acts to take the attention away from the main speaker, at least temporarily, and focus it on the interrupter's speech. Cooperative interruptions occur when one speaker wants to support or reinforce the main speaker's point without disrupting the main speaker's continuation. These types of supportive remarks are often in the form of short commentaries or clarifying questions. Such clarifying questions often support the continuing flow of the main speaker by keeping both speakers in synchrony on the topic development. Both types of interruptions may or may not involve overlapping speech, since overlapping speech is not necessarily an indication that speakers are in conflict over which speaker has the right to the floor at that moment. For example, in conversation, speaker's feedback utterances or back channel signals such umhum or yeah often overlap with the main speaker's speech, but they are not interruptions as they do not interrupt the main speaker's flow. In fact, they often contribute to the smooth flow of the main topic because of their supportive nature. Conversely, competitive interruptions can happen even when there is no overt overlapping in speakers' speech. This can occur when one speaker is not completely finished and intends to go on, but is at the end of an episode or a possible turn completion point. The other speaker may not be aware of the main speaker's plan, and may think the current speaker is completely finished, so starts to take the floor and creates an unintentional interruption, i.e. mistiming. Therefore, it is the degree of disruption to the intended continuation of the main speaker which is the critical element, and the degree of competitiveness or cooperation is determined by the actions and intentions of both speakers. Competitive Interruptions Analysis of our discourse corpus shows that competitive interruptions are typically high in pitch and amplitude. In spontaneous discourse, speakers often compete to gain control and dominance in the conversation. In competitive situations participants need a strong immediate signal to attract the attention away from the ongoing speech. In general, the more audible the signal is, the more forceful and effective it will be in overcoming the current focus and successfully taking the floor. Prosodically, this competitiveness and need for a strong signal are iconically reflected in the vocal cues of high pitch and high amplitude. Competitive interruptions are often closely tied to topic development and reflect relevance, urgency, degree of importance, and interest in the current topic. In conversation, speakers often feel the need to express something which is emotionally significant to them. Speakers often encounter moments of uncertainty and have an urgent demand for information and immediate attention at a critical moment. This urgency and immediacy are a key characteristic of interruptions and are directly related to the relevance of the current topic. Speakers often grab the opportunity while the current topic is hot to clarify something, add a pertinent fact, or express an immediate opinion. Often the high pitch and loud amplitude in competitive interruptions are caused by the emotions motivating these situations. Demand for new information or clarification The first example illustrates a typical case of competitive interruptions: (interruptions are indicated by an arrow in the transcript) (1) 13 A: So this one's better then B: Umhum 14 It's better than the regular ones. In this example the interrupter (A) comes in when the main speaker is at a slight pause in the middle of a response and at a low pitch level. The interrupter interrupts with a direct questioǹ What brand is this?' at U14 (utterance 14) using high pitch, loud amplitude and at a fast speed (see Figure 1). These prosodic features are direct results of the immediacy and urgency of the interrupter's demand for additional information for her interest. Expressing Strong Opinions (2) 149 A: It's just -hmmm 150 It's just to say that the one who speaks 151 it's just that you -you -(pause) Competitive interruptions within an ongoing topic also occur when a speaker wishes to express a strong opinion or disagreement. In the beginning section of this fragment shown in example 2, the main speaker (speaker A) is talking and speaker B mainly provides feedback. Speaker B's interruption at U151 occurs at a point where the main speaker is hesitant and pausing. Anticipating the main speaker's point, speaker B takes this opportunity to express her strong opinion on that point, and the forcefulness of her disagreement is reflected in the high amplitude and high pitch of the interruption. Comparing with the peak points for the utterances in this section (see Figure 2), we can clearly see that this interruption has a sudden pitch jump to 360Hz, and is an abrupt isolated point by comparison to the rest of the pitch points in this area, about 50Hz higher than the other points in this region. Note that the intention of the interrupter here is not to take the floor for the long term, but to make an important point at a critical moment, and this intention is indicated by the brevity of her remark and her supportive feedback thereafter. Shifting Topic The critical moment urgency of many interruptions is shown in the above example. Interruptions often occur in the normal give-andtake of conversation as participants negotiate their own interests in the conversations. Therefore one key motivation for competitive interruptions is to change topic direction. This can happen when one speaker has a topic of greater interest, wants to avoid a topic, or wants to return to an old topic. Such interruptions often occur in the form of questions, as questions obviously are a natural way to attract attention, to demand information, and to direct or guide a speaker's speech and the direction of discourse. ( In example 3, the main speaker (Speaker A) is finishing up her topic, and her intention to conclude can be inferred by her repetition of the phrase`It's just -it's just teamwork.' in U294 to tie the topic back to her beginning statement at U287. Her pitch level is getting low here. Anticipating Speaker A's completion, Speaker B comes in to shift the topic back to a previous topic. Her pitch level for this utterance (U296) is very high at 420Hz as seen in Figure 3 and Figure 4, in fact, it is one of the highest points for this speaker in the discourse. We can see that there is a dramatic and abrupt rise in pitch level. This is clearly indicated by the sharp increase of approximately 190Hz from Speaker B's previous utterance at 230Hz. Her amplitude is also loud and forceful. This interruption is followed by another lower-pitched and soft prompting interruptive question`Do you remember?' to reinforce the intended turn in topic direction. The pitch height of an interruption is closely related to the abruptness of topic shift and the intensity of expression. By contrast, for a later interruption in example 4 at U376, speaker B's pitch level is high at about 380Hz ( Figure 5), but is about 40Hz lower than the interruption to shift the topic in the previous example of U296. The reason for the higher pitch level of the previous interruption may be explained on two grounds. One is that the interest level involved, i.e. the intensity of the emotion of the speaker, is different. In the previous interruption, the speaker is bringing in a topic in which she has great interest, whereas in the current case, the interruption is just a leading question to provide an opportunity for a further topic. The second reason concerns the degree of relatedness of topics. A greater cognitive effort is involved has not been present or active for some time, when a shift is made to return to a topic which therefore requiring a stronger prosodic signal to flip back to the previous topic world, and to bring it back into the current memory of participants. In the example here, the topic shift is just one step away from the current topic which is in the participants' active memory, and thus this requires less cognitive effort, hence a less strong intonational signal. Resistance to Topic Shift Interruptions are an important element in the interactive character of discourse. This interaction comes about because of the mutual negotiating to satisfy each participant's needs in the conversation. In the above examples, topic shifts were viewed from the perspective of the interrupter, but the perspective of the main speaker also needs to be considered. When encountering interruption, a speaker may respond by yielding, by ignoring the interruption, or by continuing through forceful prosodic counter-measures. The particular response used is determined to a large degree by the existing balance of floor rights at that point in the conversation. Whether a main speaker yields or not is decided by the degree of competitiveness and urgency of the interrupter, and how related the interruption is to the ongoing topic. In resisting interruptions, the main speaker often reacts by using both loud amplitude and a high pitch level, the principle being to first grab back the floor, then proceed with content. The prosodic give-and-take of interruptions really expresses the interactively established understanding of current floor rights and participants' intensity. In this example, as the main speaker (speaker B) is coming down to the end of a subsection as signalled by the descending pitch level to a low pitch level at U77, speaker A comes in to initiate a topic of her own with a high pitch level of 310Hz. Speaker B immediately counters this interruption with a high pitch and loud amplitude. Once the threat to the floor rights is over however, speaker B immediately returns to a more normal pitch and amplitude level to resume her topic, as we can see by the dropoff in pitch and amp here. The raised pitch and differential in pitch level may be in proportion to the degree of competitiveness involved. Cooperative Interruptions The examples presented so far have mostly illustrated the discourse reasoning and the prosodic characteristics of competitive interruptions. In general, competitive interruptions are marked by a high pitch level, and a loud amplitude, expressing the participants' competition for the focus of attention. By contrast, cooperative interruptions are more supportive of the main speaker's floor rights, and the intention is to keep the attention on the main speaker's point. This difference in cooperativeness has a corresponding influence on the prosodic patterns of such supportive interruptions. Because of their non-disruptive nature, they often occur at low or medium pitch levels, and even when they are high for emotional reasons, they are generally lower in pitch than competitive interruptions. The amplitude of cooperative interruptions can vary. In our data, the amplitude is generally low in cases of acknowledging and prompting, but often high when an interruption is used to express strong opinion or emphasis. These characteristics can be seen in the following examples: The non-disruptive nature of cooperative interruption can be seen in this example. From the pitch plot ( Figure 7) we can see that speaker A is very excited in this segment, and is speaking at a very high pitch in her range. The very excited and involved state of speaker A is clearly evidenced in the fact that her pitch level is the highest point in the entire conversation, among all her utterances. This excited state is also indicated by the abrupt 105Hz pitch elevation from her previous utterance in U406 at 325Hz. By contrast, speaker B's supportive and agreeing interruption comment at U408 is said at a relatively low pitch level of about 260Hz and at a moderately low amplitude, in agreement with the implicit goal of avoiding disruption to the main speaker's progress. Figure 7: Three cases of low-pitched supportive interruptions at U408, U417, and U420. Completing An Anticipated Point Cooperative interruptions frequently occur when the main speaker is in the middle of completing a point and the other speaker already anticipates that point and is in agreement with the main speaker. In such cases, the other speaker often comes in to finish that point for the main speaker, presenting the interruption as a prompt. In our data, these instances typically occur at relatively low pitch levels, because of the certainty and confidence of the interrupter, and at a relatively high amplitude, reflecting increased emphasis. In this example, speaker A interrupts at U417 to finish for speaker B, expressing the point in an emphatic way:`There's a limit'. Speaker A's pitch is at a relatively low level of 220Hz, and that reinforces the expression of unanimity or agreement with the main speaker. The loud amplitude on this phrase signals the strong opinion and emphasis that speaker A is expressing (Figure 7). At U420, speaker A again anticipates speaker B and comes in to cooperatively develop B's point at a moderate pitch level of 270Hz and a loud amplitude, signaling the joint cooperative nature of these interruptions. Variations in Intensity One complication is that cooperative interruptions are also affected by the intensity of the accompanying emotion, and therefore may also occur at high pitch levels, as seen in the following two examples: In the first example, example (8), the interrupter is giving a strong expression of support and enthusiastic agreement, and this is evident in the semantic content`That's exactly right. You just have to cooperate. Right'. Because of the strong emotion involved, speaker B's pitch level here is very high at 385Hz, as seen in Figure 8, and the amplitude is also loud. The interruption in example (9) is also a strong interruption to support the main speaker, but adds new salient information to the ongoing topic by explicitly bringing up a notable fact 'lots of famous people'. The pitch level of this phrase (Figure 9) is high at about 380Hz and the amplitude is also great. The supportive intention of this cooperative interruption is further indicated by the continuing feedback speaker B provides, and this intention is recognized and appreciated greatly by the main speaker, as shown by her repeated echoing of speaker B's remark in U303 and U305. The Integration of Discourse Elements and the Variations of Pitch Height Our data show that the complexity of interruptions increases with the complexity of the discourse relationships. Interruptions are complex discourse phenomena. They are informationally packed, as they mediate the differing interests and knowledge states of participants in a conversation. The specific nature of each interruption is a reflection of the underlying motivation of the interrupter. The content and timing of interruptions are directly linked to the interrupter's urgent and intense emotional need for an immediate resolution. That is, it is the urgency of the emotion that is causing the interrupter to express the need to address a particular salient topic immediately at this particular time. Another factor that contributes to this complexity is that competitiveness and cooperativeness are not polar opposite characteristics of interruptions, but occur as a gradient process. The degree of competitiveness arises from the intensity of the emotions underlying the interruption. Speaker intensity is also closely linked to the degree of certainty and uncertainty inherent in the ongoing topic progression. The forcefulness of the expression also affects how the main speaker responds. An intense expression often creates a critical need for an immediate response, and speakers are more prone to stop and address the issue raised by the interrupter, hence such interruptions are more competitive. The degree of competitiveness or cooperativeness is also influenced by how related the interruption is to the ongoing topic, i.e. the degree of relatedness of topic, and the knowledge states of the participants, and by how long the interrupter intends to take the floor for. A short interruption for a clarification on the current topic is more cooperative than an interruption to change both the topic and the floor. The specific strength of signal needed to adequately overcome the ongoing topic may vary by the changing interruptability or resistance level of the topic. Because of the intentions of participants, in spontaneous discourse interruptions occur to varying degrees of intensity and varying degrees of competitiveness and cooperativeness. Interruptions thus are a complex combination of expressions of emotion, signals of attentiongetting, and signals of competitiveness, and their prosodic manifestations are directly linked to these motivations. Our data show that the pitch level of interruptions can occur at varying heights; the higher the intensity, the higher the pitch level. The specific pitch height of the interruption is determined jointly by the need to attract attention, the intensity of the emotion present, and the strength of signal needed to overcome the attention and focus on the current topic. Example 1 The prosodic patterns for this segment of 100 utterances (see Figure 10) are very revealing of the complex emotional and discourse forces at work, and also illustrate the point that intensity and degree of uncertainty and certainty are significant determinants of topic direction and prosodic structure. In this part of the conversation, speaker A is talking about a conference she attended previously and Speaker B mistook it to be the conference that she was interested in, so she initiates a series of short questions (in the form of interruptions) to confirm and clarify the information. The general cognitive pattern seen here is that the interrupter encounters an initial high unsettled state of uncertainty and gradually progresses to a more settled and certain state. This is clearly expressed in the overall downward trend in the pitch level for these utterances in the peak pitch chart. The pattern of alternating doubt and certainty is very revealing itself. At each interruption that expresses doubt and a need for clarification, there is a local rise in pitch. Those interruptions which express acknowledgement and certainty are locally lower in pitch. The specific strength of signal needed varies systematically with the resolution of the differing interests and knowledge states of participants. The overall prosodic structure of this example also provides a vivid illustration of the importance of the process of intensification and normalization in discourse. The very high pitch at U93 reflects the abrupt climax of emotional intensity and uncertainty, and as this emotional uncertainty is expressed and cognitively resolved through the sequence of interruptions, normalization in the intensity of the cognitive state and the pitch level is then achieved. Example 2 Taking a more extended view of our data shows that pitch movements of interruptions also vary according to overall patterns of topic development and intensity of speaker involvement. Analysis of the discourse text shows that the rise-fall arc seen in Figure 11 also coincides with the development of a major subtopic that both speakers actively contribute to. This involvement is signaled by the large amount of dots at varying heights of both speakers. Both speakers' involvement reaches a peak of excitement roughly at the U320-U330 section, and then gradually descends as speaker A gives more specific details in concluding the topic. As shown, the pitch levels of the interruptions of both speakers also converge and follow the same rise-fall pattern as interest in the topic increases and then is resolved. This supports the view that interruptions are a part of an overall systematic prosodic structure that integrates topic progression and speaker involvement through a process of climax and resolution. Example 3 What is happening in the conversation in Figure 12 is that one speaker (speaker B) begins to develop a topic that she is interested in but that had not been successfully communicated, and the interest level and the speaker's involvement are intensified as she attempts to overcome the mismatch as indicated by many very abrupt high pitched interruption points, whereas speaker A's pitch movements are expressed in more uniform overall descending pattern. The descending part of the curve also coincides with the resolution of an issue that speaker B had been very uncertain about throughout that section of dialogue. This reinforces our conclusion that at each level of analysis, prosody links speaker interaction, topic progression and expression of cognitive state. Implications for Dialogue Systems How can we use the above information to help build an intelligent spoken dialogue system? We can focus on 2 related aspects: Detection and Response. For example, a high pitch and amplitude would be detected as a competitive interruption of higher urgency and indicate a possible mismatch of the current state with the user's desired state. The system would respond by searching the possible topic space, adding the lexical-semantic content of the interruption to prior information to aid in the search. Ongoing monitoring of the prosody can also provide important information on the direction the dialogue is taking. For example, if user responses or interruptions follow an increasing pitch pattern, then the system can interpret this as increasing uncertainty, and modify the topic search direction. Conversely, decreasing pitch pattern can indicate that the user's certainty and progress toward a desired goal are increasing in a satisfactory way. As spoken dialogue systems become more receptive to natural human speech, the disfluencies and prosody of human speech can provide critical information to guide progress along interactively developed system paths, mirroring aspects of human-human conversational speech. Further work on adapting prosody detection to dialogue systems provides a foundation for systems which are truly interactive, taking advantage of active inputs by the user, adapting to the knowledge base of users, and providing clarifications according to search strategies that account for information presented in more natural ways, and ultimately making systems more intelligent by adapting to human motivation. Conclusion In this paper we have shown that interruptions are important elements in the interactive character of discourse and in the resolution of issues of cognitive uncertainty and planning. We have also shown that interruptions are part of the local and global coherence that is brought about through the systematic phrase-to-phrase prosodic patterns of discourse, and are an important component for speech understanding and intelligent dialogue systems.	2014-07-01T00:00:00.000Z	2001-09-01T00:00:00.000	{ "year": 2001, "sha1": "10e6b038edd8b03014e0ee83e83b3fe31bc8a31c", "oa_license": null, "oa_url": "http://dl.acm.org/ft_gateway.cfm?id=1118106&type=pdf", "oa_status": "BRONZE", "pdf_src": "ACL", "pdf_hash": "10e6b038edd8b03014e0ee83e83b3fe31bc8a31c", "s2fieldsofstudy": [ "Computer Science", "Linguistics" ], "extfieldsofstudy": [ "Computer Science" ] }
259547492	pes2o/s2orc	v3-fos-license	Dissecting dynamic plant virus synergism in mixed infections of poleroviruses, umbraviruses, and tombusvirus-like associated RNAs Mixed infections of a plant infecting polerovirus, umbravirus, and/or tombusvirus-like associated RNAs (tlaRNAs) produce unique virus disease complexes that exemplify “helper-dependence” interactions, a type of viral synergism that occurs when a “dependent” virus that lacks genes encoding for certain protein products necessary for it to complete its infection cycle can utilize complementary proteins encoded by a co-infecting “helper” virus. While much research has focused on polerovirus-umbravirus or polerovirus-tlaRNA interactions, only recently have umbravirus-tlaRNA interactions begun to be explored. To expand on the limited understanding of umbravirus-tlaRNA interactions in such disease complexes, we established various co-infection pairings of the polerovirus turnip yellows virus (TuYV), the umbravirus carrot mottle virus (CMoV), and three different tlaRNAs—carrot red leaf virus aRNAs (CRLVaRNAs) gamma and sigma, and the TuYVaRNA ST9—in the model plant Nicotiana benthamiana, then investigated the effects of these different co-infections on tlaRNA systemic movement within the host, and on virus accumulation, and aphid and mechanical transmission of each of these viruses. We found that CMoV alone could support systemic movement of each of the tlaRNAs, making this the second report to demonstrate such an interaction between an umbravirus and tlaRNAs. We also report for the first time that CMoV could also impart mechanical transmissibility to the tlaRNAs sigma and ST9, and that co-infections of either of these tlaRNAs with both TuYV and CMoV increased the efficiency with which TuYV could be mechanically co-transmitted with CMoV. Introduction Helper-dependence, also known as transcomplementation, is a particularly interesting example of virus synergism between co-infecting viruses. In such infections, the "helper" virus encodes a gene(s) product that the "dependent" virus lacks but can utilize, and in some instances this interaction is obligatory for the dependent virus to complete its infection cycle as these proteins facilitate within host movement and/or transmission between hosts (Rochow, 1972;Latham and Wilson, 2008;Syller, 2012;Alcaide et al., 2020). In many instances, such co-infections also result in significantly enhanced symptom development in the host and significantly increased accumulation of one or more of the co-infecting viruses (Murant et al., 1988;Barker, 1989;Murant, 1990;Passmore et al., 1993;Sanger et al., 1994;Li et al., 2017;Zhou et al., 2017). Mixed infections comprised of a polerovirus, an umbravirus, and/or a co-infecting tombusvirus-like associated RNA (tlaRNA) exemplify this type of viral synergism. Multiple disease complexes comprised of a polerovirus, an umbravirus, and/or a satellite virus or tlaRNA have been identified that are responsible for causing severe disease outbreaks in economically important crops Falk and Duffus, 1984;Passmore et al., 1993;Sanger et al., 1994). The groundnut rosette disease (GRD) complex, caused by co-infection of the polerovirus groundnut rosette assistor virus (GRAV), the umbravirus groundnut rosette virus (GRV), and either one of two associated satellite RNAs-which appear to be responsible for symptom severity and GRV encapsidation by GRAV produced capsids-is perhaps the most well studied example of such a viral disease complex that has caused devastating losses in crop production (Storey and Ryland, 1955;Hull and Adams, 1968;Murant et al., 1988;Murant, 1990;Taliansky et al., 1996;Naidu et al., 1998;Taliansky et al., 2000). Another notable example is the carrot motley dwarf (CMD) disease complex, comprised of the polerovirus carrot red leaf virus (CRLV), the umbravirus carrot mottle virus (CMoV), and/or one of several CRLV associated RNAs (CRLVaRNA; Watson et al., , 1998Murant et al., 1969). CMD occurs globally, anywhere that carrots are produced, and has caused epidemics resulting in severe crop losses Watson and Falk, 1994). Viruses in the genus Polerovirus (family Solemoviridae) are phloem limited and obligately vectored by aphids, often in a speciesspecific manner; they encode their own capsid proteins (CP) and are capable of in planta systemic movement (Brault et al., 1995;Mayo and Ziegler-Graff, 1996;Peter et al., 2009;Pagán and Holmes, 2010). Umbraviruses are mechanically transmissible, non-phloem limited viruses in the family Tombusviridae that can move cell-to-cell and systemically within their plant hosts but lack genes encoding for their own CP and the capacity to be independently transmitted by an insect vector (Nurkiyanova et al., 2001;Kim et al., 2007). TlaRNAs are small (~2.8 kb) putative viruses that only encode a replicase protein, making them incapable of independent movement within their plant hosts or transmission to new hosts, and as such are exclusively found co-infecting with a polerovirus, sometimes in association with an umbravirus (Falk and Duffus, 1984;Passmore et al., 1993;Watson et al., 1998;Campbell et al., 2020). One such virus complex is the polerovirus TuYV with the ST9aRNA in shepherd's purse [Capsella bursa-pastoris (C. b-p)] plants, which produces significantly enhanced symptom development and TuYV accumulation, and in which the ST9aRNA gains systemic movement and aphid transmissibility through encapsidation by TuYV capsid proteins (Falk and Duffus, 1984;Passmore et al., 1993;Sanger et al., 1994). Another pertinent example is the aforementioned carrot motley dwarf (CMD) disease complex, which also shows enhanced symptom development in various apiaceous hosts, and in which CMoV and CRLVaRNAs are known to be dependent on CRLV for aphid transmission (Murant et al., 1969;Waterhouse and Murant, 1983;Murant et al., 1985;Watson and Falk, 1994;Watson et al., 1998). Genome maps of each of these viruses and the proteins they encode are depicted in Figure 1. It has also been found that some poleroviruses can, presumably, utilize the cell-to-cell movement proteins (MPs) of a co-infecting umbravirus, allowing the polerovirus to presumably escape phloem limitation and become mechanically transmissible (Hoffman et al., 2001;Ryabov et al., 2001a;Zhou et al., 2017). While there are a good number of studies on polerovirus-tlaRNA and polerovirus-umbravirus interactions, there currently exist few studies on umbravirus-tlaRNA interactions. To further parse the various virus-virus interactions that occur in these disease complexes, we used aphid inoculation of TuYV in combination with agroinfiltration of infectious clones of CMoV and three tlaRNAs-CRLVaRNAs Gamma and Sigma, and ST9aRNA, heretofore referred to simply as Gamma, Sigma, and ST9-to generate single, double, and triple infections of each of these viruses and examine their effects on symptom development, systemic movement of tlaRNAs, and on aphid and mechanical transmission of each of these viruses. In this study, TuYV was used in place of CRLV because we did not have on hand the specific aphid vector (Cavariella aegopodii) of this virus (Murant et al., 1969;Elnagar and Murant, 1978), whereas we did have the aphid vector (Myzus persicae) of TuYV as well as an active culture of TuYV maintained in C. b-p. plants. Our results show that CMoV appears to be a driver of symptom development when co-infected with TuYV and/or Sigma or ST9. CMoV also facilitated the systemic movement of all three tlaRNAsalbeit with differing efficiencies-in the absence of TuYV, corroborating the results of a recent study that demonstrated umbravirus-facilitated tlaRNA systemic movement in the tobacco bushy top disease (TBTD) complex (Chen et al., 2022). CMoV could also facilitate mechanical transmission of Sigma and ST9 (but not Gamma) and TuYV; the transmission rate of TuYV from plants co-infected with only CMoV was very low, however when co-infected with both CMoV and either Sigma or ST9, the transmission rate of TuYV greatly increased. This is the first report to demonstrate that umbravirus co-infection can impart mechanical transmissibility to tlaRNAs and that some tlaRNAs appear to increase the efficiency with which a co-infecting polerovirus can be mechanically transmitted from plants also co-infected with an umbravirus. Effects of co-infection on the ability of CMoV and the tlaRNAs to be co-aphid transmitted with TuYV, as well as effects on the accumulation of each of these viruses were also determined. Establishing mixed infections Combined aphid and Agrobacterium tumefaciens-mediated inoculations were used to generate the various single, double, and triple infections examined in this study which are listed in Table 1. For TuYV inoculation, non-viruliferous green peach aphids (Myzus persicae) were fed on TuYV infected C. b-p plants for 18-24 h, after which they were transferred to 2-3 week old healthy Nicotiana benthamiana seedlings for a 4 day inoculation access period (IAP), after which aphids were killed by spraying with BioAdvanced 3-In-1 Insect, Disease, and Mite Control (Bayer). The plants were kept in an air conditioned room until they grew large enough for agroinoculation (4-6 leaf stage). For CMoV and tlaRNA inoculations, cultures of A. tumefaciens strain GV3101, transformed individually with infectious clones of each virus, were prepared as described by Erickson et al. (2023); cultures resuspended in infiltration buffer were adjusted to an optical density at 600 nm (O.D. 600 ) of 1.0. The A. tumefaciens cultures were infiltrated using a needless syringe into 3-4 leaves of healthy or TuYVinoculated N. benthamiana plants at the 4-6 leaf stage; for treatments including both CMoV and a tlaRNA, cultures were mixed 1:1 prior to infiltration. The plants were maintained in the air conditioned room for 4-5 days post inoculation (dpi) then transferred to a growth Aphid transmission experiments To determine how different infection combinations affected the ability of each of the viruses in this study to be aphid transmitted, aphid transmission experiments were conducted as described in the previous section, this time using as the inoculum source leaf tissue from healthy plants and plants infected with the various single and multi-virus combinations described above. For treatments with a tlaRNA alone, TuYV with any tlaRNA, or CMoV + Gamma, agroinoculated leaves were used as the inoculum source; for all other treatments non-inoculated leaf tissue was used. Inoculated plants were maintained under the same conditions as the plants described in the previous section. After 4 wpi symptoms were noted and leaf tissue was collected and stored at −80°C prior to RNA extraction and RT-qPCR analysis. The experiments were repeated twice. Mechanical transmission experiments To determine how different infection combinations affected the ability of each of the viruses in this study to be mechanically transmitted, leaf tissue from healthy plants and plants harboring each of the described virus infection treatments was ground with a mortar and pestle in 0.03 M KPO 4 + 0.1% Na 2 SO 3 (pH 7) buffer in a 1:6 ratio Frontiers in Microbiology 04 frontiersin.org (w/v), with celite added as an abrasive. For treatments with tlaRNAs alone, TuYV + tlaRNAs, or CMoV + Gamma, infiltrated leaves were used as the inoculum source, for all other treatments non-inoculated leaves were used. Using a sterile cotton swab, the homogenized plant sap was gently rubbed onto 3-4 leaves of healthy N. benthamiana plants at the 4-6 leaf stage. Plants were kept in a growth chamber held at 19°C, 70% relative humidity (RH), and 16:8 h light:dark photoperiod. At about 3-4 wpi, symptoms were noted and leaf tissue was collected and stored at −80°C prior to RNA extraction and RT-qPCR analysis. The experiments were repeated twice. Multiplexed RT-qPCR assay validation Primer and probe sequences specific to each virus used in this study were designed using the PrimerQuest Tool. 1 Standard curves were made using plasmids harboring cloned viral genome sequences of each virus to determine the amplification efficiency of each primer/ probe set (without fluorophore); temperature gradient analysis was also performed to determine an optimal annealing temperature. The assays were performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad), in a reaction mix containing 10 μL of SsoAdvanced Universal SYBR ® Green Supermix (Bio-Rad) 0.6 μL of each primer (10 μM), 2 μL of template, and 6.8 μL of nuclease free water. The thermocycling conditions were 95°C for 3 m followed by 40 cycles of 95°C for 10 s and 55°C-65°C for 30 s, and concluded with melt curve analysis; a shared optimal temperature of 60°C was selected. Each primer set was tested against non-target virus plasmids to confirm primer specificity. Next, plasmids with each virus were pooled in equimolar amounts, and standard curve analysis was repeated to confirm the pooled sample had minimal effects on primer amplification efficiency. Primer and probes (with fluorophore) were validated-first individually, then in a multiplexed reaction-in the same manner (excluding temperature gradient and melt-curve analyses), using a reaction mix containing 10 μL of iQ Multiplex Powermix (Bio-Rad), 0.04 μL of each primer (100 μM), 0.02 μL of probe (100 μM), 2 μL of template, and nuclease free water to 20 μL, and the same thermocycling conditions. The amplification efficiency of all sets was between 90% and 110%. All primers and probes used in this study are listed in Supplementary Table S1. RNA extraction and multiplexed RT-qPCR analysis Total RNA was extracted from leaf tissue samples using TRIzol™ Reagent (Invitrogen) according to the manufacturer's protocol. For the initial experiments in which the different single and multi-virus infections were established, samples were treated with RQ1 RNase-Free DNase (Promega) and cleaned by phenol/chloroform extraction prior to cDNA synthesis; this was not done for samples from the aphid and mechanical transmission experiments. RNA was used as template with the iScript™ cDNA Synthesis Kit (Bio-Rad); the synthesized 1 www.idtdna.com/PrimerQuest/Home/Index cDNA was diluted to 5 ng/μL with nuclease free water. The qPCR reaction contained 10 μL of iQ Multiplex Powermix, 0.04 μL of each primer (100 μM), 0.02 μL of each prober (100 μM), 2 μL (10 ng) of cDNA template, and nuclease free water to 20 μL, and the thermocycling conditions were as described in the previous section. The cytochrome C oxidase gene was used as a reference. Two technical replicates were performed for each sample. The 2 -∆∆ Ct method was used to calculate the relative viral accumulation in the initial set of experiments establishing the different virus infection treatments. Statistical analyses Significant differences in viral accumulation between different single and mixed virus infections were determined using ANOVA using generalized linear models with the corresponding R packages in InfoStat v2008. Normality and homoscedasticity were checked and corrected when necessary and means were separated using Fisher's least significant difference test (p < 0.05). Data was plotted in GraphPad Prism v.5.03. Symptom development No notable symptoms were observed in plants singly infected with any of the viruses, in any plants doubly infected with TuYV and any of the tlaRNAs, or in plants infected with CMoV + Gamma ( Figure 2). Plants co-infected with CMoV + Sigma or with CMoV + ST9 developed prominent leaf mosaic symptoms, which were more severe in CMoV + ST9 infected plants which displayed more prominent yellowing as well as mild leaf curling. Plants infected with TuYV + CMoV developed punctate necrotic spots on the leaves, along with mild mosaic symptoms. These same symptoms were observed in plants co-inoculated with TuYV + CMoV + Gamma. In plants co-inoculated with TuYV + CMoV + Sigma or + ST9, the observed symptoms appeared to be a combination of those that were observed in the CMoV + TuYV, CMoV + Sigma, and CMoV + ST9 plants, displaying dramatic leaf mosaic, chlorosis, and punctate necrotic lesion symptoms on leaves (Figure 3). For all non-symptomatic plants and plants co-inoculated with CMoV + Sigma or + ST9, no prominent differences in overall growth were observed ( Figure 4A). However, plants co-inoculated with TuYV + CMoV, and TuYV + CMoV + Sigma or + ST9 were severely stunted, and this effect was most severe in the TuYV + CMoV + ST9 co-infected plants ( Figure 4B). Systemic trafficking of tlaRNAs In plants singly infected with each tlaRNA, none of the tlaRNAs could be detected in the upper non-inoculated leaves, despite being detected in the inoculated leaves. This was also true for plants harboring co-infections of TuYV + Gamma or TuYV + Sigma. In a few plants, while TuYV was detected in non-inoculated leaves, it was not detected in leaves agroinoculated with the tlaRNA, which may explain the lack of tlaRNA systemic movement in these plants. However, in the majority of the plants tested both TuYV and the tlaRNAs were detected Asymptomatic virus infections in Nicotiana benthamiana plants. Depicted are asymptomatic N. benthamiana plants that have been inoculated singly with TuYV, CMoV, and tlaRNAs Gamma, Sigma, or ST9, doubly with TuYV and each of the tlaRNAs, and inoculated with CMoV + Gamma, along with a healthy, non-inoculated plant for comparison. Labels above each plant picture indicate the virus infection treatment. Photos were taken 3 wpi. non-inoculated leaves, although this interaction occurred with differing frequencies for the different tlaRNAs. In CMoV + Sigma and CMoV + ST9 co-infected plants, both tlaRNAs were systemically trafficked 100% of the time, however, in CMoV + Gamma co-infected plants, systemic trafficking of Gamma was only observed 50% of the time. Similar results were observed for plants co-infected with TuYV, CMoV, and each of the tlaRNAs. These results are summarized in Table 2. Relative virus accumulation The only co-infection treatments which were associated with a significant increase in TuYV accumulation relative to plants infected with TuYV alone were co-infections of TuYV + CMoV (5.50-fold increase), TuYV + CMoV + Gamma [7.84-fold increase in non-inoculated leaves (NILs)], and by far the most dramatic increase (172.66-fold) was observed in the NILs of TuYV + CMoV + ST9 infected plants. All other co-infection treatments resulted in non-significant changes in TuYV accumulation ( Figure 5A). CMoV accumulation increased significantly in the inoculated leaves (ILs; 17.08-and 10.10-fold) and NILs (22.36-and 39.48-fold) of CMoV + ST9 and TUYV + CMoV + ST9 inoculated plants, respectively. All other infection treatments produced minimal, non-significant changes in CMoV accumulation with respect to accumulation levels in plants inoculated with only CMoV ( Figure 5B). Gamma accumulation levels increased significantly in the ILs of TuYV + Gamma, CMoV + Gamma, and TuYV + CMoV + Gamma inoculated plants by 3.00-, 25.35-, and 37.25-fold, respectively. Interestingly in the NILs of CMoV + Gamma and TuYV + CMoV + Gamma, Gamma accumulation levels (1.45-and 2.15-fold, respectively) did not vary significantly from that in the ILs of plants inoculated with Gamma alone ( Figure 5C). Among the tlaRNAs tested in this study, Sigma accumulation varied the most in co-infected plants, relative to that in the ILs of plants inoculated with Sigma alone. While Sigma accumulation did not change significantly in the ILs of TuYV + Sigma infected plants (0.55-fold), it increased significantly both in the ILs (10.47-and 7.9-fold) and the NILs (48.66-and 30.16fold) of CMoV + Sigma and TuYV + CMoV + Sigma co-infected plants, respectively ( Figure 5D). ST9 accumulation levels were not significantly altered in the ILs of TuYV + ST9 infected plants, and only varied significantly in the ILs of CMoV + ST9 inoculated plants (1.76fold increase), however a noticeable but non-significant increasing trend was observed in the NILs of these plants (4.96-fold), as well as in the ILs (5.68-fold) and NILs (5.13-fold) of TuYV + CMoV + ST9 inoculated plants ( Figure 5E). It is interesting to note that in some co-infections with ST9, TuYV and CMoV accumulations both dramatically increased, but a compensatory increase in ST9 accumulation was not observed. Effects of co-infection on mechanical transmission As expected, when N. benthamiana plants were inoculated using tissue from plants infected with any of the viruses in this study not Frontiers in Microbiology 07 frontiersin.org known to be independently mechanically transmissible (TuYV and tlaRNAs), systemic infections were not observed by any of these viruses; when the rub-inoculated leaves were tested for these viruses, low level amplification of all but Gamma could be detected. This low level detection may simply be attributed to residual inoculum, or it may suggest that some cells could become infected with these viruses by rub inoculation but that the viruses could not move beyond the inoculated cells (data not shown); these results coincide with those from early studies on ST9 in rub inoculated C. b-p leaves (Passmore et al., 1993). CMoV was mechanically transmitted and initiated a systemic infection in 100% of the rub-inoculated plants, regardless of whether the inoculum source was infected with CMoV alone or in combination with any of the other viruses. Somewhat unexpectedly, Sigma and ST9 were both efficiently mechanically transmitted (and established systemic infections) from plants co-infected with either of these tlaRNAs and CMoV, and plants co-infected with TuYV + CMoV and either of these tlaRNAs; Sigma was transmitted to 100 and 69%, respectively, of plants when tissue from CMoV + Sigma and TuYV + CMoV + Sigma co-infected plants were used as the inoculum source, and ST9 was transmitted to 100 and 62%, respectively, of plants when tissue from CMoV + ST9 and TuYV + CMoV + ST9 co-inoculated plants, were used as the inoculum source. Gamma could not be detected in the ILs or NILS of any rub inoculated plants, regardless of whether tissue from CMoV + Gamma or TuYV + CMoV + Gamma infected plants was used as the inoculum source. TuYV was transmitted with very low efficiency (11%) from TuYV + CMoV co-infected plants. Interestingly, the efficiency with which TuYV was mechanically transmitted increased markedly from plants co-infected with TuYV + CMoV and either Sigma or ST9. When TuYV + CMoV + Sigma infected plants were used as the inoculum, the transmission rate of TuYV increased to 54%, and when TuYV + CMoV + ST9 infected plants were used as the inoculum source it increased to 77%. This data is summarized in Table 3. Effects of co-infection on aphid transmission As expected, none of the viruses in this study not known to be independently aphid transmitted (CMoV and tlaRNAs) could be detected in aphid inoculated plants, when tissue from plants infected singly or in combination with any of these viruses was used as the inoculum. Co-infection with TuYV did facilitate aphid transmission of CMoV when plants co-infected with TuYV + CMoV, TuYV + CMoV + Gamma, and TuYV + CMoV + ST9 were used as the inoculum source-CMoV was transmitted to 42%, 43%, and 64% of recipient plants, respectively, and TuYV was, respectively, transmitted to 100%, 77%, and 77% of recipient plants. Neither Gamma nor Sigma became aphid transmissible when co-infected with TuYV, despite TuYV being transmitted to 100% and 92% of recipient plants from these inoculum sources. Triple infections of each of these tlaRNAs with TuYV and CMoV did not yield different results, despite TuYV transmission efficiency remaining high (100% and 77%, respectively). Conversely ST9 was successfully aphid transmitted with low efficiency (10%) to a single recipient plant from plants co-infected with TuYV + ST9, and was aphid transmitted to 7% of recipient plants when the inoculum source came from plants also infected with TuYV Frontiers in Microbiology 08 frontiersin.org + CMoV. Transmission efficiencies of TuYV in these treatments were 100% and 77%, respectively. These results are summarized in Table 4. Discussion While there are many studies on polerovirus-tlaRNA and polerovirus-umbravirus interactions in disease complexes harboring various combinations of these viruses, there exist only two recent studies that have begun to touch upon umbravirus-tlaRNA interactions (Yoshida, 2020;Chen et al., 2022). While we investigated the effects of co-infection in all possible co-infection combinations of the polerovirus TuYV, the umbravirus CMoV, and the tlaRNAs Gamma, Sigma, and ST9-with respect to single infections of each of these viruses-perhaps the most intriguing findings we uncovered were those concerning interactions involving CMoV and tlaRNAs. With respect to symptom development in N. benthamiana plants, the most interesting result we found was that almost all co-infections that included CMoV (with the exception of CMoV + Gamma) induced enhanced symptom development in inoculated plants relative to those infected with any virus alone or plants co-infected with TuYV and any tlaRNA. Additionally, there were noticeable differences in symptom presentation depending on if CMoV was co-infected with TuYV (dispersed, punctate, necrotic lesions on leaves) or with either Sigma or ST9 (mosaic symptoms on leaves). There were also notable differences in the severity of leaf mosaic symptoms between co-infections that included Sigma (less severe) and those that included ST9 (more severe), both in double infections with CMoV and in triple Relative accumulation of TuYV, CMoV, and tlaRNAs Gamma, Sigma, and ST9 as a function of co-infection. Graphs depict log 2 changes in viral accumulation of (A) TuYV, (B) CMoV, and the tlaRNAs (C) Gamma, (D) Sigma, and (E) ST9 in mixed infections relative to accumulation of each of these viruses in single infections; the y-axis. Virus accumulation was quantified using RT-qPCR and calculated using the 2 -∆∆ Ct method. Black and gray bars represent relative viral accumulation in agroinoculated and non-inoculated leaves, respectively. Graphs depict the means ± SEs. Significant differences between treatments were determined using ANOVA with a significance value of p < 0.05; different letters indicate significant differences between treatments, whereas shared letters indicate there was not a significant difference between treatments. Frontiers in Microbiology 09 frontiersin.org infections with TuYV and CMoV. These results suggest that, in this model host and disease complex system, CMoV is the key driver of symptom development since symptoms only occurred in co-infections in which it was present. These results also demonstrate marked differences in symptom development with respect to each of the tlaRNAs; as more of these tlaRNAs are being regularly discovered, it will be interesting to further uncover the various ways they differ in the effects they have on symptom development and interactions they have with co-infecting viruses in these unique disease complexes. Until recently, it was thought that only poleroviruses were responsible for systemically trafficking tlaRNAs in these disease complexes. However, a recent study by Yoshida (2020) found that after attempting to aphid transmit CRLV, CMoV, and a CRLVaRNA from CMD affected carrot plants harboring all three of these viruses to Japanese parsley (Cryptotaenia canadensis subsp. Japonica), only CMoV and the CRLVaRNA could be detected in the recipient plant, making this the first reported evidence that an umbravirus may support tlaRNA systemic movement in the absence of a co-infecting polerovirus, although these results could also have other potential explanations. In another recent study by Chen et al. (2022), after co-agroinoculation of infectious clones of the umbravirus tobacco bushy top virus (TBTV) with the tobacco vein distorting virus associated RNA (TVDVaRNA) the authors could detect the TVDVaRNA along with TBTV in the distal non-inoculated leaves, thereby confirming an umbravirus could independently support tlaRNA systemic movement. Here, we present data demonstrating that CMoV was able to support systemic movement of all three tlaRNAs (Gamma, Sigma, ST9), however this interaction appeared to be less efficient in co-infections with Gamma, suggesting some degree of specificity in these umbravirus-tlaRNA interactions. Since neither Gamma nor Sigma moved systemically when co-infected with TuYV alone, it is possible there exists a degree of specificity in polerovirus-tlaRNA interactions. However, in TuYV + ST9 co-infected N. benthamiana plants, ST9 only moved systemically 38% of the time, which was odd as these viruses are known to naturally co-occur and form a strong helper-dependence relationship in Capsella bursa-pastoris plants (Falk and Duffus, 1984;Passmore et al., 1993;Sanger et al., 1994). We speculated this discrepancy might result from combined aphid inoculation of TuYV and agroinoculation of ST9 effectively failing to introduce these two viruses into the same cells. To address this we conducted preliminary experiments in which we coinfiltrated each of the tlaRNAs with an infectious clone of another polerovirus [barley virus G (BVG)] that we had on hand (Erickson et al., 2023). Interestingly, ST9 could be detected along with BVG in the upper NILs in 100% of co-infected plants; neither Gamma nor Sigma were detected in the upper non-inoculated leaves, suggesting that potential specificity in polerovirus + tlaRNA interactions may be driven by the tlaRNA (Supplementary Figure S1). In several of these virus disease complexes, it has been found that co-infection increases viral RNA accumulation of one or more of the co-infecting viruses (Sanger et al., 1994;Yoshida, 2020;Chen et al., 2022). Our results shows that tlaRNA ST9 appears to have a significant impact on CMoV accumulation, both in CMoV + ST9 and TuYV + CMoV + ST9 co-infections, and on TuYV accumulation in TUYV + CMoV + ST9 co-infected plants. These dramatic increases in CMoV and TuYV accumulation could potentially explain why symptom development was most severe in plants harboring these co-infection combinations. Surprisingly, TuYV + ST9 co-infection in Frontiers in Microbiology 10 frontiersin.org N. benthamiana plants did not stimulate a significant increase in TuYV accumulation, which was again unexpected given that in natural TuYV + ST9 co-infections in C. bursa-pastoris, the accumulation of both TuYV genomic RNAs and capsid proteins significantly increased (Falk and Duffus, 1984;Passmore et al., 1993;Sanger et al., 1994). This discrepancy may again indicate a requirement for certain host factor(s) to facilitate TuYV + ST9 interactions. Interestingly, the relative accumulation of Gamma increased significantly in the ILs of CMoV + Gamma and TuYV + CMoV + Gamma co-infected plants, but minimal differences in Gamma accumulation were observed in the NILs. A somewhat similar effect was observed for ST9 in CMoV + ST9 infected plants, wherein a significant increase was observed in ILs but not in NILs of CMoV + ST9 inoculated plants, however there was a notable increasing trend of ST9 in the NILs. The opposite was observed for Sigma, wherein a non-significant increasing trend in Sigma accumulation was observed in the ILs of CMoV + TuYV and TuYV + CMoV + ST9 inoculated plants, while a significant increase was observed in the NILs; this overall increase in accumulation of Sigma may partially explain the enhanced mosaic leaf symptoms observed in these co-infected plants. The mechanisms responsible for increased accumulation of some viruses as a result of co-infection aren't precisely known, however there are three main ways this is thought-and in some instances has been demonstrated-to occur (Rochow, 1972;Latham and Wilson, 2008;Syller, 2012;Alcaide et al., 2020). One is that co-infection functions to increase the replication of one or more co-infecting viruses, resulting in more viral copies per cell, as has been found in co-infections of the plant infecting reoviruses southern rice-black streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV), and for TuYV and ST9 in C. b-p. plants (Passmore et al., 1993;Li et al., 2017). Another possibility is that umbravirus encoded movement proteins interact with co-infecting heterologous viral RNAs to impart them with cell-to-cell and systemic movement within the plant thereby resulting in more cells being infected with the dependent virus, as has been observed in co-infections of the polerovirus potato leafroll virus (PLRV) with the umbravirus pea enation mosaic virus 2 (PEMV2; Ryabov et al., 2001a). This could explain the increase of TuYV accumulation in the presence of CMoV, since on its own TuYV is phloem limited and co-infection may help it break this phloem limitation. Weak interactions between Gamma RNAs and CMoV movement proteins may explain the differences in accumulation of Gamma between the ILs and NILs of plants co-infected with CMoV, as well as the reduced efficiency of systemic transport of Gamma. A third possibility is that one or more of the co-infecting viruses have different host defense mechanisms that can suppress host defense systems against which the other co-infecting virus(es) may be susceptible. For example, the P0 protein of some poleroviruses, including TuYV, functions as a suppressor of the RNA interference (RNAi) system of the host plant (Baumberger et al., 2007;Bortolamiol et al., 2007;Csorba et al., 2010). TlaRNA ST9 was found to have a structural feature in the 3′ untranslated region (UTR) of its genomic RNA that functions to stall host XRN1 degradation (Campbell et al., 2022), which may explain the dramatic effect ST9 appeared to have on TuYV and CMoV accumulation in co-infected plants. The long distance movement protein (encoded by ORF3) of umbraviruses has been shown to form protective ribonucleoprotein complexes with both umbravirus and heterologous virus RNAs (Ryabov et al., 2001b;, and has also been shown in PEMV2 to protect (May et al., 2020). Perhaps the combined effects of the TuYV P0 RNAi silencing suppressor (RSS) activity, putative CMoV derived NMD resistance, and stalling of XRN1 degradation by ST9 could explain the drastic increases of TuYV and CMoV in co-infections of these three viruses and the severe symptom development. It is likely that a combination of these various viral functions interplay to produce the variable effects on viral accumulation and disease presentation observed in the different co-infections of the viruses used in this study. There are multiple examples of umbraviruses conferring mechanical transmissibility to a co-infecting polerovirus. Falk et al. (1979) showed that TuYV [formerly referred to as beet western yellows virus (BWYV)] was occasionally mechanically transmitted from plants co-infected with the umbravirus, lettuce speckles mottle virus (LSMV), and PLRV has been observed to gain mechanical transmissibility as a result of co-infection with PEMV2 (Falk et al., 1979;Ryabov et al., 2001a). In the latter study, it was also found that PLRV became mechanically transmissible when co-infected with a cucumber mosaic virus (CMV) vector engineered to express the GRV ORF4 protein, suggesting that this protein likely plays an important mechanistic role in mechanical transmission. However, when PLRV was coinoculated with a GRV-ORF4 expressing CMV vector that had a defective 2b RSS, mechanical transmissibility of PLRV was lost, further highlighting the likely role virus encoded host defense suppressors may play in such interactions. However, there are other examples of polerovirus-umbravirus co-infections that did not confer mechanical transmissibility to the polerovirus, as has been observed in co-infections of tobacco bushy top virus (TBTV) and tobacco vein distorting virus (TVDV; Chen et al., 2022). While it has been speculated, no studies have been published on whether an umbravirus can confer mechanical transmissibility to a tlaRNA, until now. In this study, we found that both Sigma and ST9 could be mechanically transmitted from plants co-inoculated with CMoV; Gamma, conversely, did not gain mechanical transmissibility. When TuYV was co-infected with CMoV alone, it became mechanically transmissible, but with extremely low efficiency (11%). However, when TuYV was co-inoculated with CMoV and either Sigma or ST9, the efficiency of TuYV mechanical transmission greatly increased. The observed increase in TuYV accumulation in these plants may partially explain the increased rate of mechanical transmission of TuYV. Conversely, the low accumulation of Gamma may help to explain why this tlaRNA could not be mechanically co-transmitted with CMoV. We also found that CMoV became aphid transmissible when co-infected with TuYV, except from plants co-infected with TuYV + CMoV + Sigma. This supports similar findings that demonstrate CMoV could be transmitted by M. persicae aphids when co-infected with either of the poleroviruses potato leaf roll virus (PLRV) or beet western yellows virus (BWYV), along with other examples of compatible interactions between non-naturally co-occurring poleroviruses and umbraviruses, which could have important epidemiological implications for the development of novel disease complexes or transmission of umbraviruses to novel hosts (Abraham et al., 2014;Zhou et al., 2017). CMoV was transmitted with the greatest efficiency (64%) from plants co-infected with TuYV + CMoV + ST9. Whether this increase in CMoV co-transmission rate in the presence of ST9, or the lack of CMoV co-transmission observed when Sigma was present, are indicative of potential synergistic and antagonistic effects, respectively, requires further investigation. Similar to other findings in this study, while ST9 did become aphid transmissible when co-infected with TuYV, the transmission rate was lower than expected, again highlighting the potential need of host specific factors for this interaction. Together, these findings add to the growing body of knowledge on disease complexes involving these types of viruses and provide novel insights into the virus-virus interactions that occur. Such information could potentially be employed in programs aimed at preventing or controlling outbreaks of such disease complexes and perhaps could be used in the development of novel virus-based gene delivery systems. 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. Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1223265/ full#supplementary-material SUPPLEMENTARY FIGURE S1 RT-PCR data of from BVG + tlaRNA co-inoculation experiments. Panel (A) shows the products obtained from RT-PCR based detection of BVG in Nicotiana benthamiana plants that were agroinoculated with BVG alone, or co-inoculated with BVG and CMoV, or BVG and tlaRNAs Gamma, Sigma, or ST9. Expected product size for BVG is 390 bp. Panel (B) shows products from RT-PCR based detection of CMoV, Gamma, Sigma, and ST9 in N. benthamiana plants co-inoculated with BVG and each of these viruses. Expected product sizes are as follows: CMoV=532 bp; Gamma=534 bp; Sigma=399 bp; ST9=430 bp. We suspect the faint bands in the gel image for Gamma detection are likely from minor cross contamination or nonspecific primer binding. RT+: reverse transcription positive control; PCR+: plasmids used as PCR positive controls -some of these did not amplify, we suspect too much plasmid was used in the reaction; NTC: no template control.	2023-07-11T15:51:25.659Z	2023-07-06T00:00:00.000	{ "year": 2023, "sha1": "ea58e16cf7a621d97b02cf81087bafc1088ac03e", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fmicb.2023.1223265/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "922dfdd02c7cecc3794288618ba40f37a57a4e4f", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [] }
26111847	pes2o/s2orc	v3-fos-license	A Truncated Form of p23 Down-regulates Telomerase Activity via Disruption of Hsp90 Function* The Hsp90-associated protein p23 modulates Hsp90 activity during the final stages of the chaperone pathway to facilitate maturation of client proteins. Previous reports indicate that p23 cleavage induced by caspases during cell death triggers destabilization of client proteins. However, the specific role of truncated p23 (Δp23) in this process and the underlying mechanisms remain to be determined. One such client protein, hTERT, is a telomerase catalytic subunit regulated by several chaperone proteins, including Hsp90 and p23. In the present study, we examined the effects of p23 cleavage on hTERT stability and telomerase activity. Our data showed that overexpression of Δp23 resulted in a decrease in hTERT levels, and a down-regulation in telomerase activity. Serine phosphorylation of Hsp90 was significantly reduced in cells expressing high levels of Δp23 compared with those expressing full-length p23. Mutation analyses revealed that two serine residues (Ser-231 and Ser-263) in Hsp90 are important for activation of telomerase, and down-regulation of telomerase activity by Δp23 was associated with inhibition of cell growth and sensitization of cells to cisplatin. Our data aid in determining the mechanism underlying the regulation of telomerase activity by the chaperone complex during caspase-dependent cell death. Telomerase is a specialized reverse transcriptase responsible for the maintenance and preservation of telomere ends in germ cells, immortalized cells, and cancer cells (1). hTERT, 3 the reverse transcriptase subunit of telomerase, possesses catalytic activity, whereas the associated RNA component, human telomerase RNA, serves as a template for the synthesis of telomeric sequences (2). Expression analyses of hTERT and human telomerase RNA components in heterologous systems have enhanced our understanding of the biochemical features of telomerase. Human telomerase activity has been reconstituted in a variety of in vitro systems, including yeast, baculovirus, rabbit reticulocyte, wheat germ, and human cell extracts (3)(4)(5). In each system, the essential roles of hTERT and human telomerase RNA in active telomerase complexes have been confirmed. Recent studies have identified other proteins associated with the telomerase holoenzyme. For instance, Hsp90 and its co-chaperone, p23, bind hTERT and contribute to telomerase activity (6). The Hsp90 chaperone complex, which includes Hsp90, p23, Hsp70, p60, and Hsp40/ydj, is required for the assembly of human telomerase both in vitro, in a cell-free rabbit reticulocyte lysate system, and in vivo, in human cells. Among the Hsp90 partners, the acidic protein p23 is the smallest and has a relatively simple structure (7,8). p23 is ubiquitously expressed in all eukaryotes, from yeast to humans. Initially discovered as part of the Hsp90 complex with the progesterone receptor (9), p23 has since been identified in complexes containing a variety of Hsp90-associated proteins, including other steroid receptors (10), the heme-regulated kinase HRI (11), Fes tyrosine kinase, heat shock transcription factor, aryl hydrocarbon receptor (12), and polymerases such as telomerase (6) and hepatitis B-reverse transcriptase (13). Although it is known that p23 binds directly to Hsp90 in an ATP-dependent manner, it is unclear whether it interacts with client proteins or solely with Hsp90. Recent studies have confirmed that Hsp90 undergoes a conformational change upon binding to ATP, which promotes formation of additional dimer contacts near the N terminus (14). At this stage, ATP becomes trapped by Hsp90 and is committed to hydrolysis (15,16). p23 then binds Hsp90 and stabilizes this conformational state. Early studies on steroid receptor complexes indicate that p23 stabilizes the mature complex with Hsp90 in a state in which the receptor is active and able to bind hormones (17)(18)(19). Intensive studies using biochemical and crystallographic analyses revealed that p23 contains two domains: a stable, folded core domain and an unstructured, highly acidic C-terminal tail of ϳ30 -50 amino acids (8). Human p23 is a 160amino acid protein that contains eight ␤-strands (residues 1-110) and a C-terminal tail (residues 111-160). The tail is required for chaperone activity but does not participate in binding to Hsp90 (7). Nevertheless, C-terminal truncation of p23 decreases hormone-binding activity to the receptor (8). Inter-estingly, recent reports have shown that several apoptotic stimuli induce p23 caspase-mediated cleavage at the C-terminal tail but do not directly alter interactions with Hsp90 (20 -22). Active caspases 3, 7, and 8 cleave specifically at aspartic acid 142, generating a truncated form of p23 (⌬p23) with 18 fewer amino acids than the wild-type protein. Recombinant ⌬p23 displays an affinity for Hsp90 similar to full-length protein but loses its own chaperone activity in vitro. However, it remains to be determined whether cleavage of p23 alters the chaperone activity of Hsp90. In the present study, we found that p23 cleavage was associated with a reduction in hTERT expression and telomerase activity during apoptosis. Moreover, overexpression of ⌬p23 induced a decrease in the amount of hTERT protein without affecting hTERT mRNA levels, suggesting that the loss of p23 function destabilizes the hTERT protein. Furthermore, ⌬p23 suppressed phosphorylation at specific serine residues in Hsp90, and phosphoserine-defective mutants of Hsp90 failed to enhance telomerase activity. These data indicate that downregulation of telomerase activity during apoptosis correlates with hTERT destabilization via loss of Hsp90 chaperone activity. To our knowledge, this is the first report of caspase-mediated regulation of the Hsp90 chaperone complex. These results further our understanding of the mechanisms that regulate telomerase activity in apoptotic cells. EXPERIMENTAL PROCEDURES Cell Lines and Constructs-HeLa and 293 cells were cultured in minimum essential medium or Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA) in 5% CO 2 . Human p23 cDNA was kindly provided by Dr. David Toft (Mayo Clinic, Rochester, MN). p23 cDNA was subcloned into pEGFP-C1 expression vector (BD Biosciences Clontech, San Diego, CA) and pcDNA4/HisMax vector (Invitrogen), following amplification. The human Hsp90␣ cDNA fragment (a kind gift from Dr. Takayuki Nemoto, Nagasaki, Japan) was removed from pGEX-2T by digestion with BamHI/SmaI and subcloned into pcDNA4/HisMax for expression in human cells. Two potential Hsp90 phosphorylation site mutants (S231A/S263A and S231E/S263E) were generated using the QuikChange TM site-directed mutagenesis kit (Stratagene, La Jolla, CA). All constructs were verified by restriction enzyme digestion and DNA sequencing. Protein Purification and in Vitro Binding Assay-To generate p23 depleted of the 18 C-terminal amino acids (⌬p23), we constructed point mutants in which translation was terminated at 142 residues. T7-inducible pET-p23 vectors encoding fulllength human p23 or ⌬p23 were produced. Bacterial cultures were grown according to the manufacturer's instructions. After 3-h induction with 0.2 mM isopropyl-␤-D-thiogalactopyranoside, bacteria were pelleted and washed with phosphate-buffered saline. Next, bacterial cells were resuspended in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM monothioglycerol (Sigma), and sonicated. The soluble extract was applied to a DEAE-cellulose column. Elution with 0 -0.5 M KCl resulted in the isolation of highly purified p23 and ⌬p23. Protein-containing fractions were identified by immunoblotting. Fractions containing p23 or ⌬p23 were dialyzed into 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 3 mM MgCl 2 , and 10 mM monothioglycerol. Purified proteins were concentrated using an Amicon filtration unit to ϳ1 mg/ml, flash frozen, and stored at Ϫ70°C. Hsp90 binding was measured by combining 2 g of native purified Hsp90 (Assay Designs Stressgen) with 2 g of p23 in a final volume of 200 l binding buffer (10 mM Tris-HCl, 50 mM KCl, 8 mM MgCl 2 , 2 mM dithiothreitol, 4 mM ATP, 10 mM Na 2 MoO 4 , 0.01% Nonidet P-40, pH adjusted to 7.5), including an ATP-regeneration system consisting of 10 mM phosphocreatine and 7 units of creatine phosphokinase, as described previously (23). After incubation for 60 min at 30°C, samples were chilled on ice and subjected to immunoprecipitation with anti-p23, as described above. In Vitro Phosphorylation Assay-Rabbit reticulocyte lysate (40 l, Promega, Madison, WI) was incubated with 10 g of recombinant p23 or ⌬p23 in the presence of 50 mM HEPES (pH 7.4) and 2 l of [␥-32 P]ATP (3000 Ci/mmol, 10 mCi/ml, PerkinElmer Life Sciences, Boston, MA) for 60 min at 37°C. The reaction was chilled on ice and subjected to immunoprecipitation with anti-Hsp90, as described above. Immunoprecipitates were subjected to SDS-PAGE and autoradiography. The quantity of immunoprecipitated Hsp90 was monitored by Coomassie Blue staining and Western blotting. Metabolic Labeling with [ 32 P]Orthophosphate-HeLa cells were grown to near confluency in 10-cm tissue culture dishes in minimum essential medium with 10% fetal bovine serum at 37°C. Dishes were washed once with phosphate-free minimum essential medium and incubated for 15 min at 37°C in phosphate-free minimum essential medium with 2% dialyzed bovine serum (Invitrogen). The medium in each dish was replaced with 5 ml of phosphate-free medium containing 0.5 mCi of carrierfree [ 32 P]orthophosphate (8500 -9120 Ci/mmol, 10 mCi/ml, PerkinElmer Life Sciences). Cells were incubated for 60 min at 37°C, washed in cold phosphate-buffered saline, and lysed in M-PER reagent containing 1ϫ protease inhibitor mixture for 30 min on ice. Immunoprecipitation with anti-Hsp90 was performed as described above. Immunoprecipitates were subjected to SDS-PAGE and autoradiography. The quantity of immunoprecipitated Hsp90 was monitored by Coomassie staining and Western blotting. Evaluation of Apoptosis-Apoptosis was determined by staining cells with annexin V-phycoerythrin (BD Biosciences Pharmingen, San Diego, CA), as described previously (24). Briefly, detached cells were washed with phosphate-buffered saline, and resuspended in binding buffer (BD Biosciences Pharmingen) at a concentration of 1 ϫ 10 6 cells/ml. After incubation with annexin V-phycoerythrin, cells were analyzed with FACScan flow cytometer (BD Biosciences, San Jose, CA). Telomerase Activity Assay-Telomerase activity was determined with the telomeric repeat amplification protocol (TRAP, TRAP-eze Telomerase Detection Kit (Chemicon), including a 36-bp internal standard to facilitate quantitation of activity), as described previously, with minor modifications (6). After telomerase extension for 30 min at 30°C, products were amplified by three-step PCR (94°C for 30 s, 59°C for 30 s, and 72°C for 60 s) for 30 cycles in a thermocycler. Amplified products were separated by electrophoresis on a 10% polyacrylamide gel and stained with ethidium bromide. RESULTS Stability and Binding Activity of ⌬p23-The p23 protein is cleaved by caspases during apoptotic cell death induced by several stimuli (20 -22). The caspases responsible for cleavage of p23 are dependent on the cell type and the nature of the apoptotic stimulus. The C-terminal Asp-142 residue of p23 is predicted to be the most susceptible site for cleavage. To determine whether p23 cleavage affects the regulation of Hsp90 client proteins, we confirmed p23 cleavage and caspase dependence under our experimental conditions. Tunicamycin, an inhibitor of protein glycosylation, induced p23 cleavage in HeLa cells (Fig. 1A). Preincubation of HeLa cells with the pan-caspase inhibitor z-VAD-fmk abolished tunicamycin-induced p23 cleavage following tunicamycin treatment (Fig. 2), indicating that caspases are responsible for cleavage. In in vitro cleavage assays, p23 was most effectively cleaved by caspase-7, but was also cleaved, although to a lesser extent, by caspases 3, 8, and 9 (data not shown). Treatment with the caspase inhibitor z-DEVD-fmk, and replacement of Asp-142 with glutamate abolished p23 cleavage by caspase-7 (data not shown). Previous studies have shown that both native and truncated p23 bind to Hsp90 (20,22). Here, we confirmed that truncated p23 interacts with Hsp90 both in vitro and in vivo. For in vitro binding assays, we purified recombinant fulllength p23 and ⌬p23, as described under "Experimental Procedures." As expected, both wild-type p23 and ⌬p23 co-immunoprecipitated with purified human Hsp90 (Fig. 1B). Geldanamycin (GA), a benzoquinone ansamycin antibiotic, is a known Hsp90 inhibitor that specifically disrupts Hsp90-p23 interactions (25). Hsp90 did not co-immunoprecipitate with p23 in the presence of GA. To demonstrate that ⌬p23 and Hsp90 interact in vivo, we performed reciprocal co-immunoprecipitation experiments on HeLa cell lysates using p23-and FIGURE 1. Truncated p23 is stable and retains the ability to bind Hsp90. A, HeLa cells were treated with 40 M tunicamycin (Tuni) for the indicated times. p23 and its truncated form were analyzed by Western blotting with an anti-p23 antibody. B, Hsp90 (2 g) was incubated with p23 (2 g) or ⌬p23 (2 g) in the presence or absence of 1 M GA. DMSO was used as a vehicle control for GA treatment. Proteins in complexes were immunoprecipitated with anti-p23, and Hsp90 in immunoprecipitates was analyzed by Western blotting. C, HeLa cells were treated with 60 M tunicamycin for 24 h, and cell lysates were immunoprecipitated with anti-p23 or anti-Hsp90 antibodies. Hsp90 and p23 in immunoprecipitates were analyzed by Western blotting. D, HeLa cells transfected with GFP, GFP-p23, or GFP-⌬p23 were treated with 100 g/ml cycloheximide (CHX), and protein turnover at the indicated times was analyzed by Western blotting with an anti-GFP antibody. E, HeLa cells transfected with GFP, GFP-p23, or GFP-⌬p23 were treated with 10 M MG132 for 8 h or left untreated. Cell lysates were analyzed by Western blotting. p53 accumulation was assessed as a positive control for MG132 activity. ␤-Actin was used as the loading control. Next, we tested if ⌬p23 is destabilized and degraded through the ubiquitin-proteasome proteolytic pathway. HeLa cells were treated with tunicamycin for 6 -72 h, and the amount of ⌬p23 was monitored over time. As shown in Fig. 1A, ⌬p23 was first detected in lysates prepared from cells treated with tunicamycin for 12 h, and displayed a time-dependent increase during the treatment period. Decreases in ⌬p23 were not observed for at least 72 h after exposure to tunicamycin. After blocking protein synthesis with cycloheximide, no significant differences in stability were observed between native and ⌬p23 (Fig. 1D). A recent report by Mollerup and Berchtold suggested that ⌬p23 is targeted to the proteasome (21). However, following treatment of HeLa cells expressing green fluorescent protein (GFP), GFPtagged p23 (GFP-p23), or GFP-tagged ⌬p23 (GFP-⌬p23) with the proteasome inhibitor MG132, GFP-p23 and GFP-⌬p23 levels were similar to those of control GFP (Fig. 1E). Thus, it seems unlikely that ⌬p23 is more sensitive to the proteasomal degradation system than its native counterpart, although we cannot exclude the possibility that the GFP tag in our experiments compromised accessibility of ⌬p23 to the proteasome. Because ⌬p23 was not immediately degraded and remained stable in the cytosol, we speculated that it has roles in the regulation of Hsp90 chaperone machinery that are distinct from those of full-length p23. Down-regulation of Telomerase Activity by ⌬p23-It has been suggested that Hsp90 confers stability to client proteins through interactions between the heterocomplex and the client protein, which are further stabilized by p23 (17,23). Disruption of these heterocomplexes by Hsp90 inhibitors, such as GA, promotes proteolytic degradation of client proteins (25)(26)(27). Thus, if p23 loses its co-chaperone activity following caspase cleavage, depletion of client proteins may occur, regardless of Hsp90-binding activity. To examine this possibility, we determined the levels of several client proteins by Western blot analysis using lysates from HeLa cells treated with tunicamycin for 24 h (Fig. 2). Treatment with 10 M tunicamycin for 24 h was sufficient to induce p23 cleavage, whereas z-VAD-fmk completely abolished p23 cleavage, even after exposure to concentrations as high as 60 M. Notably, after tunicamycin treatment, hTERT levels were markedly reduced in association with p23 cleavage (Fig. 2). In addition, a greater degree of p23 cleavage was observed in cells exposed to 60 M tunicamycin, coincident with a depletion of cellular hTERT. The decrease in hTERT was completely prevented by z-VAD-fmk. Because expression of hTERT is tightly linked to the regulation of telomerase function in normal and tumor cells (2), we next examined telomerase activity in tunicamycin-treated HeLa cells using a TRAP assay. As shown in Fig. 3A, telomerase activity was inhibited in tunicamycin-treated cells but was completely restored upon z-VAD-fmk pretreatment. Consistent with previous results, changes in hTERT expression levels paralleled changes in telomerase activity. Previous reports have demonstrated that full-length p23 is an essential component of the Hsp90 chaperone complex and is necessary to establish the active telomerase holoenzyme, both in vitro and in vivo (6). Accordingly, we determined whether ⌬p23 performs a similar telomerase-activity regulatory function. Expression of GFP-p23 in HeLa cells led to enhanced telomerase activity, as expected. In contrast, expression of GFP-⌬p23 significantly reduced telomerase activity and hTERT expression compared with control GFPexpressing cells (Fig. 3B). Because GFP-⌬p23-transfected HeLa cells express endogenous p23 (data not shown), it is possible that GFP-⌬p23 functions as a dominant-negative telomerase inhibitor. The observed dose-dependent GFP-⌬p23-mediated inhibition of telomerase activity and decreased hTERT expression support the specificity of the inhibitory effect (Fig. 3C). Our data collectively indicate that ⌬p23 induces hTERT depletion, and thereby inhibits telomerase activity. Ubiquitination-mediated Degradation of hTERT by ⌬p23-To examine whether ⌬p23 regulates hTERT protein expression through transcriptional regulation of the hTERT gene, we used reverse transcription-PCR analysis to estimate hTERT mRNA levels in GFP-⌬p23-transfected HeLa cells or in cells treated with tunicamycin to induce formation of ⌬p23. Expression of GFP-⌬p23 did not affect hTERT mRNA levels compared with those in cells transfected with GFP only (Fig. 4A), indicating that the effect of ⌬p23 on hTERT levels was not mediated at the transcriptional level. Interest- ingly, tunicamycin treatment induced suppression of hTERT mRNA levels. However, pretreatment with z-VAD-fmk, which prevents tunicamycin-induced reduction in hTERT protein level, did not restore hTERT transcript levels (Figs. 3A and 4A), suggesting that tunicamycin effects on hTERT tran-scription are functionally unrelated to the mechanism under investigation. To extend these observations, we next tested whether ⌬p23 inhibits telomerase activity by destabilizing the hTERT protein. Prior studies have suggested that disruption of Hsp90 chaperone function induces ubiquitination and proteasome-mediated degradation of hTERT (28). To determine whether hTERT is ubiquitinated prior to degradation, we immunoprecipitated lysates from HeLa cells transfected with GFP, GFP-p23, or GFP-⌬p23 using an anti-ubiquitin antibody, and evaluated immunoprecipitates by immunoblot with an anti-hTERT antibody. Overexpression of ⌬p23 markedly increased the level of ubiquitinated hTERT protein (Fig. 4B). To confirm that the observed reduction in hTERT levels was due to proteasome-dependent degradation, we treated GFP-⌬p23-expressing cells with the proteasome inhibitor MG132. As shown in Fig. 4C, treatment with MG132 restored hTERT levels in GFP-⌬p23-expressing cells. Hsp90 Phosphorylation Is Enhanced by p23, but Not by ⌬p23-Hsp90 is constitutively phosphorylated at serine residues, although phosphorylation of threonine and tyrosine residues has also been reported (29 -31). Prior studies have suggested that the pool of phosphorylated Hsp90 plays an important role in the functional regulation of client proteins (32,33). On the basis of this finding, we examined the phosphorylation status of Hsp90 in GFP-⌬p23-expressing HeLa cells. Serine phosphorylation of Hsp90 was significantly decreased in GFP-⌬p23-expressing cells compared with cells expressing GFP or GFP-p23 (Fig. 5A). Neither phosphothreonine nor phosphotyrosine was detected in the same blot (data not shown). Upon immunoprecipitation of lysates with an anti-GFP antibody, serine phosphorylation was evident only in Hsp90 co-immunoprecipitated from GFP-p23-expressing cells (Fig. 5B, bottom). To examine the possibility that ⌬p23 functions as a phosphatase for Hsp90, we incubated recombinant ⌬p23 with phosphorylated Hsp90 purified from HeLa cell , or were transfected with GFP, GFP-p23, or GFP-⌬p23. RNA was isolated, and hTERT mRNA levels were analyzed by reverse transcription-PCR using specific primers (5Ј-TGAACTTGCGGAAGACAGTGG-3Ј and 5Ј-ATGCGTGAAACCTGTACGCCT-3Ј). B, cells were transfected with GFP, GFP-p23, or GFP-⌬p23. After 24 h, cell lysates were immunoprecipitated with an anti-ubiquitin (Ub) antibody. hTERT in immunoprecipitates was analyzed by Western blotting. C, HeLa cells transfected with GFP, GFP-p23, or GFP-⌬p23 were treated with 10 M MG132 for 8 h or left untreated. Cell lysates were analyzed by Western blotting. p53 accumulation was assessed as a positive control for MG132 activity. ␤-Actin was used as the loading control. lysates. However, the phosphoserine status of Hsp90 bound to ⌬p23 was unchanged relative to that of Hsp90 bound to fulllength p23 (Fig. 5C). To investigate whether full-length p23 participates in Hsp90 phosphorylation, we reconstituted the complete chaperone machinery of Hsp90 using a rabbit reticulocyte lysate system. Interestingly, incubation of rabbit reticulocyte lysates with recombinant p23 and ␥-[ 32 P]ATP resulted in significant labeling of Hps90 (Fig. 5D). No radiolabeled band appeared when ␣-[ 32 P]ATP was substituted for ␥-[ 32 P]ATP in the reaction, and treatment with a serine phosphatase inhibitor increased the intensity of the labeled band in the presence of ␥-[ 32 P]ATP, consistent with the identity of the band as phospho-Hsp90 (Fig. 5E). Although the molecular mechanisms underlying Hsp90 phosphorylation and dephosphorylation during the chaperone cycle are not clearly understood, it has been suggested that CKII and protein phosphatase 5 (PP5) are involved in the phosphorylation and dephosphorylation of Hsp90 serine residues, respectively (29,31). To determine whether CKII is involved in p23-induced Hsp90 phosphorylation, we preincubated the reaction mixture with 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole, an inhibitor of CKII. Phos-phorylation of Hsp90 by full-length p23 was completely abolished by 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole, indicating that CKII participates in this process (Fig. 5E). To confirm that p23 stimulates Hsp90 phosphorylation in vivo, we grew HeLa cells in the presence of [ 32 P]orthophosphate and then immunoprecipitated Hsp90 with an anti-Hsp90 antibody. 32 P-Labeled Hsp90 was observed in cells transfected with GFP-p23 but not in those transfected with GFP-⌬p23 (Fig. 5F). Regulation of Telomerase Activity via Hsp90 Phosphorylation-To clarify the mechanism of p23-mediated Hsp90 phosphorylation, we examined the interaction between Hsp90 and CKII in cells expressing GFP-p23. CKII is comprised of ␣ and ␤ subunits, of which the ␣ subunit directly binds to Hsp90 (34). Co-immunoprecipitation analyses revealed that the interaction between Hsp90 and CKII␣ was enhanced in GFP-p23-expressing cells (Fig. 6A). Because Hsp90 binds to and enhances CKII kinase activity (34), we conclude that p23 accelerates Hsp90 phosphorylation by promoting its association with CKII. Next, we tested whether phosphorylation of Hsp90 directly affected telomerase activity. Two Hsp90 serine residues, Ser-231 and Ser-263, have been reported to be phosphorylation targets (29). Constructs encoding phosphorylation-defective (S231A/ S263A) and phospho-mimetic (S231E/S263E) Hsp90 mutant proteins were prepared and introduced into HeLa cells. The double mutant S231A/S263A displayed decreased Hsp90 serine phosphorylation, indicating that these residues are authentic targets for phosphorylation (Fig. 6B). Importantly, the S231A/S263A mutant did not enhance telomerase activity; in contrast, the S231E/S263E phospho-mimetic mutant enhanced telomerase activity to an extent similar to that of wild-type Hsp90 (Fig. 8C). We next examined the mechanism of ⌬p23-induced regulation of Hsp90 phosphorylation. We initially assumed that ⌬p23 did not affect the interaction between Hsp90 and CKII␣ but found instead that ⌬p23 increased Hsp90 binding to CKII␣ (Fig. 6A). PP5 is a known component of the Hsp90 chaperone complex (35). The yeast ortholog of PP5, Ppt1, has been identified as a serine phosphatase of yeast Hsp90 (31). Accordingly, we tested whether ⌬p23 affected the interaction between Hsp90 and PP5. As shown in Fig. 7A, overexpression of GFP-⌬p23 effectively increased the binding of PP5 to Hsp90. Likewise, co-immunoprecipitation experiments using purified recombinant proteins confirmed that PP5 bound more tightly to Hsp90 in the presence of ⌬p23 than in the presence of p23 (Fig. 7B). To determine whether Hsp90 dephosphorylation influences telomerase activity, we assessed the effects of PP5 knockdown using small interfering RNAs. As expected, PP5small interfering RNA decreased PP5 protein levels in HeLa cells and markedly increased telomerase activity (Fig. 7C). Small interfering RNAmediated depletion of p23 protein led to a decrease in telomerase activity, consistent with the reported role of p23. These results indicate that PP5 is a negative regulator of telomerase. Accordingly, we conclude that ⌬p23 inhibits telomerase activity by promoting the interaction between PP5 and Hsp90 and further propose that fulllength p23 induces serine phosphorylation of Hsp90, which is essential for enhancing telomerase activity. In contrast, ⌬p23 fails to enhance phosphorylation of Hsp90, resulting in destruction of the maturing hTERT peptide. Effects of ⌬p23 on Cell Growth and Death-To address the physiological relevance of p23-mediated regulation of telomerase activity, we generated 293 cell lines stably expressing GFP, GFP-p23, or GFP-⌬p23. Isolated clones were assayed for GFP, GFP-p23, or GFP-⌬p23 expression by immunoblot and immunofluorescence analyses (Fig. 8A, middle panel, and data not shown). Telomerase activity and hTERT expression in stably transfected 293 cells were regulated in a manner similar to that in HeLa cells transiently expressing these proteins (Figs. 3B and 8A). Telomerase is essential for the maintenance of genomic integrity in rapidly growing cells, and inactivation of telomerase through depletion of human telomerase RNA rapidly inhibits the growth of human cancer cells expressing hTERT (36). Therefore, we reasoned that suppression of hTERT by ⌬p23 could block proper progression of cell growth. Consistent with this idea, we observed that the rate of growth in the GFP-⌬p23 cell line was retarded compared with that in control and GFP-p23 cell lines (Fig. 8B). A recent study showed that depletion of hTERT facilitates the induction of apoptotic cell death by genotoxic agents, particularly cisplatin (37). We thus evaluated whether overexpression of ⌬p23 influenced the chemosensitivity of cells to cisplatin. Analysis of apoptosis using annexin-V-phycoerythrin staining revealed a substantial increase in apoptotic cells in the cisplatin-treated GFP-⌬p23 cell line compared with cisplatin-treated control and GFP-p23 cells (Fig. 8C). Inhibition of cisplatin-induced apoptosis in the GFP-p23 cell line is consistent with a previous model suggesting FIGURE 6. Regulation of telomerase activity via Hsp90 phosphorylation. A, HeLa cells were transfected with GFP, GFP-p23, or GFP-⌬p23. After 24 h, cell lysates were immunoprecipitated with an anti-Hsp90 antibody, and immunoprecipitates were analyzed by Western blotting with anti-Hsp90 and anti-CKII␣ antibodies. B, HeLa cells were transfected with empty vector, Hsp90, or His-tagged Hsp90 mutant (S231A/S263A). After 24 h, lysates were immunoprecipitated with an anti-pS antibody, and immunoprecipitates were analyzed by Western blotting. C, HeLa cells were transfected with empty vector, Hsp90, or His-tagged Hsp90 mutants (S231A/ S263A or S231E/S263E). After 24 h, cell lysates were assayed for telomerase activity by TRAP analysis. FIGURE 7. Truncated p23 decreases telomerase activity by enhancing the interaction of Hsp90 with PP5. A, HeLa cells were transfected with GFP, GFP-p23, or GFP-⌬p23. After 24 h, lysates were immunoprecipitated with an anti-Hsp90 antibody, and immunoprecipitates were analyzed by Western blotting with anti-Hsp90 and anti-PP5 antibodies. B, recombinant GSTtagged PP5 (2 g, Novus Biologicals, Littleton, CO) was incubated with purified Hsp90 (2 g) in the presence of 2 g of p23 or ⌬p23. Reactions were immunoprecipitated with glutathione-agarose, and immunoprecipitates were analyzed by Western blotting. C, small interfering RNAs targeting p23 or PP5 (Ambion, Austin, TX) were introduced into HeLa cells. After 48 h, cell lysates were examined by Western blotting and TRAP analysis. that hTERT is a novel endogenous inhibitor of apoptosis (37). Restoring hTERT expression in the GFP-⌬p23 cell line by transfection with a hemagglutinin-tagged hTERT gene decreased cisplatin-induced apoptosis, confirming that sensitization of the GFP-⌬p23 cell line to cisplatin resulted from suppression of hTERT expression (Fig. 8D). DISCUSSION Telomerase activity is tightly regulated in cells, and hTERT is a key molecule in this process (2). Although hTERT protein levels are primarily controlled by transcriptional regulation in normal human cells, recent reports suggest the additional involvement of ubiquitination and proteasome-mediated degradation due to Hsp90 malfunction (28). The role of Hsp90 and its co-chaperone p23 in mediating the functional maturation of hTERT is well documented (6), but the precise mechanisms are not fully understood. Here, we propose that p23 regulates Hsp90 phosphorylation, a process that is modulated by caspasedependent cleavage of p23. By disrupting Hsp90 phosphorylation and function, truncated p23 induces proteasomal degradation of hTERT and down-regulates telomerase activity. The findings presented here provide support for the idea that Hsp90 co-chaperones regulate the post-translational modification of Hsp90 and help to determine the mechanisms underlying the regulation of telomerase activity during cell death. As part of this mechanism, p23 enhances CKII binding to Hsp90. We found that partial proteolysis of p23 by active caspase did not compromise CKII binding but did promote the binding of PP5 to Hsp90 (Fig. 9). ⌬p23 apparently binds Hsp90 by displacing pre-bound p23, consistent with our preliminary data showing that ⌬p23 has a greater affinity for Hsp90 than does full-length p23 (data not shown). It is currently unclear if cleavage of p23 increases PP5 recruitment into the Hsp90 chaperone complex, or if it blocks release of PP5 from the complex. Because PP5 is an essential component of the Hsp90 chaperone complex (35), the latter mechanism is more likely. However, further investigation is necessary to clarify this issue. Hsp90 is a phosphoprotein that is primarily phosphorylated at serine residues but can be phosphorylated to a lesser extent on tyrosine and threonine residues (29 -31). Earlier studies reported that a number of kinases are capable of phosphorylating Hsp90, including CKII, double-stranded DNA-activated kinase, and Akt (29,38,39). However, proteins that regulate Hsp90 phosphorylation by these kinases have yet to be identified. In a cell-free reticulocyte lysate system, we found that p23 alone had no effect on Hsp90 phosphorylation status, but was able to increase phosphorylation of Hsp90, an effect that was blocked by a CKII inhibitor (Fig. 5, C and E). In addition, expression of p23 in intact cells enhanced the interaction between Hsp90 and CKII. Collectively, these data indicate that full- length p23 stimulates CKII-mediated phosphorylation of Hsp90. Interestingly, overexpression of ⌬p23 also enhanced binding of CKII to Hsp90. Because CKII is a client protein of Hsp90 (34), it is possible that the interaction between CKII and Hsp90 in the presence of ⌬p23 is not an enzyme-substrate interaction, but rather a client-chaperone interaction, thus, enhanced binding of CKII to Hsp90 by ⌬p23 overexpression might not increase Hsp90 phosphorylation. To our knowledge, CKII is the only kinase capable of phosphorylating Hsp90 at conserved serine residues located in the charged region. Although this region is apparently dispensable in Escherichia coli and yeast, it has been suggested that phosphorylation in this region influences Hsp90 chaperone function by modulating the interactions between Hsp90 and client proteins (40 -42). Here, we present evidence that phosphorylation of the charged region regulates the interaction between Hsp90 and the client protein, hTERT. Because the charged region of yeast Hsp90 does not contain any conserved serine residues that can be phosphorylated by CKII, we would not expect p23mediated Hsp90 phosphorylation to be replicated in the yeast system. The length of this charged region is increased from six amino acids in E. coli to 67 in human Hsp90, suggesting that this region and its phosphorylation sites have been evolutionary targets for gain of function in higher organisms. To date, more than a dozen distinct Hsp90 co-chaperones have been identified (43). Some of these co-chaperones facilitate activation of a specific set of substrate proteins. In this context, p23 participates in the maturation of steroid hormone receptors and hTERT. Interestingly, most client proteins are released from Hsp90 after completion of protein folding, although p23 and Hsp90 remain associated with mature hTERT (44). Our results clearly demonstrate that ⌬p23 induces proteasomal degradation of hTERT protein by blocking Hsp90 phosphorylation. Although it is possible that p23-regulated Hsp90 phosphorylation only affects the stability of hTERT, we cannot exclude the possibility that the levels of other client proteins, such as steroid hormone receptors, are also regulated by p23 cleavage. Further experiments are required to investigate this possibility. Research from several laboratories confirms that p23 is a substrate of active caspases (20 -22). The mechanisms by which caspase-cleaved p23 acts in dying cells are unclear, but two hypotheses have been proposed. One possibility is that the loss of p23 function, independent of its role as an Hsp90 co-chaperone, is the central factor. In this scenario, the resulting increase in ⌬p23 reduces anti-aggregating activity, and the depletion of p23 sensitizes cells to endoplasmic reticulum stress; thus, the loss of p23 function potentially plays a protective role against endoplasmic reticulum stress, passive chaperone activity, and cytosolic prostaglandin E2 synthetase activity (8,22,45). The second possibility involves the loss of p23 co-chaperone functions, dependent on Hsp90 interactions. Several reports demonstrate that hTERT requires the co-chaperone function of p23 as well as Hsp90 for full activity (6,46); Hsp90 promotes the proper folding of hTERT and subsequent binding to the telomeric primer, whereas p23 is involved in primer dissociation from telomerase. The p23-regulated serine phosphorylation of Hsp90 is essential for telomerase activity, and p23 cleavage leads to down-regulation of telomerase activity, as demonstrated here. In addition, telomerase activity is a prerequisite for cancer cell survival, and hTERT plays a protective role against certain apoptosis-inducing stimuli. Consistent with the role of full-length p23 in this process, we found that overexpression of ⌬p23 inhibited the growth of transformed cells and increased their sensitivity to anticancer drugs. Thus, reducing telomerase activity by enhancing p23 proteolysis may be an attractive strategy for cancer treatment. Further research is required to generalize the effects of p23 cleavage during cell death. In view of the protective roles of p23 in stress environments, it is reasonable to assume that the cleaved fragment of p23 in apoptotic cells undergoes rapid degradation. However, our results show that the p23 proteolytic fragment remained stable during cell death. It also displayed a binding activity to Hsp90 similar to that of full-length p23. Thus, the cleaved form of p23 is not merely apoptotic debris that has to be eliminated, but may be a functional molecule that turns off telomerase activity, FIGURE 9. Model for down-regulation of telomerase activity by p23 cleavage. Interaction of p23 and Hsp90 increases serine phosphorylation at the linker region of Hsp90. Active caspases cleave p23 after Asp-142, generating truncated p23 (⌬p23). ⌬p23 binds to Hsp90 instead of p23 and then promotes PP5-mediated dephosphorylation of Hsp90. hTERT levels decrease due to ubiquitination-dependent degradation. and is required for apoptotic progression. In a recent report, Dix and colleagues showed that more than one-third of proteolytic fragments of newly characterized caspase substrate proteins are stable for at least 4 h (47). Additional research is required to determine the general significance of this finding and to identify additional substrates regulated by caspase-mediated proteolysis. During the chaperone cycle, Hsp90 undergoes continuous phosphorylation and dephosphorylation (32). Evidently, dephosphorylation of Hsp90 is also important for the maintenance of chaperone activity. Another recent report showed that the serine/threonine phosphatase PP5/Ppt1 directly dephosphorylates Hsp90 and modulates the maturation of Hsp90 client proteins (31). ⌬p23 strengthened the interaction between Hsp90 and PP5, suggesting that the truncated protein deregulates the Hsp90 phosphorylation cycle (Fig. 7, A and B). Although PP5 specifically dephosphorylates CKII-phosphorylated Hsp90 in vitro (31), we found no evidence to suggest that PP5 directly dephosphorylates Hsp90 at CKII-phosphorylated serine residues in intact cells. Further studies are needed to confirm that PP5 bound to ⌬p23-containing Hsp90 complexes directly dephosphorylates serine residues located in the charged region of Hsp90. PP5 interacts with Hsp90 via the tetratricopeptide repeat domain (35). The tetratricopeptide repeat domain-binding region of Hsp90, located at the C terminus, is the binding site for several co-chaperones. Recent structural data show that the acidic C-terminal tail of p23 becomes well ordered upon binding to Hsp90. The mechanism by which ⌬p23 enhances the interaction of Hsp90 with PP5 is currently unclear. However, we speculate that the structure of the tetratricopeptide repeat acceptor site of Hsp90 is altered depending on the presence or absence of the p23 C-terminal tail.	2018-04-03T04:50:45.697Z	2009-09-09T00:00:00.000	{ "year": 2009, "sha1": "21bcb005662295d32abdf7f29edf16f6e688ea92", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/284/45/30871.full.pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "a3442357829a8bc9ac7f6be0a4d44d90aa812582", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
88511695	pes2o/s2orc	v3-fos-license	On Intrinsic Geometric Stability of Controller This work explores the role of the intrinsic fluctuations in finite parameter controller configurations characterizing an ensemble of arbitrary irregular filter circuits. Our analysis illustrates that the parametric intrinsic geometric description exhibits a set of exact pair correction functions and global correlation volume with and without the variation of the mismatch factor. The present consideration shows that the canonical fluctuations can precisely be depicted without any approximation. The intrinsic geometric notion offers a clear picture of the fluctuating controllers, which as the limit of the ensemble averaging reduce to the specified controller. For the constant mismatch factor controllers, the Gaussian fluctuations over equilibrium basis accomplish a well-defined, non-degenerate, flat regular intrinsic Riemannian surface. An explicit computation further demonstrates that the underlying power correlations involve ordinary summations, even if we consider the variable mismatch factor controllers. Our intrinsic geometric framework describes a definite character to the canonical power fluctuations of the controllers and constitutes a stable design strategy for the parameters. Introduction Stable design of controllers is one of the most interesting research issue since the proposition of the robust controllers. Such a principle has been extremely successfully which applies in both the domestic and industrial application [1]. This follows because of the fact that it is easy in implementation and requires less number of parameter tuning. Further, the stably designed controllers are the best alternative to the existing controllers. This follows from the fact that the intrinsic geometric design takes an account of the model uncertainties and specifically allows for a clear-cut determination of the controller settings and parameters. Such an observation is supported from the fact that a class of controllers remains insensitive to the time delay and parametric deviation for the first order systems [1]. Global Stability phenomenon are the subject matter of [2]. Also, the parametric approach robust controllers have been brought out in the picture with the notions of [3]. An important filter design taking an account of the load disturbance is proposed [4] in order to improve the speed of the tuning response function. The stability of such a class of controllers depends only on the domain of the controller parameters and so the construction of a nominal plant. In addition, although the system can reached control input saturation, but the stability of the designed controller can be maintain to only depend on the parameters of the intrinsically designed controller and that of the plant, as per the outlines of the Ref. [5]. Similar direct model reference adaptive controllers [6,7,8] are explored in diverse applications. Parametric model controller design is one of the best methods to improve the robust recital of the controller because of the fact that a polynomial approach is involved in the performance improvement of the controller. The parameters concerning the speed of the controller depend on the model parameters and mismatch factor of the low pass filter circuit. This is because the robustness of the controllers and their performance depend on the model parameters {a, b} and the mismatch factor f of the filter. In the non-linear domain of the above parameters, our intrinsic geometric analysis provides a stable characterization of the controllers. From the viewpoint of the present interest, Ref. [9] leads us to provide an appropriate mathematical design and parameterization for the controllers. The parameterized equation controller block diagram is further brought out into the present attention. From the out-set of the above reference [9], we can track the desired trajectory and minimize the plant error. The key issues concerning the controllers are the speed response, low pass filter and geometric model. The robust performance of such a controller is based on these parameters [10,11]. A novel approach is thus made possible in the history of controllers via the present investigation. The method as outlined above is very general in its own and it leaves different possible versions to be explored further. One of the key issues is an appropriate design of the controller circuits. To design the parametric internal model, one traditionally assumes that one of the parameters of the controller is in the subset of parametric family of the controller. It is sometime called finite dimensional parametric model. The filtering operations in these controllers are associated with the parameter involved. The distribution of parameters in the parametric model can be taken to be finite dimensional. Furthermore, the model reference parameterizations are taken into an account [12] and the associated parameterizable methods are used to obtain the parametric controllers. Based on the conventional design method, we offer intrinsic stability analysis of the controllers. The linear parametric polynomial approach which improves structures of the bounded parametric controller integralderivative (id) designs, is explicitly presented. Improvements in the limiting linear parametric controllers are shown. It turns out that the present intrinsic geometric notion is particularly well suited for the practical applications. The mathematical design procedure thus taken can be extended for any controllers. In fact, we have a clear picture of further investigation. This has been a real bestow for stabilization of the conventional controllers. The present analysis of controller can further be used to explore the intrinsic nature of the unmodeled part of the plant and the associated bounded disturbances arising from the fluctuations of parameters and mismatch factor. Intrinsic geometric modelings involving equilibrium configurations of the extremal and the non-extremal black holes in string theory [13,14,15,16,17,18,19,20] and M -theory [21,22,23,24] possess rich intrinsic geometric structures [25,26,27,28,29]. There has been much well focused attention on the equilibrium perspective of black holes, and thereby explicates the nature of concerned parametric pair correlations and associated stability of the solutions containing a large number of branes and antibranes. Besides several general notions which have earlier been analyzed in the condensed matter physics [30,31,32,33,34,35], we consider specific controller configurations thus mentioned with equilibrium parameters and analyze possible parametric pair correlation functions and their correlation relations. Basically, the investigation entails an intriguing feature of the underlying fluctuations which are defined in terms of the parameters. Given a definite covariant intrinsic geometric description of a consistent controller configuration, one can expose (i) for what conditions the considered configuration is stable?, (ii) how its parametric correlation functions scale in terms of a set of chosen fluctuating circuit parameters? In this process, one can enlist a complete set of non-trivial parametric correlation functions of the controller configurations [9]. It may further be envisaged that similar considerations remain valid over the black hole solutions in general relativity [36,37,38,39], attractor black holes [40,41,42,43,44,45] and Legendre transformed finite parameter chemical configurations [46,47], quantum field theory and the associated Hot QCD backgrounds [48]. Thus, the differential geometry plays an important role in the thermodynamic study of the controllers. In this paper, we analyze the stability of the controllers under the fluctuation of the parameters and the mismatch factor. The controller under consideration is depicted in the Fig.[1]. The stability is demonstrated for a suitable design and its parameterizations. From the general parametrization equation of the controller, the stability of block diagram is drawn into attention. To the best of authors' knowledge, this approach is made possible for the first time towards the intrinsic geometric stability analysis of controllers. The proposed method is very general and different Legendre transformed versions of the present analysis are possible. By employing the standard notion of the intrinsic Riemannian geometry, the rest of the sections are devoted to the local and global stability properties of fluctuating controllers. Fluctuations in the Controllers The controller circuit of the present interest is depicted in the Fig.[1]. Although the analysis of the present investigation remains for any controllers, nevertheless we illustrate it for a class of controllers, which are of an immediate interest as shown in the Fig.[1]. Here, s is a complex signal having a modulus and an angle of phase. In the subsequent analysis, we denote a locally constant signal by the corresponding uppercase notation S. Furthermore, we show that the investigation of the stability analysis is valid for the general controllers, and demonstrate that our approach remains consistent with the other existing ones. Given the controller, we consider the intrinsic geometric stability of the underlying low pass filter system. The parametric stability criterion offers a proficient method to determine the parameters of the circuit and thereby to design the controller as per ones requirement. Ref [49] implements the principle for as associated class of controllers. From a close perspective, such an analysis of the controllers takes an account of the model uncertainties. Specifically, it allows a straightforward relation of controller settings with the associated model parameters. Notice further that the first order consideration of the controllers is insensitive to time delay and parameter deviation, and the output is approximately equal to the PI controllers [1]. The response of the controller is sluggish, although it does not have important overshoot effects [50] and the integral action of such a controller is used to eliminate the offset of the system. To explore the stability of the first order controllers [51], we design stable controllers from the perspective of the intrinsic geometry. Having mentioned the domains of the parameters, we consider the following two specification of the controllers, viz., constant mismatch factor controllers, and variable mismatch factor controllers. For arbitrary n th order low pass filter, the controllers of the intrinsic interest are Notice further that the aim of the present paper is to expose the power of the intrinsic geometry. In this concern, the controller described by the Eqn.(1) serves only as an example of the present consideration. Subsequently, our analysis as the exposition of the intrinsic geometric investigation remains valid for any smooth controller and thus the above class of the controllers. In order to begin the subsequent intrinsic geometric analysis of fluctuations, we introduce the correlation in the controller arising from the fluctuations of the circuits parameters. Thus, we consider an ensemble of controllers fluctuating over the limiting Gaussian ensemble. In this analysis, we consider that the controllers can have non-zero fluctuations due to the vibrations of frequencies, residual ripple factors in the filter circuits, and possible other practical uses. This follows from the fact that we do not restrict ourselves in the specific domains of filter circuit used in the controller. Consequently, we allow an ensemble of limiting configurations with finite fluctuations in an arbitrary non linear domain of the parameters and thereby analyze the nature of a class of generic controllers. We also take an account of the variable mismatch factor defining the speed response tuning parameter of the controller. Notice further that the analysis of the present exposition is valid for all range of the parameters of the controllers. Physically, their deviation from the origin signifies a contribution of the non-linear effects of the controller. Specifically, the values a = 0 and b = 0 of the parameter signify a purely linear model controller. Subsequently, Figure 1: A class of controllers as the function of parameters a and b, describing fluctuations of the controller with a given mismatch factor f . the stability of the controller can be analyzed in the non large frequency domains. This is because the present analysis is devoted for the controllers which are defined as the n th -order low pass filter circuit. Such a notion is observed, when there are ripples in the filter circuits of the controller. Constant Mismatch Controllers Let us first describe the intrinsic stability of the controller with a given mismatch factor. The correlations are described by the Hessian matrix of the controller, defined with a set of desired corrections over a chosen model mismatch factor f under the tuning response function. Following Eqn.(1), the components of the metric tensor defined as Hess(G con (a, b)) reduce to the following expressions: In this framework, we observe that the geometric nature of parametric pair correlations divulges the notion of fluctuating controllers. Thus, the fluctuating controllers may be easily determined in terms of the intrinsic parameters of the underlying circuit configurations. Moreover, it is evident for a given controller that the principle components of the metric tensor signify self pair correlations, which are positive definite functions over a range of the parameters. Physically, this signifies a set of heat capacities against the intrinsic interactions on the configuration (M 2 (R), g) of the controller. It is worth mentioning that the controllers turn out to be well-behaved for the generic values of the parameters. Over the domain of the circuit parameters {a, b}, we notice that the Gaussian correlations form stable correlations, if the determinant of the metric tensor remains a positive function on the parametric surface (M 2 (R), g). What follows further is that we specialize ourselves for the physical values of the parameters, and subsequently, we analyze the stability for a = 0, b = 0 corresponding to the linear controllers Fig.[2]. Under such a limiting specification of the parameters, the local correlation functions reduce to the set of following expressions: For a = 0, b = 0, the determinants of the metric tensor reduce to the following expression: The behavior of the determinant of the metric tensor shows that such controllers become unstable for specific values of the mismatch factor. For S = 1 and n = 1, the nature of the determinant of the metric tensor is depicted in the Fig.[3]. It is worth mentioning further that the constant mismatch controllers become highly unstable in the limit of vanishing mismatch factor. Specifically, the system acquires a throat in the regime of \|f \| ≤ 0.7. In order to explain the nature of transformation of the parameters {a, b} forming an intrinsic surface, we examine the functional behavior of the associated Christoffel connections. A direct computation yields that the limiting non-trivial Christoffel connections reduce to the following expressions: (1 + f S) 2n + 6(1 + f S) n + 1 The present investigation shows that a typical controller is globally un-correlated over all possible Gaussian fluctuations of the parameters {a, b}. As the matter of fact, the correlation length of underlying nearly equilibrium system is global characterized by the scalar curvature of (M 2 , g). This follows from the fact that the scalar curvature, arising from the definition of the Gaussian fluctuations over the parameters {a, b}, vanishes identically for the constant mismatch factor controllers. For the above type of controllers with a chosen mismatch factor, we see that the Riemann Christoffel curvature tensor vanishes identically over the entire {a, b} surface. Thus, the present intrinsic geometric analysis anticipate that a constant mismatch controller is always a non-interacting system over the surface of fluctuating parameters {a, b}. Fluctuating Mismatch Controllers In the present section, we explore the nature of an ensemble of generic controllers generated with a variable mismatch factor. To consider the most general case, we chose the controller as the function of mismatch factor along with the other system parameters. When the mismatch factor is allowed to fluctuate, we exploit the definition of the Hessian function Hess (G con (a, b, f )). Following Eqn.(1), we see for the variable mismatch factor controllers have a set of interesting properties. It follows that the pure pair correlations {g aa , g ab , g bb } between the parameters {a, b} remain the same as depicted in the Eqn.(2) for the constant mismatch factor controllers. The remaining parametric pair correlations, involving the variation of the mismatch factor f , are given by the following set of equations: We see that the fluctuations of the mismatch factor controller comply physically expected conclusions. In particular, the heat capacities, defined as the self-pair correlations, remain positive quantities for well-defined controllers. A straightforward computation further demonstrate the overall nature of the parametric fluctuations. A variable mismatch factor controller is stable under the set of Gaussian fluctuations, if the associated principle minors {p 2 , p 3 } remain positive functions on the manifold (M 3 , g). Subsequently, an explicit computation shows that the stability constraint on the ab-surface is given by where the polynomials m i (S) are given as The stability constraint on the entire configuration is determined by the determinant of the metric tensor Notice that the co-ordinate charts on (M 3 , g) are described by the parameters {a, b} and mismatch factor f of the controller. In Enq (10) take a negative value over (M 3 , g). In Eqn. (11), it turns out that {h k (S)} reduce to the following polynomials: It is not difficult to compute an exact expression for the scalar curvature describing the global parametric intrinsic correlations. By defining set of controller functions, we find explicitly that the most general scalar curvature can be presented as (13) where the co-efficients {r i (a, b)} appearing in the numerator can be written as the following polynomials The weights {w i } occurring in the summation of the numerator of Eqn. (13) are given by the sequence wi := {−6(n − 1), (9n − 1), (10n + 16), (8n − 18), (16n + 18), (n + 1)} (15) Furthermore, it turns out that D(a, b) appearing in the denominator of the scalar curvature, Eqn. (13), is expressed as the following function Consequently, we may easily expose the associated important conclusions for the specific considerations of the variable mismatch factor controllers. The global nature of phase transitions can be thus explored over the range of parameters describing the controllers of interest. For the limiting linear controllers corresponding to the values a = 0, b = 0, the limiting intrinsic scalar curvature simplifies to the following ratio of series R = 5 k=0 t k (1 + f S) kn ((n + 1)(1 + f S) 2n + 2n(1 + f S) n + n − 1) 2 (17) where t i (n) are defined by the following sequence Eqn. (17) shows that the global interactions exist for the limiting linear values of the variable mismatch factor controllers. This follows from the fact that the coefficients of the scalar curvature Eqn. (17), signifying the global correlation volume of controller, remain non-zero in the linear limit. In the limit of n = 1, the Eqn. (17) shows further that the scalar curvature diverges on the f S-surface of the root of (2 + f S) 2 . The graphical views of the curvature scalar of the variable mismatch factor controllers are depicted in the two and three dimensions. The intrinsic characterization offered in (i) Fig.[4] is over the three dimensions and (ii) Fig.[7] is over the two dimensions. This describes the precise global behavior of the parametric fluctuations over the entire intrinsic manifold (M 3 , g) for the limiting linear variable mismatch controllers Fig.[2]. For the limiting linear controllers, it is worth mentioning that the local pair correlations, as the components of the metric tensor, have an expected behavior. The pure pair correlations reduce as the Eqn.(4). The others involving a variation of the mismatch factor reduce to the following equations: In the case of the limiting linear controllers, we observe further that the determinant of the metric tensor reduces to the following expression: It is important to notice that the global stability of the controllers may be determined by observing the sign of the determinant of the metric tensor. For n = 1, we find that the determinant of the metric tensor reduces to the following expression: The behavior of the determinant of the metric tensor shows that the variable mismatch factor controller becomes unstable for the specific values \|f \| ≤ 1.2 of the associated mismatch factor. For the general value the S, the nature of the determinant of the metric tensor is depicted in the Fig.[5]. For S = 1 and n = 1, the corresponding surface nature of the determinant of the metric tensor is depicted in the Fig.[6]. In contrast to the constant mismatch factor controllers, we notice in the present section that the determinant of the metric tensor reduces to the cusp form in the regime of f → 0. In this domain of the mismatch factor, we observe that the variable mismatch factor controllers are relatively less stable than the constant mismatch factor controllers. The corresponding surface behavior of the scalar curvature is depicted in the Fig.[7]. This describes the global phase properties of the variable mismatch factor controllers. It is shown that the non-zero value of intrinsic scalar curvature further demonstrate the existence of a finite correlation volume. Specifically, the phase stability of typical controllers with a variable mismatch factor can thus be easily determined by analyzing the nature of the scalar curvature in the domain of interest. This has been depicted in the Figs. [4,7], in which we show the global nature of the parametric correlations. The further observation of the Fig.[4] shows that the variable mismatch factor controller systems have no phase transitions on the parametric manifold (M 3 , g). Subsequently, the global nature of variable mismatch factor controllers is well explicable against the local fluctuations of the model parameters {a, b} and mismatch factor f of the controller. Conclusion The intrinsic geometric design of controllers is offered under the fluctuations of the model parameters and mismatch factor. Such fluctuations are expected to arise due to non-zero heating effects, chemical reactions and possible conventional corruptions associated with the controller under the application. The intrinsic geometric method is used to improve the structure of the bounded parametric controller id thus designed. It is pictorially presented for the limiting polynomial approach corresponding to the limiting linear parametrization. The stability analysis thus introduced is most generic for the fluctuations of the parameter and the mismatch factor the controllers. The present analysis is well suited for practical applications. The intrinsic geometric design procedure is presented for the controllers with a (i) constant and (ii) variable mismatch factor. In this concern, the Fig. (3) and Fig.(6) show the respective determinants of the metric tensor for the constant and variable mismatch factor controllers. These figures illustrate that the typical instability appears as (i) a throat for the constant mismatch factor controllers and (ii) a cusp for the variable mismatch factor controllers. Subsequently, a straightforward comparison may be made between the stability properties of the constant and the variable mismatch factor controllers. In the first case, it turns out that the associated controllers correspond to a non-interacting system, while in the second case such a controller configuration corresponds to an interacting system. This follows from the fact that the manifold of parameters is flat in the first case, while it becomes curved for the variable mismatch factor controllers. In the limit of f → 0, we have shown in the first case that the determinant of the metric tensor acquires a throat, whereas the determinant of the metric tensor of the variable mismatch factor controllers acquires a cusp in this limit. Thus, the present investigation predicts that the controller systems with the constant mismatch factor are relatively more stable and better-behaved than those with the variable mismatch factor. In addition, our model is well suited for the robust controllers. Such controllers are very popular now a days, because of their high performance and low maintenance needs. From the commercial viewpoints, such a robust controller is very lucrative. It is worth mentioning that the use of the intrinsic geometric principle is rapidly growing in robust controller design in recent years. Based on the definition of the controller, the intrinsic stability analysis remains compatible for parametrically stable designs of the controller and their modern appliances. The present analysis thus provides the intrinsic geometric front to the stability analysis of existing controllers and their possible future generations. It may be also used, in order to model in a suitable fashion the un-modeled part of the plant and the bounded disturbances. Finally, it is envisaged that our analysis offers perspective stability grounds, when applied to the electrical plants. It is expected further that the present investigation would be an important factor in an appropriate design of the safety guards, which can work as the indicators under fluctuations of the parameters, mismatch factor and the other possible components.	2011-04-14T14:59:58.000Z	2011-04-14T00:00:00.000	{ "year": 2011, "sha1": "d2532a0c95a3e72d14a7ee629c07df23adcc0602", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "d2532a0c95a3e72d14a7ee629c07df23adcc0602", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics", "Physics" ] }
256911814	pes2o/s2orc	v3-fos-license	Long-term cilostazol treatment reduces gliovascular damage and memory impairment in a mouse model of chronic cerebral hypoperfusion Chronic cerebral hypoperfusion is a major cause of age-related vascular cognitive impairment. A well-characterised mouse model has shown that hypoperfusion results in gliovascular and white matter damage and impaired spatial working memory. In this study, we assessed whether cilostazol, a phosphodiesterase III inhibitor, could protect against these changes. Adult, male C57Bl/6J mice were subjected to bilateral common carotid artery stenosis or a sham operation and fed normal or cilostazol diet for three months. Cilostazol treatment reduced the impairment in working memory and white matter function after hypoperfusion. Endothelial adhesion molecules and gliosis, increased after hypoperfusion, were ameliorated with cilostazol treatment. Interestingly, the improvement in working memory was closely correlated with reduced microglia and endothelial adhesion molecules. Further, the number of stroke lesions after hypoperfusion was reduced in the cilostazol-treated group. Altogether cilostazol showed potential to ameliorate the gliovascular damage and working memory impairments after hypoperfusion possibly via endothelial protection supporting its potential use in the treatment of vascular cognitive impairment. To date there remains no effective treatments for VCI and instead current therapies are targeted to ameliorate the cognitive impairments via anti-cholinesterase approaches. Current drug developments are focussed on strategies to improve vascular health and the gliovascular unit 6,7 . Since multiple mechanisms may be involved in VCI, drugs that have pleiotropic effects may be particularly useful. One such drug, cilostazol, a phosphodiesterase III inhibitor, is known to have neuro-glio-vascular protective effects through multiple mechanisms. Cilostazol is approved as an antiplatelet agent for the prevention and treatment of cerebral infarction and peripheral arterial disease. Cilostazol increases cAMP in vascular cells and can exert multiple beneficial effects on the vasculature such as endothelial protection 8,9 , maintenance of microvascular integrity 10 , vasodilation, anti-oxidation, anti-inflammation, regulation of smooth muscle cells 11 and increase in cerebral haemodynamics 12 . There is now emerging evidence to suggest cilostazol may have beneficial effects in models of VCI and protect against white matter damage and cognitive impairment. However, to date the majority of these studies have focussed on the short-term effects of cilostazol over weeks [13][14][15][16] . The present study assessed whether cilostazol could protect long-term against the degenerative changes within the gliovascular unit and cognitive impairment induced by chronic cerebral hypoperfusion and whether it could exert beneficial effects on microvascular inflammation. Results Cilostazol had modest effects on spatial working memory after hypoperfusion. Spatial working memory has been shown to be selectively impaired in response to hypoperfusion 3,17 . Similarly, the hypoperfused-control mice showed a clear impairment in this task and committed more revisiting errors (Fig. 1a) and performed less novel entries (Fig. 1a) than the sham mice throughout the test. Noteworthy, the hypoperfused-cilostazol mice showed a learning process similar to shams up to block trial 4 (day 12 of test) before they plateaued (Fig. 1a) suggesting that the spatial memory impairment, whilst reduced, was not completely ameliorated with cilostazol treatment. Statistical analysis of the number of revisiting errors and novel entries indicated an overall Figure 1. Cilostazol improved spatial working memory and white matter function: (a) Spatial working memory was assessed using an 8-arm radial arm maze. Statistical analysis of the number of revisiting errors and novel entries indicated an overall significant difference between the 3 groups (F (2, 34) = 5.169; p = 0.029 and F (2, 34) = 5.718; p = 0.022, respectively). Hypoperfused-control mice exhibited significantly more revisiting errors and less novel entries than the sham operated mice (p < 0.05 and p < 0.05), while hypoperfused-cilostazol mice showed a learning process similar to that of sham mice before they plateaued. Data represents the average of 4 trials for block 1 and 2 and the average of 2 trials for blocks 3-6. (b) In the corpus callosum, evoked compound actions potentials (CAPs) were recorded at various distances from the stimulating electrode. Peak latencies of CAPs were significantly different between the three groups (F (2,12) = 14.78; p = 0.0048). The peak latency of CAPs in hypoperfused-control mice was overall significantly increased compared to sham mice (p < 0.01) but this was ameliorated with cilostazol treatment. Data represents mean ± SEM. *p < 0.01, Hypoperfused-control vs Sham. significant difference between the 3 groups (F (2,34) = 5.169; p = 0.029 and F (2,34) = 5.718; p = 0.022, respectively). Post-hoc analysis indicated that there was a significant difference in the number of revisiting errors and novel entries between the hypoperfused-control and the sham group (p < 0.05), but there was no difference between the hypoperfused-cilostazol and sham group (p > 0.05). This data suggests that the chronic treatment with cilostazol has modest beneficial effects on spatial working memory after long term hypoperfusion. Cilostazol improved white matter function. We have previously demonstrated that chronic cerebral hypoperfusion causes diffuse white matter changes 3 . To build on this work, we determined whether the functional integrity of white matter was altered in response to hypoperfusion and if this was ameliorated by cilostazol. Peak latencies of compound action potentials (CAPs) were significantly different between the three groups (F (2, 12) = 14.78; p = 0.0048) Post-hoc analysis indicated that the peak latency of CAPs in hypoperfused-control mice were significantly increased compared to shams (p < 0.01). However, the peak latencies of CAPs in the hypoperfused-cilostazol group were not significantly different to those of sham mice (p > 0.05) (Fig. 1b). These results suggest that hypoperfusion significantly impedes the conduction velocity of myelinated fibers and that cilostazol treatment is able to reduce this deficit. Cilostazol did not significantly restore white matter integrity. Since white matter function was found to be compromised with hypoperfusion and restored with cilostazol treatment, we next evaluated white matter integrity using diffusion tensor imagingimaging (DTI) and pathological analysis (myelin associated glycoprotein, MAG, immunostaining). There was no significant difference in fractional anisotropy (FA) in the corpus callosum between the groups (F (2,33) = 2.513; p = 0.097) (Fig. 2a). However it was noted that there was a modest reduction in FA between the sham and hypoperfused-control groups (0.47 ± 0.01 vs. 0.40 ± 0.03), consistent with a previous report 18 . Cilostazol treatment did not have any effect on FA (0.40 ± 0.02). Interestingly, there was a significant negative correlation between FA and the number revisiting errors made on the radial arm maze test with mice committing more errors having lower FA values which are indicative of a more disrupted white matter architecture ( Fig. 2b) (Spearman r = −0.583, p = 0.0003). There was a significant difference in MAG staining between the three groups (p = 0.0003). Post-hoc analysis indicated that there was a significant loss of axon-glial integrity (identified by MAG immunostaining) in the hypoperfused-control compared to the sham mice in the corpus callosum (p < 0.001). However, the loss of axon-glial integrity was also found to be significantly different in the hypoperfused-cilostazol mice as compared to sham mice (p < 0.05) (Fig. 2c) indicating that cilostazol was unable to improve axon-glial disruption after hypoperfusion. Cilostazol improved cellular neuroinflammation. A prominent feature of hypoperfusion is a robust cellular neuroinflammatory response. Consistent with this, microglial activation (identified by the extent of Iba1 immunostaining) was increased in the corpus callosum after hypoperfusion ( Fig. 3a) but in this study we found no significant overall effect between the 3 groups (F (2, 33) = 2.866; p = 0.072). However the results did indicate that there was an increase in the density of Iba1 positive staining following hypoperfusion which was lower with cilostazol treatment (8.26 ± 1.47 vs 6.61 ± 1.05) (Fig. 3b). Notably, there was a robust association between the extent of Iba1 staining in the corpus callosum and revisiting errors (Fig. 3b, Pearson r = 0.712, p < 0.0001), indicating that higher levels of cellular neuroinflammation correlate with poorer spatial working memory. Similarly, in the corpus callosum there was an overall difference in the extent of reactive astrogliosis (glial fibrillary acidic protein, GFAP, immunostaining) (p = 0.04). It was found that GFAP immunostaining was significantly reduced after hypoperfusion with cilostazol treatment (p < 0.05) ( Fig. 3c and d). To explore potential dynamic changes in gliovascular unit, alterations in the protein aquaporin 4 (AQP4) were investigated. AQP4 is a water channel typically confined to the endfeet of vascular associated astrocytes and is reported to maintain osmotic balance and to lose the polarization to the perivascular endfeet related to the development of microinfarcts 19,20 . Mislocalisation of the water channel AQP4 (as measured by % area AQP4) was observed in the hypoperfused-control mice compared to sham mice consistent with our previous study 5 and cilostazol tended to revert this redistribution (Sham; 0.67 ± 0.25, Hypoperfused-control; 4.31 ± 3.21, Hypoperfused-cilostazol; 2.20 ± 0.56, p = 0.12) (Fig. 3d). Cilostazol significantly suppressed endothelial adhesion molecule expression. In order to elucidate the mechanism by which cilostazol reduces glial activation, we measured endothelial activation, previously shown to be linked to beneficial effects of cilostazol [8][9][10] . Intercellular adhesion molecule-1 (ICAM1), a marker of endothelial cell activation 21 , was investigated. There was a significant difference in ICAM between the three groups (F (2, 33) = 13.19; p < 0.0001). A significant increase in ICAM1 immunostaining was determined in the corpus callosum in the hypoperfused-control group compared to both the sham and hypoperfused-cilostazol groups (p < 0.001 and p < 0.01) ( Fig. 4a and b). Supportive of a close relation between endothelial cell activation, microgliosis and memory, a significant association between ICAM1 and Iba1 immunostaining was determined (Pearson r = 0.375, p = 0.029), and a significant association between ICAM1 and the number of revisiting errors (Pearson r = 0.412, p = 0.016) (Fig. 4c) was determined in the corpus callosum. Cilostazol reduced stroke lesions but did not affect baseline CBF. Following on from the finding that cilostazol had a beneficial effect on endothelial cell activation, we determined whether this may also impact on baseline cerebral blood flow (CBF) using arterial spin labelling (ASL). There was an overall difference in CBF between the three groups in the corpus callosum (F (2, 25) = 11.0; p = 0.0004) and thalamus (F (2, 25) = 33.05; p < 0.0001). CBF was significantly reduced in hypoperfused-control and hypoperfused-cilostazol mice compared to sham mice in the corpus callosum (p < 0.001 and p < 0.001) and the thalamus (p < 0.001 and p < 0.001) ( Fig. 5a and b). Stroke lesions (ischemic and hemorrhagic), detected on T2-MRI (Fig. 6a), were present after hypoperfusion but absent from all sham mice. Interestingly, cilostazol was found to decrease the number of the ischemic stroke lesions (present in only 2 of 13 (15%) hypoperfused-cilostazol mice versus 6 of 11 (55%) hypoperfused-control mice) (Fig. 6b). Discussion The current study is of the first to report the beneficial long-term effects of cilostazol in a model of chronic cerebral hypoperfusion relevant to the pathophysiology of VCI. Cilostazol exerted a protective effect against white matter dysfunction and modestly improved spatial working memory at 3 months after cerebral hypoperfusion. These improved functional outcomes were closely related to reduced gliovascular damage, cellular inflammation and endothelial activation. Previous studies have indicated that cilostazol protects against hypoperfusion-induced white matter damage [13][14][15][16] contrary to the present study in which cilostazol was found to have minimal impact on white matter integrity. The effects of cilostazol have been predominantly studied in more severe models of cerebral hypoperfusion such as a rat 2-vessel occlusion (2VO) model and within 1 to 5 weeks hypoperfusion [13][14][15] . The rat 2VO model shows marked BBB disruption, as early as 3 days post-operation 22 , leading to prominent white matter lesions, glial activation and profound impairment of various aspects of cognition 15,23,24 . In contrast, our mouse model of cerebral hypoperfusion shows gradual and progressive temporal changes of white matter dysfunction, gliovascular unit disruption and glial activation leading to working memory impairment 3, 5 . In our model, there is minimal disruption of the BBB until 6 months post-hypoperfusion 5 whereas there is marked BBB damage in the rat 2VO models which may enhance movement of cilostazol directly to the brain 25 . There are also differences in the severity of hypoperfusion, BBB and white matter damage between the different laboratories that study the mouse model of cerebral hypoperfusion 16 . We have previously shown that there are subtle changes in the distribution of paranodal-nodal proteins with hypoperfusion 4 that were not measured in the present study. This redistribution of the molecular architecture of myelinated axons can impact on white matter function. In support of this, despite the lack of protection against white matter changes, cilostazol was able to improve white matter function as assessed using electrophysiological approaches and improve spatial working memory which is dependent on frontal cortical circuitry. Increased microglial density accompany hypoperfusion-induced damage to white matter 3-5, 15, 16 and may contribute to the damage via pro-inflammatory mechanisms. Notably increased microglia were determined in the corpus callosum of hypoperfused mice and reduced by cilostazol-treatment. Furthermore, it was found that the extent of microgliosis in white matter relates to the impairment in working memory. A previous study has also There was a trend towards an increase in Iba1 stained micoglia that was reduced with cilostazol after hypoperfusion. There was a robust association between microgliosis and the number of revisiting errors (Pearson r = 0.712, p < 0.0001). (c) Representative images of immunofluorescence for Collagen IV, AQP4 and GFAP in the corpus callosum. (d) There was an increase in the area of GFAP positive astroglia in the hypoperfused-control mice. There was a significant reduction with cilostazol treatment as compared to control hypoperfused. Astrocyte-endfoot displacement tended to be observed in the hypoperfused-control mice and less in the cilostazol treated mice. Data represents mean ± SEM. p < 0.05, Hypoperfused-control vs Hypoperfused-cilostazol. shown beneficial effects of cilostazol on microglia and anti-inflammatory pathways 26 . Thus neuroinflammation is proposed to be strongly associated with white matter dysfunction and working memory impairment (Fig. 7). Endothelial dysfunction is widely considered to be one of the pivotal mechanisms of the structural and functional brain-vessel alterations in small vessel disease (SVD) and evidence has accumulated to support the hypothesis that early endothelial failure is a major precipitant of sporadic SVD 27,28 . Endothelial damage can lead to increased permeability, damage to vessel wall, dysregulation of vascular tone, inflammation, demyelination, gliosis and at a late stage, luminal narrowing and thrombosis 21,27 . White matter lesions are characterized by the expression of adhesion molecules such as ICAM1 28 , which are induced by proinflammatory cytokines that promote the adherence of monocytes. The increased expression of ICAM1 may reflect endothelial dysfunction or microvascular inflammation; ICAM1 expression is recognized mainly in the endothelial cells but can also be found in microglia and leukocytes. In the current study, ICAM1 staining, as an index of endothelial dysfunction, was markedly increased with hypoperfusion and restored with cilostazol treatment. Additionally endothelial dysfunction was significantly correlated with neuroinflammation and spatial working memory impairment. Thus the data suggest that cilostazol might restore endothelial dysfunction without apparent BBB disruption leading to dampening of glial activation, improved white matter function and cognition (Fig. 7, large arrows). However whether this is a direct effect of cilostazol on endothelial cells or secondary to other effects, for example on microglia 26 , remains to be determined. Interestingly cilostazol has been shown to have effects on spatial learning in control mice via effects on growth factors 29 . Thus cilostazol appears to have multiple beneficial effects. Future studies could define whether endothelium dependent vasodilator agents afford similar protection in the hypoperfusion model in vivo in comparison to cilostazol treatment. Similarly, in animal models relevant to Alzheimer's disease There was a significant difference in ICAM staining between the three groups (F (2, 33) = 13.19; p < 0.0001). ICAM1 immunostaining was significantly greater in the hypoperfused-control group compared to the sham (p < 0.001) and this was reduced with cilostazol treatment (p < 0.01). (c) There was a significant association between ICAM1 positive areas and microgliosis (Pearson r = 0.375, p < 0.05), and the number of revisiting errors (Pearson r = 0.412, p < 0.05). Data represents mean ± SEM. (AD), increased endothelial adhesion molecules have been highlighted as markers of endothelial dysfunction and vascular inflammation 30 . Endothelial dysfunction may be a common target in the treatment of AD, VCI and vascular dementia. Our present study supports the utility of cilostazol in the treatment of VCI. A previous study has also indicated beneficial effects of cilostazol in a mouse model relevant to AD through increased amyloid β clearance due to improved cerebrovascular function 31 . Retrospective clinical studies indicated the potential for cilostazol treatment to suppress cognitive decline in elderly patients with mild dementia or cognitive impairment 32,33 . However the current study suggests that whilst cilostazol improves hypoperfusion-induced memory deficits these are not completely restored, the improvement in memory plateaued after 12 days of testing. The current studies were also conducted in mid-age rodents and further studies should include aged cohorts where the pathological and functional impairments to hypoperfusion may be exacerbated and be affected differently by cilostazol. Although cilostazol is well known for its vasodilatory effect via up-regulation of cAMP, there was no effect of cilostazol determined on CBF measures in the corpus callosum and the thalamus at 3 months post-operation. These results are consistent with other studies 13,14 , which indicated that CBF was not significantly influenced by cilostazol despite demonstrations that white matter damage is restored in the cilostazol treated mice 14 . Similarly, although there was a reduction in the number of stroke lesions with cilostazol this was not paralleled by an increase in CBF. In this study it is difficult to precisely relate the CBF measures with stroke lesions since the CBF measures using ASL were conducted at one brain level whereas the stroke lesions were found throughout different levels of the brain and in different brain regions. There may be other effects of cilostazol that are independent of perfusion. For example, restoration of endothelial dysfunction by cilostazol directly or indirectly via microglia 33 might reduce the number of stroke lesions without an effect on perfusion (Fig. 7). Vasomotor reactivity or neurovascular coupling, which are recognized to be associated with cognitive impairment 34,35 might also be impaired by hypoperfusion and remain to be elucidated. Our study showed that cilostazol ameliorated gliovascular damage and working memory impairment possibly via endothelial protection in a mouse model of chronic cerebral hypoperfusion. This evidence indicates that cilostazol, originally a drug for stroke and peripheral arterial disease, might be a useful candidate for the treatment of VCI. However, these beneficial effects of cilostazol are modest in this model of VCI and thus other more potent drugs need to be considered. A proposed mechanism by which cilostazol may improve cognitive impairment induced by chronic cerebral hypoperfusion. Cilostazol reduced the number of stroke lesions and significantly ameliorated the spatial working memory impairment and white matter dysfunction induced by chronic cerebral hypoperfusion. Remarkably, cognitive deficits seem to be closely associated with the hypoperfusion-induced neuroinflammatory processes which are significantly decreased with cilostazol treatment. Cilostazol might restore endothelial dysfunction without apparent BBB disruption leading to recovery of neuroinflammation and white matter dysfunction (large arrow). via bilateral common carotid artery stenosis 3-5 using microcoils fitted on both common carotid arteries under isoflurane anaesthesia (induced at 5% and maintained at 1.5%). Sham mice underwent the exact same surgical procedure with the exception that coils were not applied to the arteries. Mice were fed with either pelleted chow containing 0.3% cilostazol (cilostazol-treated mice) or standard pelleted chow only (control-treated mice) from one week before the surgery. Methods Animal cohorts and exclusions. Mice were coded and randomly divided into different groups (1) sham-control, (2) hypoperfused-control, and (3) hypoperfused-cilostazol. Two different cohorts were studied: Cohort 1 in which the mice were assessed using Radial Arm Maze, Magnetic Resonance Imaging (MRI) with endpoints of pathology; Cohort 2; mice were studied using electrophysiology. Investigators were blinded to the surgical or drug intervention until the final analysis. Cohort 1 Initial numbers at the outset of study were: sham-control n = 10, hypoperfused-control n = 12, hypoperfused-cilostazol n = 13. One mouse in the hypoperfused-control group was culled due to poor recovery before the MRI scan. The final numbers for behaviour, were sham-control n = 10, hypoperfused-control n = 12, hypoperfused-cilostazol n = 13. The final numbers for MRI (T2/DTI) were sham-control n = 10, hypoperfused-control n = 11, hypoperfused-cilostazol n = 13. ASL was assessed in a subset of these since one mouse in sham-control, four mice in hypoperfused-control and three mice in hypoperfused-cilostazol group were excluded due to poor quality of ASL. Thus the final group sizes for ASL were: sham-control n = 9, hypoperfused-control n = 7, hypoperfused-cilostazol n = 10. Cohort 2: Initial numbers at the outset were sham-control n = 4, hypoperfused-control n = 6, hypoperfused-cilostazol n = 5. Electrophysiology recordings could not be made in two mice: one hypoperfused control and one hypoperfused cilostazol. Thus the final numbers for electrophysiology were sham-control n = 4, hypoperfused-control n = 5, hypoperfused-cilostazol n = 4. Radial Arm maze. Spatial working memory was assessed using an 8-arm radial maze. Mice were food deprived to reduce their initial body weight by 10-15% and the restricted feeding was maintained until the end of behavioural testing. Two pretraining days were undertaken in order to familiarize the animals with the experimental environment, the apparatus and the task itself (see supplementary methods). For the training procedure one food pellet was placed at the end of each of the 8 arms of the maze. At the beginning of every trial (one trial/ day) the animal was placed on the central platform with all arm doors open and allowed to make an arm choice. An arm choice was recorded when the centre of the mouse (as tracked by the AnyMaze software) was 5 cm into the arm. Once the mouse had entered one of the arms, the doors to the other 7 arms were closed automatically. When the animal exited the visited arm it was confined on the central platform for 5 sec by closing the remaining door. After the 5 sec delay it was allowed to make a new choice. A trial ended when the mouse had retrieved all 8 pellets or 25 min had elapsed. The behavioural testing lasted 16 days. For each trial, the number of correct arm entries within the first eight visits, the number of revisited arms (working memory errors) and the time taken to complete the task were recorded. Data were expressed as the mean of 4 trial blocks for the first 8 days and 2 trial blocks for the rest of 8 days. Magnetic Resonance Imaging. Neuroimaging was conducted on completion of the behavioural assessment. Mice were anaesthetised and placed in an MRI compatible holder (Rapid Biomedical, Wurzburg, Germany). Rectal temperature and respiration were monitored and controlled throughout to ensure normal physiological parameters. Structural T2-weighted and DT-MRI data were collected using an Agilent 7 T preclinical MRI scanner (Agilent Technologies, Yarnton, UK); with a 72 mm volume coil and a 2 channel phased array mouse brain coil (Rapid Biomedical). Arterial Spin Labelling (ASL) was performed using a Look-Locker FAIR single gradient echo (LLFAIRGE) sequence (see supplementary methods for details). T2-weighted MRI slices of the mouse brain (1.0 to − 4.6 mm Bregma) were examined for (i) the presence and type of cortical and/or subcortical primary ischaemic lesions and (ii) primary haemorrhages and anatomical location 3 (supplementary methods). The number of ischaemic and hemorrhagic lesions were determined and the percentage of mice with lesions in each cohort calculated. Fractional Anisotropy (FA) was generated using 'in-house' custom software. FA and ASL techniques were processed by regions of interest (ROIs) analysis. ROIs were set in the thalamus and the corpus callosum at the levels of the striatum and the hippocampus for FA and at the level of hippocampus for ASL. Immunohistochemistry. Mice were transcardially perfused with 4% paraformaldehyde (PFA) under deep anaesthesia (5% isoflurane in oxygen enriched air) and brains were carefully removed, postfixed in 4% PFA for 24 hours and processed for cryostat sectioning. Coronal brain sections were cut using a cryostat and mounted onto superfrost slides (10 µm) or preserved in cryoprotective medium at −20 °C (30 µm). All sections except for ICAM1 staining were heat-retrieved with either EDTA pH 8 or citrate pH 6 and primary antibodies were incubated overnight at 4 °C in blocking buffer (see supplementary data for further details). Subsequently, the sections were incubated with biotinylated secondary antibodies (1:100; Vector Labs) and a solution of streptavidinbiotin-peroxidase complex or alternatively with fluorescent secondary antibodies (Alexa Fluor, Thermofisher; 1:500). Peroxidase activity was localised using 3,3′ diaminobenzadine tetrahydrochloride (DAB) as a chromagenic substrate (Vector Labs). DAB immunolabeled sections were analyzed using an optical microscope Olympus BX51 and fluorescent immunolabeled sections were analyzed using a laser scanning confocal microscope Leica SP5 C. All measurements were done in the corpus callosum using imageJ. To assess microglial activation, expression of endothelial adhesion molecule and activation of astrocytes, positively stained area of Iba1, ICAM1 and GFAP were measured respectively. The loss of axon-glial integrity of the corpus callosum (disorganised white matter fibres, presence of myelin debris and vacuolation) was assessed using MAG antibody and graded from 0 (none) to 3 (extensive) as previously described 3 . Percentage area stained by AQP4 and COL4 as well as their respective colocalization (Manders coefficient) was determined after being equally thresholded across all sections for the respective antibody. The measure of AQP4 out with vessels was defined as the difference between percentage area stained by AQP4 and COL4, respectively. Electrophysiology. Three months after surgery, mice were sacrificed and their brains were quickly removed on top of a frozen petri-dish with a few drops of sucrose artificial cerebrospinal fluid (aCSF) (189 mM Sucrose, 10 mM D-glucose, 26 mM NaHCO3, 3 mM KCl, 5 mM MgCl2, 0.1 mM CaCl2, 1.25 mM NaH2PO4). The brain was then rapidly dissected, placed in a cell strainer and submerged in ice-cold oxygenated sucrose aCSF for 2-3 minutes. Sections were then cut at 400 μm using a vibrating blade microtome (Hydrax V50, Zeiss, Cambridge, UK). A single coronal slice (−1.2-1.7 mm Bregma) was then transferred to a warmed (34 °C) incubation chamber (BSK1, Digitimer, Welwyn Garden City, UK) with oxygenated aCSF (124 mM NaCl, 5 mM KCL, 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 1.3 mM MgSO 4 , 2 mM CaCl 2 , 10 mM D-Glucose). Following at least a 1 hour period of post-slicing recovery slices were carefully transferred to a recording chamber (RC27L, Harvard Apparatus, Cambridge, UK) and mounted on an Olympus BX51 microscope where they were superfused with oxygenated aCSF (2-3 ml/min) at 24 °C. This temperature was employed to enable discrimination of compound actions potentials (CAP) arising from myelinated and unmyelinated fibres. Slices were supported by a slice anchor and then allowed to rest for 30 minutes prior to recording. A bipolar stimulating electrode (WPI Inc, Hertfordshire, UK) with manually blunted tips was lowered into the corpus callosum ~1 mm lateral to the midline using a manual manipulator and constant current stimulus-isolated square wave pulses (NL800, Digitimer, Welwyn Garden City, UK) were applied to evoke CAPs. Recording electrodes were pulled from borosilicate glass capillaries (1.5/0.84 mm OD/ID, 100 mm long, WPI Inc., Hertfordshire, UK) and back filled with aCSF to give a final resistance of 1-3 MΩ. These were connected to the amplifier's headstage via an Ag/AgCl wire and then lowered into the corpus callosum (Patchstar micromanipulator, Scientifica, East Sussex, UK) adjacent to the midline 2.5 mm contralateral to the stimulating electrode. For analysis of the CAP peak amplitude standardised input-output functions were generated by varying the intensity of stimulus pulse (200 μs duration, 0.2 Hz) in 0.25 mA increments from 0.25-4 mA. To enhance the signal to noise ratio, all electrophysiology analysis was conducted on averaged waveforms of four successive sweeps. Evoked CAPs were amplified (x500) and filtered (bandpass = DC to 10 kHz) using an Axopatch 200 A amplifier in voltage following mode (Molecular Devices, Berkshire, UK). Responses were digitized at 200 kHz, and recorded for offline analysis using pClamp software (v10, Molecular Devices, Berkshire, UK). CAPs were evoked at the maximal stimulation of 4 mA and the distance between the stimulating and recording electrode altered in 0.5 mm increments from 1-2.5 mm by moving the recording electrode. The post-stimulus latency of the CAP peak was then measured.	2023-02-17T14:53:49.351Z	2017-06-27T00:00:00.000	{ "year": 2017, "sha1": "f5fe45b74a0b7209b6dc4b17d6a4429e7e3cbffe", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-017-04082-0.pdf", "oa_status": "GOLD", "pdf_src": "SpringerNature", "pdf_hash": "f5fe45b74a0b7209b6dc4b17d6a4429e7e3cbffe", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [] }
569872	pes2o/s2orc	v3-fos-license	Recombinant DNA production of spider silk proteins Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. Introduction Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Spider silks are remarkable natural polymers that consist of three domains: a repetitive middle core domain that dominates the protein chain, and nonrepetitive N-terminal and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX) polypeptides alternate. At least seven different types of silk proteins are known for one orb-weaver species of spider (Lewis, 2006a). Silks differ in primary sequence, physical properties and functions (Hu et al., 2006). For example, dragline silks used to build frames, radii and lifelines are known for outstanding mechanical properties including strength, toughness and elasticity (Gosline et al., 1984). On an equal weight basis, spider silk has a higher toughness than steel and Kevlar (Vepari and Kaplan, 2007;Heim et al., 2009). Flageliform silk found in capture spirals has extensibility of up to 500%. Minor ampullate silk, which is found in auxiliary spirals of the orb-web and in prey wrapping, possesses high toughness and strength almost similar to major ampullate silks, but does not supercontract in water. Figure 1 depicts the location and structural elements of MaSp, MiSp and Flag silks. Finally, there are other silk types such as aciniform, pyriform, aggregate and tubuliform (egg case) with unusual primary structure, composition and properties. Diverse and unique biomechanical properties together with biocompatibility and a slow rate of degradation make spider silks excellent candidates as biomaterials for tissue engineering, guided tissue repair and drug delivery, for cosmetic products (e.g. nail and hair strengthener, skin care products), and industrial materials (e.g. nanowires, nanofibres, surface coatings). Recent advances in genetic engineering have provided a route to produce various types of recombinant spider silks (Prince et al., 1995;Fahnestock and Bedzyk, 1997;Rabotyagova et al., 2009;Xu et al., 2012). However, production of spider silk proteins at a larger scale remains challenging. Moreover, recombinant silk threads do not recapitulate the full potential of native fibres in terms of mechanical properties. Different heterologous host systems have been investigated to develop suitable production systems. In this review, we discuss recent advances in the production of recombinant spider silks in heterologous host systems with the main focus on microbial production. In particular, we focus on dragline silks. Current cloning strategies, expression systems and purification strategies will be discussed to help researchers to engineer customized synthetic spider silk-like proteins for various needs, including biomaterials and material science applications. Structure of silk proteins Spider silks are fascinating polymers, as is the spinning process that members of Araneidae family use to make these exceptional materials. Spiders use complex spinning to rapidly transform water soluble, high molecular weight, silk proteins into solid fibres at ambient temperature and pressure, giving rise to an environmentally safe, biodegradable and high performance material (Asakura et al., 2007;Lewicka et al., 2012;Teulé et al., 2012a). The details on anatomy and physiology of the spider spinning apparatus (N. clavipes) can be found elsewhere (Knight and Vollrath, 2001;2002;Eisoldt et al., 2011;Rising et al., 2011). In order to understand the challenges and needs associated with biotechnological production of recombinant spider silks, primary protein motifs, composition and secondary structural elements must be discussed. As mentioned earlier, one spider is capable of producing up to seven different types of silks with varying mechanical properties. In spite of different mechanical and physiological properties, the majority of spider silks share a common primary structural pattern comprised of a large central core of repetitive protein domains flanked by non-repetitive N- Fig. 1. A. An adult female orb weaver spider Nephila clavipes and her web. B. Schematic overview of N. clavipes web composed of three different spider silk proteins and their structures. The coloured boxes indicate the structural motifs in silk proteins. An empty box marked '?' indicates that the secondary structure of the 'spacer' region is unknown. Note: MaSp1 or MaSp2: major ampullate spidroin 1 or 2; MiSp1 and 2: minor ampullate spidroin1 and 2; Flag: flagelliform protein. The photo was taken by Olena and Artem Tokarev in the Florida Keys. and C-terminal domains. The most investigated silk is dragline silk, which shows a remarkable combination of strength and elasticity. The golden orb-weaver spider, N. clavipes, produces dragline silk in the major ampullate gland (Knight and Vollrath, 2001). Dragline silk is the protein complex composed of major ampullate dragline silk protein 1 (MaSp1) and major ampullate dragline silk protein 2 (MaSp2). Both silks are approximately 3500 amino acid long. MaSp1 can be found in the fibre core and the periphery, whereas MaSp2 forms clusters in certain core areas. The large central domains of MaSp1 and MaSp2 are organized in block copolymer-like arrangements, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX) polypeptides alternate in core domain. The main difference between MaSp1 and MaSp2 is the presence of proline (P) residues accounting for 15% of the total amino acid content in MaSp2 (Hu et al., 2006), whereas MaSp1 is proline-free. By calculating the number of proline residues in N. clavipes dragline silk, it is possible to estimate the presence of the two proteins in fibres; 81% MaSp1 and 19% MaSp2 (Brooks et al., 2005). Different spiders have different ratios of MaSp1 and MaSp2. For example, a dragline silk fibre from the orb weaver Argiope aurantia contains 41% MaSp1 and 59% MaSp2 (Huemmerich et al., 2004). Such changes in the ratios of major ampullate silks can dictate the performance of the silk fibre (Vollrath and Knight, 1999). Specific secondary structures have been assigned to poly(A)/(GA), GGX and GPGXX motifs including β-sheet, 3 10-helix and β-spiral respectively (Humenik et al., 2011). The primary sequence, composition and secondary structural elements of the repetitive core domain are responsible for mechanical properties of spider silks; whereas, non-repetitive N-and C-terminal domains are essential for the storage of liquid silk dope in a lumen and fibre formation in a spinning duct (Ittah et al., 2006). The primary amino acid sequence, composition and secondary structural elements of other silk types are reviewed elsewhere (Lewis, 2006b;Humenik et al., 2011). Production of recombinant silk proteins Spiders cannot be farmed, in contrast to silkworms, due to their aggressive behaviour and territorial nature (Kluge et al., 2008). Collecting silk from webs is a time-consuming task. It took 8 years to make a golden spider silk cape from 1.2 million golden orb webs (Chung et al., 2012). Therefore, biotechnological production of recombinant spider silks is the only practicable solution to harvest silks on a larger scale and to meet growing needs of medicine and biotechnology. A variety of heterologous host systems have been explored to produce different types of recombinant silks (Table 1 and Table 2). Recombinant partial spidroins as well as engineered silks have been cloned and expressed in bacteria (Escherichia coli), yeast (Pichia pastoris), insects (silkworm larvae), plants (tobacco, soybean, potato, Arabidopsis), mammalian cell lines (BHT/ hamster) and transgenic animals (mice, goats). Unicellular organisms as heterologous host systems. Unicellular organisms, such as bacteria and yeast, have been investigated as host systems for recombinant silks. A gram-negative, rod-shaped bacterium E. coli is a wellestablished host for industrial scale production of proteins. Therefore, the majority of recombinant spider silks have been produced in E. coli (Lewis et al., 1996;Fahnestock and Irwin, 1997;Wang et al., 2006;Rabotyagova et al., 2009;Rabotyagova et al., 2010;An et al., 2011;An et al., 2012;Teulé et al., 2012a). E. coli is easy to manipulate, has a short generation time, is relatively low cost and can be scaled up for larger amounts protein production. The recombinant DNA approach enables the production of recombinant spider silks with programmed sequences, secondary structures, architectures and precise molecular weight (Rabotyagova et al., 2011). There are four main steps in the process: (i) design and assembly of synthetic silk-like genes into genetic 'cassettes', (ii) insertion of this segment into a DNA vector, (iii) transformation of this recombinant DNA molecule into a host cell and (iv) expression and purification of the selected clones. Figure 2 summarizes the recombinant DNA approach used to prepare silk-like proteins. The monomeric silk-like gene sequences can be synthesized as short single-stranded oligonucleotides (up to 100 bp) by commercial oligonucleotide synthesis or used directly as polymerase chain reaction products from cDNA libraries. Large repetitive sequences can be constructed by using concatemerization, step-by-step directional approach and recursive ligation (Fig. 3). Concatemerization is a useful method when a library of genes of different sizes is desired but has limitations in the preparation of genes with specific sizes (Meyer and Chilkoti, 2002). To overcome limitations of concatemerization, recursive directional ligation or a step-by-step ligation is employed (Meyer and Chilkoti, 2002;Wright and Conticello, 2002). Recursive directional ligation allows for facile modularity, where control over the size of the genetic cassettes is achieved. Moreover, recursive directional ligation eliminates the restriction sites at the junctions between monomeric genetic cassettes without interrupting key gene sequences with additional base pairs that makes it different from the step-by-step ligation approach (Higashiya et al., 2007). For example, we have employed step-by-step directional ligation to produce various partial recombinant spider silks as well as engineered silk-like proteins based on the sequences of dragline silk originated from N. clavipes (Prince et al., 1995;Wang et al., 2006; Rech and D. L. Kaplan et al., 2007;Rabotyagova et al., 2009;Mieszawska et al., 2010;Gomes et al., 2011;Numata et al., 2012). As one example, spider silk block copolymers were generated in E. coli (Rabotyagova et al., 2009;. In the first cloning step, a commercially available pET30a(+) vector (Novagen, San Diego, CA, USA) was modified with an adaptor sequence, carrying NheI and SpeI restriction sites. The adaptor was inserted into XhoI and NcoI sites of a pET30a(+) to generate pET30L. The coding sequences of two spider silk-like monomers A (hydrophobic block) and B (hydrophilic) were designed to carry SpeI and NheI restriction sites at the ends of the sequences. This allowed ligation of the domains into a pET30L vector. By using a step-by-step directional ligation approach, direct control over the assembly of monomeric genes into complex sequences was achieved. Six different constructs were cloned and transformed into the bacterial host for expression. An N-terminal His-tag was used for protein purification by immobilized metal affinity chromatography (Rabotyagova et al., 2009). Another genetic engineered strategy has been proposed by Lewis Laboratory to assemble long repetitive spider silk genes (Teule et al., 2009). This cloning strategy employs a one-step head-to-tail ligation that can produce large inserts in precise manner (Lewis et al., 1996;Brooks et al., 2008;Teule et al., 2009;Teulé et al., 2012a). The spider silk synthetic genes were optimized for codon usage in E. coli and were cloned into a plasmid vector pBluescriptII SK(+) (Stratagene). Each silk module was carrying compatible XmaI and BspEI restriction sites at the ends on the coding sequences. The vector also contained a unique restriction site (ScaI) in the ampicillin resistance gene. By simultaneously performing two double digestion reactions ScaI -XmaI and ScaI -BspEI two fragments each containing a copy of a silk monomer gene were obtained. The fragments were ligated together using T4 ligase resulting in the doubling of the size of silk genes and restoring the ampicillin resistance of the plasmid (Fig. 4). Several round of cloning were performed to obtain repetitive sequences of a desired size. Next, the multimeric synthetic genes were subcloned into an expression pET19b vector using NdeI and BamHI restriction sites. Since the expression vector was carrying NdeI and BamHI sites, the liberated inserts were cloned in-frame with pET19b. Similar to pET30L, silk genes in pET19b are under control of the T7 promoter and require the addition of isopropyl-β-D-1-thiogalactopyranoside to initiate protein expression. The expressed proteins can be purified by immobilized metal affinity chromatography (IMAC) due to the presence of an N-terminal His-tag. Several recombinant spider silk proteins from different species were produced using this genetic engineering strategy including silks from N. clavipes (Teule et al., 2009) Argiope aurantia (Brooks et al., 2008). Recombinant spider silk proteins from Nephylengys cruentata, Parawixia bistriata and Avicularia juruensis were produced employing this cloning strategy (Leopoldo et al., 2007) (US patent 20 100 311 645). Figure 4 summarizes the strategy. A three module cloning strategy based on the sequences of ADF-3 and ADF-4 was developed by Scheibel research group (Huemmerich et al., 2004), designed so that multiple modules can be combined. Moreover, additional coding sequences such as N-or C-terminal domains can be added if needed. The purification protocol is based on heat resistance of silk proteins followed by an ammonium sulphate precipitation that is different from Ni-NTA IMAC. Different purification strategies have been employed recently to optimize small and large-scale production of recombinant silks. Most of the spider silk proteins are produced with an N-or C-terminal His-tags to make purification simple and produce enough amounts of the protein. However, the presence of this tag can affect protein secondary structure and interfere with the process of spider silk fibre formation. Dams-Kozlowska et al. (2012) proposed two strategies to purify spider silks from lysates without the use of a His-tag. These protocols are based on thermal treatment and organic acid resistance of silk proteins and do not require the presence of the His-tag. After purification, silk proteins based on MaSp1 gene sequence were formed into films that subsequently were used to grow murine fibroblast cell culture. The results demonstrated that silk films were non-toxic to the cells (Dams-Kozlowska et al., 2012). Because of the highly repetitive core sequence of spider silk genes, frequent homologous recombination, deletions, transcription errors, translation pauses, accumulation in inclusion bodies and low yields were observed during the production of recombinant silks in E. coli. Moreover, when the protein size was increased from 43 kDa to higher (the size of native spidroins is between 300 and 350 kDa), protein yields decreased dramatically. Codon optimization for the specific host expression system helped maximize the translation of the foreign gene transcripts and thus, improved protein yields Bedzyk, 1997, Lewis, 2006b). It was also suggested that depletion of tRNA pools upon protein expression resulted in transcription and translation errors (Rosenberg et al., 1993). Recently, Xia et al. (2010) employed a metabolic engineered strategy to enhance the production of recombinant spider silks. The authors reported production of full length (284.9 kDa) recombinant N. clavipes dragline silk proteins that were rich in glycine (43-45%). Production of these silk proteins was enhanced by the use of the metabolically engineered expression host within which the glycyl-tRNA pool was elevated. The fibres spun with the native-sized recombinant spider silk protein showed tenacity, elongation and Young's modulus of 508 MPa, 15% and 21 GPa, respectively, comparable to those of native spider dragline silk (Xia et al., 2010). Through extensive proteomic analysis, serine hydroxymethyltransferase (GlyA) and β-subunit of glycly-tRNA synthetase (GlyS) were found to be upregulated to meet the high cellular demand for glycly-tRNA when expressing glycine-rich silk proteins. Increased glycine biosynthetic flux by overexpressing glycyl-tRNA synthetase elevated the total tRNAGly pool and resulted in enhanced production of high molecular weight recombinant spider silks. Recently, large spider recombinant egg case silk protein from Nephila antipodiana, 378 kDa, was engineered using E. coli, where gene multimers were chemically linked by cysteine disulfide bonds. The recombinant silk sequence consisted of two silk proteins: tubuliform spidroin 1 (TuSp1) and C-terminal domain of MisP1. Nonrepetitive C-terminal domain of MiSp1 was chosen due to its higher water solubility and stability compared with the C-terminal domain of TuSp1. A disulfide linkage between two C-terminal domains was formed by introducing a point mutation (S76 to S76C). This link allowed the formation of a hybrid DNA construct that was expressed in E. coli (DE3). The recombinant protein was expressed in E. coli. Moreover, the artificial fibres spun from this protein showed higher tensile strength and Young' modulus than natural egg case protein (Lin et al., 2013). The highly repetitive silk gene arrangement and the unusual mRNA secondary structure result in inefficient translation that limits the size of the silks produced in E. coli. To minimize the presence of truncated silk proteins and allow the extracellular secretion of silks, the mythylotropic yeast P. pastoris has been used. Fahnestock and Bedzyk (1997) produced N. clavipes spider dragline silks in yeast P. pastoris. Synthetic genes were expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein. Results demonstrated that P. pastoris can be used to successfully produced produce long repetitive proteins (Fahnestock and Bedzyk, 1997). Spider silks from Araneus diadematus (ADF-1, 2 and 3) have also been expressed using the type III secretion system of a gram-negative, non-spore-forming, enterobacterium Salmonella. The authors reported yield values range from 90 to 410 nmol L −1 h −1 that is similar to 10 mg L −1 h −1 for a protein the size of ADF-2. The results demonstrated the feasibility to use Salmonella for the large-scale spider silk production (Widmaier et al., 2009). Mammalian cell lines, such as bovine mammary epithelial alveolar and baby hamster kidney cells, were used to express MaSp1 and MaSp2 (Lazaris et al., 2002). The cells expressed recombinant proteins; however, as size of silk gene increased, the yield decreased dramatically due to inability of mammalian cells to cope with large repetitive sequences. Several factors have attributed to the decreased yields including, but not limited to, inefficient transcription, insufficient secretion, low copy numbers and translational limitations. The produced silk proteins were spun into fibres, and their mechanical properties were tested. It was noted that those recombinant silks that were produced without a His-tag demonstrated better mechanical properties compared with fibres made of silk proteins with a His-tag (i.e. fibres were brittle). Similar problems (i.e. transcription and translation limitations) have been reported when green monkey kidney fibroblastlike cell lines (COS-1) were used to express a 636-base pair gene fragment of MaSp1 from the African spider Euprosthenops sp. (Grip et al., 2006). Table 1 summarizes genetic engineering approaches, cloning strategies, and production yields of recombinant silk proteins produced in unicellular heterologous host systems. Multicellular organisms as heterologous host systems. Due to the low production rate and instability (i.e. frequent homologous recombination, deletions, transcription errors, translation pauses) of spider silk repetitive genes in unicellular organisms, multicellular organisms such as insects, plants and mammals have been studied for production of recombinant spider silk proteins. Silkworms (B. mori) can be farmed and produce cocoons containing large quantities of silkworm silk known as fibroin (Vepari and Kaplan, 2007;Hu and Kaplan, 2011). Moreover, to produce a solid thread, silkworms employ a spinning process that is similar to that used by spiders to make dragline silk. The presence of a natural silk production system in silkworms makes them excellent candidates to investigate as heterologous hosts for spider silk production. There have been several reports of the transfer of silk genes from spiders to silkworms (Motohashi et al., 2005;Zhang et al., 2011;Teulé et al., 2012b). Baculovirus-based expression systems have been used to introduce silk genes into a heterologous host. Baculovirus infects silkworms and allows for production of large quantities of heterologous proteins in a short period of time (Motohashi et al., 2005). Using this expression system, MaSp1 from N. clavipies linked with an enhanced green fluorescent protein (EGFP) fusion protein was cloned and expressed in the B. mori cell line (BmN) and larvae (Zhang et al., 2008). The authors reported successful production of a recombinant EGFP-MaSp1 fusion protein in both systems. In the silkworm larvae, a total of 6 mg of fusion protein was expressed, whereas in the BmN cells, 5% of the cell total protein was occupied by this recombinant silk. The major limitations of this expression system were low solubility of silk proteins and inability to assemble spider silk fibres. It was shown that more than 60% of the fusion proteins formed aggregates via self-assembly. To overcome solubility issues, MaSp1 C-terminal domain is to be incorporated due to its role to prevent aggregate formation. To produce fibres, germlinetransgenic silkworms (B. mori) were produced by injecting silkworm eggs with a piggyBac transformation vector carrying MaSp1 sequence (Wen et al., 2010). The insects were capable of spinning fibres and forming cocoons containing recombinant spider silk. However, the mechanical properties of the fibres were lower than dragline MaSp1 silk due to the low ratio of MaSp1 in the total silk protein. In a recent effort to develop tough fibres, transgenic silkworms encoding chimeric silkworm/spider silk proteins were produced using piggyBac vectors (Teulé et al., 2012b). The vector, used previously by the Tamada group (Kojima et al., 2007) included the B. mori fibroin heavy chain promoter and enhancer, a genetic sequencing encoding a 78 kDa synthetic spider silk protein, and an EGFP tag. Strong EGFP signals were observed by fluorescence (Fig. 5). The composite fibres were tougher than the parental silkworm silk fibres and as tough as native dragline spider silk fibres. These results demonstrate that silkworms can be engineered to generate composite silk fibres containing stably integrated spider silk protein sequences, which significantly improved overall mechanical properties. Transgenic plants have also been investigated as heterologous host systems to produce recombinant spider silks. Advances in genetic engineering technology and transformation methods make it possible to produce non-plant proteins in plants (Yang et al., 2005;Rech et al., 2008). Moreover, one plant offers several different expression systems, such as seeds, leaves, tubers and roots with potential for organelle-specific accumulation of recombinant proteins (Scheller and Conrad, 2005). Stable transgenic tobacco and potato lines were engineered to express MaSp1 genes from N. clavipes ranging from 420 to 3600 bp (Scheller et al., 2001). Recombinant spider silk proteins were found in the endoplasmic reticulum (ER) of tobacco and potato leaves at the accumulation of 2% of total soluble protein. Moreover, the production levels were independent of the size of silk genes. Purification was performed using high temperature treatment followed by acidification and ammonium sulphate precipitation. Additionally, recombinant MaSp1-like proteins were also produced in the leaves and seeds of Arabidopsis (small flowering plants related to cabbage) as well as in somatic soybean embryos (Barr et al., 2004). The expression of recombinant silks was driven by the 35S promoter in leaves and the β-conglycinin α' subunit promoter in seeds and somatic soybean embryos. The results demonstrated that recombinant spider silk proteins had higher accumulation levels in seeds than in the leaves. Recently, a native-sized FLAG protein from N. clavipes was cloned and expressed in the ER of tobacco plant (Nicotiana benthamiana) leaf cells using an intein-based posttranslational protein fusion technology (Hauptmann et al., 2013). This method avoids the need for highly repetitive transgenes resulting in a higher genetic and transcriptional stability. Additional details on production of fibrous proteins in plants can be found elsewhere (Scheller and Conrad, 2005). Transgenic production of recombinant silk proteins in mammary glands and secretion of them into milk has been investigated in mice and goats (Williams, 2003;Xu et al., 2007). In case of transgenic mice production, MaSp1 and MaSp2 synthetic genes (40 and 55 kDa) were synthesized and cloned into the pBC1 expression vector (Invitrogen, Carlsbad, CA, USA) together with a goat β-casein signal sequence. The chimeric gene construct was microinjected into pronuclei of fertilized eggs of Kunming white mice (Xu et al., 2007). Southern blot analysis was used to identify mice containing transgene construct as well as a copy number of transgene. The expression of dragline silk in milk was confirmed by Northern blot followed by Western blot analysis. The results revealed that transgenic mice were capable of expressing recombinant silk proteins in their milk. Geneti-cally engineered (transgenic) goats capable of expressing spider silk proteins based on the sequences of MaSp1 and MaSp 2 were produced by Nexia Biotechnologies, and later by the Lewis group (Lazaris et al., 2002;Service, 2002). Silk protein expression was controlled by the β-casein promoter and was expressed in the milk of transgenic goats. Silk proteins were observed only in mammary tissues as confirmed by Western blot (Steinkraus et al., 2012). Maximum yields observed for the recombinant silk production in transgenic animals were low (11.7 mg l −1 ) when compared with bacterial expression (Table 1 and Table 2). Today, the large-scale production of recombinant silk proteins from transgenic animals is relatively expensive and challenging in terms of animal breeding. Future outlook Over the last decade there has been considerable progress in understanding the genetic organization encoding spider silks. Cloning, expression and purification of spider silks has improved, and the self-assembly and processing of spider silk into many material formats is now better understood. Recently a native-sized (285 kDa) recombinant protein of the spider N. clavipes was produced and spun into a fibre displaying mechanical properties comparable to those of the native silk, indicating a breakthrough in standard recombinant production of spider silks. Moreover, a variety of heterologous host systems have been explored to produce different types of recombinant silks. For example, transgenic silkworm/spider silk production systems have been developed to produce tough fibres. It is possible to mix and match key modules via recombinant approaches, providing additional insights into the role of individual modules and effects of neighbouring elements on properties. This approach should lead to the development of custom structures built from specific silk elements. Future challenges will include development of tailor-made production systems for recombinant silks keeping in mind differences in chemical and physical properties of individual silk modules, scaling up silk production, prevention of the formation of aggregates and matches to the mechanical properties of silk fibres.	2016-05-12T22:15:10.714Z	2013-10-11T00:00:00.000	{ "year": 2013, "sha1": "3b70ef29e73a23768e8f5d23a9e146cc27536542", "oa_license": "CCBY", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1751-7915.12081", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "3b70ef29e73a23768e8f5d23a9e146cc27536542", "s2fieldsofstudy": [ "Biology", "Materials Science" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
14050253	pes2o/s2orc	v3-fos-license	Differential fast fixed-point algorithms for underdetermined instantaneous and convolutive partial blind source separation This paper concerns underdetermined linear instantaneous and convolutive blind source separation (BSS), i.e., the case when the number of observed mixed signals is lower than the number of sources.We propose partial BSS methods, which separate supposedly nonstationary sources of interest (while keeping residual components for the other, supposedly stationary,"noise"sources). These methods are based on the general differential BSS concept that we introduced before. In the instantaneous case, the approach proposed in this paper consists of a differential extension of the FastICA method (which does not apply to underdetermined mixtures). In the convolutive case, we extend our recent time-domain fast fixed-point C-FICA algorithm to underdetermined mixtures. Both proposed approaches thus keep the attractive features of the FastICA and C-FICA methods. Our approaches are based on differential sphering processes, followed by the optimization of the differential nonnormalized kurtosis that we introduce in this paper. Experimental tests show that these differential algorithms are much more robust to noise sources than the standard FastICA and C-FICA algorithms. I. INTRODUCTION B LIND source separation (BSS) methods [1] aim at restoring a set of unknown source signals from a set of observed signals . The latter signals are in many cases linear instantaneous or convolutive mixtures of the source signals. Convolutive mixtures read (1) where and are the source and observation vectors, denotes the convolution operator, and the mixing matrix is composed of the impulse responses of unknown mixing filters. This general framework includes linear instantaneous mixtures. Then, reduces to a constant scalar mixing matrix and the observations read (2) Here, we assume that the signals and mixing matrix are realvalued and that the sources are zero-mean and mutually statis-Manuscript received August 1, 2006;revised November 8, 2006. The associate editor coordinating the review of this manuscript and approving it for publication was Prof. Simon J. Godsill tically independent, and we propose BSS methods based on independent component analysis (ICA). Moreover, we consider the underdetermined case, i.e., , and we require that . Various analyses and BSS methods have been reported for these difficult mixtures, most often for linear instantaneous ones [2]- [15] but also in the convolutive case [16]- [20] or even for nonlinear mixtures [21]. Many of them assume source sparsity [5], [6], [9]- [13], [15]- [18], [20], [21]. Some other methods set other restrictions on the sources [8], [13], [14], [16], [19] or on the mixing conditions [2], [19]. Discrete sources are especially considered in some cases [3], [4], [7]. Here, we aim at avoiding all these constraints. In [22], we introduced a general differential BSS concept for processing underdetermined mixtures. In its standard version, we consider the situation when (at most) of the mixed sources are nonstationary while the other sources (at least) are stationary. The nonstationary sources are the signals of interest in this approach, while the stationary sources are considered as "noise sources." Then, our differential BSS concept achieves the "partial BSS" of the sources of interest, i.e., it yields output signals which each contain contributions from only one of these sources, still superimposed with some residual components from the noise sources (this is described in [22]). However, we point out that this partial separation can be considered as a pseudocomplete separation task in situations when one does not aim at separating the noise sources since they do not include information. This method can be of practical use for the noisy "cocktail party" scenario, when some stationary noise sources are present in addition to the speech signals to be separated. One may also use our approach in multiple-input-multiple-output (MIMO) communication systems, in which received signals are often disturbed by stationary noise sources in real applications. This method may also be applied to biomedical signals, which often contain various stationary noise components. Although we first defined this differential BSS concept in a quite general framework in [22], then we only applied it to a simple but restrictive BSS method, which is especially limited to mixtures, only involving two strictly causal moving average (MA) mixing filters (i.e., no instantaneous mixing), and based on slow-convergence algorithms. Here, we introduce much more powerful BSS criteria and associated algorithms, based on differential BSS, for both linear instantaneous and convolutive mixtures. Our instantaneous method is obtained by extending to underdetermined mixtures the kurtotic separation criterion [23] and the associated, fast converging, fixed-point, FastICA algorithm [24]. In the convolutive case, we extend to underdetermined mixtures the time-domain fast fixed-point algorithm restricted to overdetermined convolutive ICA (i.e., ) that we recently presented in [25]. We thus keep the attractive features of the latter algorithm. This paper is organized as follows. In Section II, we describe our fast fixed-point algorithm for underdetermined linear instantaneous mixtures derived from the differential source separation concept. In Section III, first we summarize the principles of our previous method, that achieves fast fixed-point separation for (over)determined convolutive mixtures, and then, derive from this a differential extension that aims at achieving the partial separation of the sources of interest. Section IV consists of experimental results which compare our differential instantaneous and convolutive algorithms with the associated standard ones. Conclusions are derived from these investigations in Section V. A. New BSS Criterion Based on Differential Kurtosis The standard FastICA method [24], which is only applicable to linear instantaneous mixtures with (or ), extracts a source by means of a two-stage procedure. The first stage consists in transferring the observation vector through a real matrix , which yields the vector In the standard FastICA method, is selected so as to sphere the observations, i.e., so as to spatially whiten and normalize them. Then, the second stage of that standard method consists in deriving an output signal as a linear instantaneous combination of the signals contained by , i.e., where is a vector, which is constrained so that . This vector is selected so as to optimize the (nonnormalized) kurtosis of , defined as its zero-lag fourth-order cumulant (5) Now, consider the underdetermined case, i.e., . We again derive an output signal according to (3) and (4). We aim at defining new criteria for selecting and , in order to achieve the previously defined partial BSS of the sources of interest. To this end, we apply the general differential BSS concept that we described in [22] to the specific kurtotic criterion used in the standard FastICA method. Therefore, we consider two times and , then introduce the differential (nonnormalized) kurtosis that we associate with (5) for these times. We define this parameter as (6) Let us show that, whereas the standard parameter depends on all sources, its differential version only depends on the nonstationary sources. Equations (2)-(4) yield (7) where the vector (8) includes the effects of the mixing and separating stages. Denoting with , the entries of , (7) implies that the output signal may be expressed with respect to all sources as (9) Using cumulant properties and the assumed independence of all sources, one derives easily (10) where is the kurtosis of source , again defined according to (5). The standard output kurtosis (10), therefore, actually depends on the kurtoses of all sources. The corresponding differential output kurtosis, defined in (6), may then be expressed as (11) where we define the differential kurtosis of source in the same way as in (6). Let us now take into account the assumption that sources are nonstationary, 1 while the other sources are stationary. By , we denote the set containing the indices of the unknown nonstationary sources. The standard kurtosis of any source with then takes the same values for the times and , so that . 2 Then, (11) reduces to (12) This shows explicitly that this differential parameter only depends on the nonstationary sources. Moreover, for given sources and times and , it may be seen as a function of the set of variables , i.e., is equal to (13) where the parameters are here equal to the differential kurtoses of the nonstationary sources. The type of function defined in (13) has been widely studied in the framework of standard kurtotic BSS methods, i.e., methods for the case when , because the standard kurtosis used as a BSS 1 The number N of nonstationary sources is assumed to be equal to the number P of observations hereafter, except in Section III-E. 2 Note that the "complete" stationarity of the sources s (n) with q = 2 I is sufficient for, but not required by, our method: We only need their differential kurtoses (and their differential powers below) to be zero for the considered times. Apart from the stationarity case, the differential kurtoses are zero for instance when the peakednesses (measured by the normalized kurtoses defined as kurt (n)=Efs(n) g ) and the squared powers of the sources vary inversely proportionally. criterion in that case may also be expressed according to (13). 3 The following result has been established (see [1, p. 173] for the basic two-source configuration and [23] for a general proof). Assume that all parameters with are nonzero, i.e., all nonstationary sources have nonzero differential kurtoses for the considered times and . Consider the values of the function in (13) on the -dimensional unit sphere, i.e., for such that (14) The results obtained in [1] and [23] imply in our case that the maxima of the absolute value of on the unit sphere are all the points such that only one of the variables , with , is nonzero. Equation (9) shows that the output signal then contains a contribution from only one nonstationary source (and contributions from all stationary sources). Thus, we reach the target partial BSS for one of the nonstationary sources. The last aspect of our method that must be defined is how to select the matrix and to constrain the vector (which is the parameter controlled in practice, unlike ) so that the variables meet condition (14). First, we define the differential power of a signal between the two times and by (15) By using the independence of the source signals, it may be shown easily that, similarly to the differential kurtosis (12), we have (16) The BSS scale indeterminacy makes it possible to rescale the differential powers of the nonstationary sources up to positive factors. Therefore, provided these differential powers are strictly positive for the considered times and , they may be assumed to be equal to 1 without loss of generality. Then, (16) reads (17) so that the constraint (14) can be expressed in terms of unit differential power for the output signal . Then, we introduce a differential extension of the sphering stage of standard kurtotic methods, i.e., we aim at deriving a matrix so that the normalization of the differential power of can be done by normalizing the extraction vector . To this end, we define the differential correlation matrix of as 3 In standard approaches, the summation for q 2 I in (13) is performed over all P = N sources and the parameters are equal to the standard kurtoses kurt (n) of all these sources. However, this has no influence on the later discussion, which is based on the general properties of the type of functions defined by (13). where is the standard correlation matrix of at time . Let us now consider the Schur decomposition [26, p. 393] of the real symmetric matrix (19) where is a real orthogonal matrix and is a diagonal matrix. It may be shown as follows that the entries of are strictly positive. Equations (2) and (18) yield (20) where is a diagonal matrix due to source independence and its entries with indices are null due to the stationarity of the corresponding sources. It may be checked easily that the columns of with indices yield no contributions in the right-hand term of (20), so that we also have (21) where consists of the columns of with indices and the diagonal matrix contains the differential powers of the nonstationary sources for times and . While (20) involves nonsquare matrices, (21) only contains matrices. If we assume that is invertible, [26,Th. 4.2.1,p. 141] implies that is positive definite if and only if is also positive definite. Therefore, if we again assume the differential powers of the nonstationary sources to be strictly positive (for times and ), (19) and (21) show that all diagonal entries of are strictly positive. Moreover, if these differential powers are rescaled to unity, then and (21) becomes (22) Since only has positive entries, it has a real-valued square root, so that we can define by (23) Then, with defined by (3), we have (24) Let us now consider the output signal defined by (4). We have (25) By considering the relations (17) and (25), we obtain (26) By constraining our vector to have unit norm, we hence constrain the vector to be on the unit sphere. Then, as explained previously, the maxima of the absolute value of on the constraint surface rigorously correspond to the partial separation points. B. Summary of Proposed Methods The first practical method which results from the previous analysis consists of the following steps. Step 1) Select two nonoverlapping bounded time intervals for estimating the statistical parameters (kurtosis, correlation, and power) at two times and . In most cases, we obtained better results with intervals such that the differential powers of the sources (which can be roughly evaluated by the differential powers of the observations) are high. These intervals must be such that all nonstationary 4 sources have positive differential powers [defined as in (15)] and nonzero differential kurtoses. 5 The signs of the differential powers of the sources can be obtained by estimating the differential correlation matrix and by computing its Schur decomposition . Indeed, if all its eigenvalues are strictly positive, we proved that the sources of interest all have strictly positive differential powers (if all these eigenvalues are strictly negative, we can permute the two time intervals so as to make them all positive). Step 2) Derive the matrix from the previously defined matrices and . This matrix performs a "differential sphering" of the observations, i.e., it yields a vector defined by (3) which meets (24). Step 3) Create an output signal defined by (4), where is a vector which satisfies and which is adapted so as to maximize the absolute value of the differential kurtosis of , defined by (6). Various algorithms may be used to achieve this optimization, especially by developing differential versions of algorithms which were previously proposed for the case when . The most classical approach is based on gradient ascent [1]. Here, 4 "Nonstationary" here means "long-term nonstationary." More precisely, all sources should be stationary inside each of two "short" time intervals associated with times n and n , so that their statistics may be estimated for each of these intervals, by time averaging. This corresponds to "short-term stationarity." Then, the aforementioned "sources of interest" (respectively, "noise sources") consist of source signals whose statistics are requested to vary (respectively, not to vary) from one of the considered time intervals to the other one, i.e., sources which are "long-term nonstationary" (respectively, "long-term stationary"). 5 Note that the hypothesis Dkurt (n ; n ) 6 = 0; 8q 2 I does not require the source distributions to have different peakednesses at the two times n and n . Indeed, the peakedness is measured by the normalized kurtosis defined as kurt (n)=Efs(n) g , so that a source can have different nonnormalized kurtoses but the same peakedness at the two times as soon as this source has different powers at these times. Then, except in the very specific case when the normalized kurtoses and the squared powers of the sources vary inversely proportionally, we are sure that the sources have nonzero differential kurtoses. we preferably derive an improved method from the standard fixed-point FastICA algorithm [24], which yields two advantages with respect to the gradient-based approach, i.e., fast convergence and no tunable parameters. Briefly, as the standard Fas-tICA algorithm, our linear instantaneous differential fixed-point ICA algorithm, denoted LI-DFICA hereafter, takes advantage of the fact that if an extraction vector optimizes the criterion , then the gradient of has the same direction as (see [1, p. 178]). Therefore, our update expression uses the gradient of the differential kurtosis which is derived in Appendix A, i.e., it is based on the assignment (27) More precisely, starting from a random unit-norm vector , our LI-DFICA algorithm then consists in iteratively performing the following operations: 1) differential update of (28) where the statistical parameters are estimated by time averaging; 2) normalization of , to meet condition , i.e., Step 4) The nonstationary source signal extracted as in Step 3) is then used to subtract its contributions from all observed signals. The resulting signals are then processed by using again the previously described complete procedure, thus extracting another source, and so on until all nonstationary sources have been extracted. This corresponds to a deflation procedure, as in the standard FastICA method [24], except that a differential version of this procedure is required here again. This differential deflation operates in the same way as the standard deflation, except that the statistical parameters are replaced by their differential versions in order to estimate the entries of the mixing matrix . Indeed, we prove in Appendix B that the scale of the contribution of an extracted source in the th observation can be obtained by estimating the differential correlation defined by (30) which is shown to be equal to the entry of the mixing matrix . Instead of the previously described deflation-based version of our LI-DFICA method, a differential extension of the symmetric approach described in [24] can be considered. The resulting symmetric LI-DFICA method consists of the Steps 1) and 2), followed by iterations on the following operations: 1) differential update of vectors , with , according to (28); 2) symmetric orthogonalization of the vectors , i.e., (31) where . C. Convergence Proofs Let us consider the update expressions (28) and (29) of our differential deflation-based algorithm. The output signal may be expressed with respect to the sources according to (7) with and . Moreover, we proved that the differential kurtosis of only depends on the sources of interest (whose indices are in the set ) and on the associated mixing submatrix . Therefore, when considering the overall component of corresponding to the nonstationary sources, we can make the change of variable where the matrix combines the mixture and the differential whitening processes for the nonstationary sources. Then, we have (32) where the column vector only contains the values of the nonstationary sources, i.e., with . For the sake of legibility, we here omit the considered two times and in the notation for differential kurtosis. Then, all the components but so that quickly become small compared to . With the normalization which implies because of the orthogonality of the matrix , we conclude that and . Thus, we proved that converges towards a vector with only one nonzero entry (equal to ). This yields the partial separation of the sources of interest, as . Thus, we proved the global convergence [i.e., whatever ] of our algorithm. Moreover, this convergence is cubic (as with the (over)determined FastICA algorithm [24]), which means very fast convergence. For our symmetric algorithm, the same approach as in [27] can be used in the differential case. Indeed, in the (over)determined case, Oja proved that when the updates obey the rules (44) for the th component of each extraction vector , these updates combined with the symmetric orthogonalization stage imply cubic convergence to the separation points and the stability of these separation points, as in the deflation algorithm. By noticing that our differential algorithm yields (41), which is equivalent to (44) with , except that the standard kurtoses are replaced by their differential versions , we thus prove rigorously the cubic convergence of our symmetric differential algorithm. A. Mixture Model and Goal Here, we consider convolutive mixtures defined by a set of unknown filters with impulse responses which form a matrix function . The overall relationship between the source and observation vectors and then reads Each source is here assumed to be expressed as (46) where is a filter impulse response and is the innovation process of . Denoting , we can then express the mixing (45) as (47) where with . We make the following assumptions concerning the aforementioned mixture model. • The process is real-valued, zero-mean, temporally independent and identically distributed (i.i.d.) and spatially independent, i.e., its components are statistically independent from each other and do not necessarily have the same distribution. • The function matrices and thus correspond to causal and finite impulse response (FIR) filters and are nonsingular. Note that infinite impulse response (IIR) systems can also be approximated by equivalent (high-order) FIR models. Convolutive BSS typically aims at estimating the contributions of all sources in each observation, i.e., . In convolutive deflation-based methods such as [28], this is achieved as follows. 1) Extract the innovation process of a source from the observations. 2) Identify coloring filters and apply them to in order to recover the contributions of in each observation. 3) Subtract these contributions from all the observations. 4) Set . If , go back to Step 1) in order to extract another source. B. Previously Reported Approach for (Over)Determined Convolutive Mixtures Recently, we proposed a time-domain fast fixed-point algorithm for determined (i.e., ) and overdetermined (i.e., ) convolutive mixtures [25]. This algorithm, denoted C-FICA hereafter, may be seen as a convolutive extension of FastICA and keeps its attractive features. Here, we briefly describe the principles of the kurtotic version of this previous approach, as we will then apply our differential BSS concept to that algorithm in order to introduce a new partial separation method for underdetermined mixtures involving some nonstationary sources. The first step of our (over)determined approach performs a "convolutive sphering" of the observations, defined as follows. At any time , we consider the column vector (48) which contains entries. We derive the -entry column vector defined as where is an matrix chosen so that With respect to , operation (49) may, therefore, be considered as conventional sphering, which consists of principal component analysis and normalization. Now, with respect to the original observations , this may be interpreted differently: Equations (48) and (49) show that the signals are convolutive mixtures of the signals . Equation (50) then means that the signals are created so as to have unit variances and to be mutually uncorrelated, which may be seen as a spatio-temporal whitening and normalization of the observations . Let us denote by the extracted signal (51) where is an -entry extended column vector of extraction coefficients which, together with (48) and (49), yields a convolutive combination of the observations. The power of reads . By constraining so as to meet (50), we get . Our method then consists in maximizing the absolute value of the nonnormalized kurtosis of defined by (51) under the constraint so that has unit power. We proved in [25] that this constrained optimization yields an estimate of a delayed and scaled source innovation process , under some conditions. Therefore, this C-FICA algorithm based on our modified vector extracts one source innovation process as follows. • Initialize to a value , e.g., using the approaches presented below. • Repeat the following Steps 1) and 2) until convergence 1) 2) The aforementioned initial value of may be selected randomly. An improved approach was derived in [25] from the relationship which exists between our vector and the coefficients of the FIR filters of Tugnait's approach [28], which derives an output signal as where are noncausal FIR filters in practice. Indeed, in [25], we proved that (55) where the row vector consists of the impulse response coefficients of the filters to . This relation lets us initialize our vector as in Tugnait's method, i.e., with unit filters , so that is the sum of all observations . That corresponds to defined as This initialization of provided better experimental results than a random one and is also used in Section III-C for underdetermined convolutive mixtures. As stated previously, this extraction stage provides an estimate of a source innovation process up to a delay and a scale factor. Then, we can color it to obtain each contribution of the th source in the th observation . This can be done by deriving the noncausal coloration filters which make the signals be the closest to in the mean square sense [29]. This was achieved in [25] by noncausal FIR Wiener filters [30], whose impulse response coefficients form vectors defined by (58) where is the autocorrelation matrix of the signal and is the cross-correlation vector of the signals and . Note that the autocorrelation matrix has a highly regular Toeplitz structure and there are a number of efficient methods [30] for solving the linear matrix equation (58). After subtracting the contributions from all observations, we obtain another mixture configuration with sources. The extraction stage must then be iterated as explained in Section III-A to extract the innovation process of another source. C. Extension of Convolutive Approach to Underdetermined Mixtures Here, we aim at extending our C-FICA algorithm for convolutive BSS to the underdetermined case, using the differential BSS concept that we introduced in [22]. The resulting algorithm is therefore denoted C-DFICA hereafter. Let us again define by (48), (49), and (51) where and will be selected as explained further in this section. As in Section II, we denote by and , respectively, the nonnormalized kurtoses of for two times and , and we define its nonnormalized differential kurtosis by (6). Combining the definition (48), (49), and (51) of with (47) shows that is a linear combination of delayed versions of the processes i.e., where and are derived from the orders of the FIR filters involved in (47) and (48). The processes are here still assumed to be real-valued, zero-mean, and mutually and temporally independent. of them are now assumed to be long-term nonstationary (i.e., not identically distributed) and correspond to the sources of interest, while the other are stationary and correspond to the "noise sources." Using the multilinearity of the kurtosis for independent random variables, we derive from (6) and (59) (60) where (61) is defined as in (6). As in the linear instantaneous case, let us denote by the set containing the indices of the unknown nonstationary sources. The previous hypotheses of our differential concept mean that only the processes with are assumed to have the same kurtosis for different times, i.e., (62) Thus, we have (63) Hence, we see that the noise sources (whose indices do not belong to the set ) are invisible in our differential extraction criterion. As an extension of the linear instantaneous case, we here consider the values of (63) on the dimensional unit sphere 6 where , i.e., such that (64) First, it may be shown easily that the differential power of , again defined by (15), here reads (65) Each process with is assumed to exhibit nonstationarity from time to . However, it is here assumed to be identically distributed for all times when is varied from to . In practice, this means that we select and such that , and that should exhibit long-term nonstationarity between the times and but short-term stationarity inside each time window with or . Then, the terms in (65) do not depend on , i.e., (65) only involves a single differential power for each nonstationary source. These differential powers may again be rescaled by positive factors because of the BSS scale ambiguity. Hence, when they are strictly positive, they may be assumed to be equal to 1 so that (65) 6 Convolutive mixtures entail a slight approximation concerning which points of this sphere may be reached, as explained in Section III-E. so that due to (66) (69) Therefore, varying so that yields a simple method in the underdetermined convolutive case to constrain our vector to be on the unit sphere (64). The optimization of the absolute value of defined in (63) under the constraint (64), therefore, belongs to the same generic problem as in (13)-(14), with a set of variables here denoted instead of . Applying again the results of [1] and [23] that are used in Section II-A here guarantees that the maxima of on the unit sphere correspond to all the points such that only one of the considered variables , with , is nonzero. Equation (59) shows that the output signal then only consists of a delayed and scaled version of the process associated to a nonstationary source (and contributions from all stationary sources). We then color to estimate its contributions in all observations. This is done by adapting the Wiener solution that we used in [25], i.e., by introducing a differential Wiener filtering process, that we define in Section III-D. D. Summary of Proposed Method We propose the following procedure to achieve the fast partial separation of underdetermined convolutive mixtures. This procedure is based on an adapted fixed-point algorithm and on our differential Wiener filtering process in order to recover the contributions of the sources in the observations in the framework of a deflation approach. Step 1) Select two nonoverlapping bounded time intervals using the same type of approach as in Section II-B. Step 2) Compute an estimate of the differential correlation matrix of the expanded observation vector . Then, perform the symmetric Schur decomposition of that matrix. This yields a matrix whose columns are the unit-norm eigenvectors of the estimate of and a diagonal matrix which contains the eigenvalues of the estimate of . Then, derive the matrix . We thus obtain a vector defined by (49) which meets (67). Step 3) Initialize the extraction vector , using (57) as in the C-FICA algorithm but with the new matrix as defined in Step 2). Create an output signal defined by (51), where is a normalized vector which is adapted so as to maximize the absolute value of the differential kurtosis of . The same approach as in Step 3) of Section II-B may be used to this end. This here yields the following C-DFICA algorithm: a) differential update of (70) b) normalization of , to meet condition , i.e., Step 4) The nonstationary process extracted as in Step 3) is then colored by means of our differential Wiener filtering process so as to recover the contributions of the corresponding source in all observations. This filter minimizes the differential power of the difference between an observation and a noncausal filtered version of the signal . In Appendix C, we show the consistency of this minimization and we derive the expression of the coloration filters which achieve it, i.e., As with the kurtotic and power statistical functions, Appendix C proves that this coloration process does not depend on the noise sources. The resulting signals are then subtracted from the observations. Steps 2)-4) are then iterated until we have extracted all source innovation processes. We do not introduce here a symmetric version for this differential algorithm intended for convolutive mixtures. Indeed, as detailed in Section III-E, the considered convolutive mixtures may also be expressed in terms of instantaneous mixtures and therefore correspond to a considerably higher dimensionality than in Section II-B for our optimization space. Then, we save major computational and memory resources by extracting the innovation processes one by one. E. Convergence Proof Inside each of the mixed signals of defined by (48), we here only consider the overall component which corresponds to the nonstationary sources, since the above BSS criterion only depends on this component. The analysis in [25] shows that these convolutive mixtures can be interpreted as linear instantaneous mixtures of multiple delayed and scaled versions of the processes with . In particular, that analysis entails that if denotes the order of in (47), and if (73) is met, where is the number of lags, is the number of mixtures and is the number of nonstationary sources, then is an (over)determined instantaneous mixture of delayed and scaled versions of the processes with . The convergence proof obtained in Section II-C in the instantaneous case can then be applied to our differential convolutive algorithm and, therefore, guarantees global cubic convergence here again. If (73) is not met, which is especially the case when , the reformulated instantaneous mixture is underdetermined. This underdetermination is related to the finite order of the equivalent extraction filters in (54). However, when the ratio associated with (73) tends to 1 (which is the case when and is large), our configuration is nearly determined i.e., the equivalent instantaneous mixing matrix is almost square. Then, one still almost obtains an estimate of one process by maximizing the absolute value of the differential nonnormalized kurtosis under unit differential power constraint. 7 IV. EXPERIMENTAL RESULTS Here, we aim at comparing the performance of our new methods for underdetermined mixtures with the nondifferential ones i.e., the FastICA and C-FICA algorithms. First, we tested our deflation method LI-DFICA for linear instantaneous mixtures in the case and , where the two nonstationary sources correspond to some bass and piano sound signals [31] with 100 000 samples on each of the time domains and . The three noise sources are stationary signals corresponding to uniform, Gaussian, and Laplacian distributions with the same power. Their scale factors and resulting powers are varied in our tests in order to investigate the evolution of performance with respect to the input signal-to-noise ratio (SNR ) of the observations. Our SNR criterion reads where is the contribution of the th source on the th sensor. We average the two SNR values associated with the two used time domains. For each scale factor applied to the normalized noise sources, we made 100 Monte Carlo simulations by varying the coefficients of the mixing matrix with a uniform distribution in . The performance of our BSS method is measured by its output signal-to-interference ratio (SIR ), which may be compared to the input SIR (SIR ) associated to the observations. The definitions of these parameters are provided in Appendix D. Fig. 1(a) shows that whatever the SNR , the SIR is higher for our differential algorithm compared to the standard one. Moreover, performance begins to fall significantly for SNR smaller than 10 dB for our differential algorithm, instead of 35 dB for the standard one. Besides, for our differential version, the SIR is greater than 30 dB for the first extracted source for SNR down to about 0 dB, instead of 20 dB for the standard algorithm. These results are obtained for a mean SIR of 5 dB. We also compared the symmetric version of our instantaneous differential algorithm with the standard symmetric FasICA algorithm in the same mixture configuration as previously. Another criterion is commonly used to evaluate performance for symmetric algorithms: We can directly consider the estimated nonstationary sources and compare them with the normalized original sources. This can be done by using the performance matrix which is the product of the mixing 7 The approximation that we mentioned in footnote 6 concerning the points of the unit sphere which may be reached in the convolutive case is related as follows to the previous discussion. In the differential linear instantaneous case, The resulting is averaged with respect to the index so as to obtain a unique criterion for a given performance matrix . Fig. 1(b) shows the same advantages as previously described for our differential algorithm compared to the standard one. This time, the performance index begins to fall significantly for SNR smaller than 15 dB (respectively, 35 dB) for the differential algorithm (respectively, the standard algorithm) and this criterion stays higher than 33 dB for SNR down to 0 dB (respectively, 16 dB). In this figure, the SIR of the source contributions, estimated by the differential correlation (30) as a postprocessing stage, is also represented. This shows for these symmetric algorithms that the criterion involving power normalization yields higher performance figures than the SIR criterion, which requires source contribution estimation. Note that these SIR of source contributions are slightly greater than those obtained with the deflation algorithm. In the convolutive case, we tested our C-FICA and C-DFICA algorithms for two long-term nonstationary artificial tenth-order colored sources driven by Laplacian processes. They are short-term stationary on two time windows of 100 000 samples. We used real mixing filters whose sixty fourth-order impulse responses were measured at the ears of a dummy head [32]. As in the linear instantaneous case, the noise sources are scaled uniform, Gaussian, and Laplacian signals whose scale factors are varied in order to change the SNR defined by (74). For each scale factor applied to the noise sources, we considered 153 different filter sets associated with different source positions. We only constrained the two sources of interest not to be very close to each other (the angular difference was taken greater than or equal to 20 ) and placed the noise sources at positions associated with the angles 90 , 30 , and 150 . As with the deflation versions of the instantaneous algorithms, we express performance in terms of SIR of source contributions (measured as explained in Appendix D), this time obtained by using the differential Wiener process (72). Fig. 2(a) represents the SIR of the standard and differential fixed-point ICA algorithms depending on the input SNR. As in the linear instantaneous case, the performance of our differential algorithm stays higher than the C-FICA algorithm whatever SNR . More precisely, it begins to fall significantly for SNR smaller than 15 dB (respectively, 35 dB) for our differential version (respectively, the standard one). For the first extracted source, the SIR stays greater than 15 dB for SNR down to 8 dB (respectively, 22 dB). In the last experiment, we aim at evaluating the robustness of our differential algorithm for convolutive mixtures depending on the number of samples available in each time domain. To this end, we fixed the scale factor applied to the stationary noises so as to obtain a mean SNR of 11 dB over all source positions and for each number of samples , we tested our differential algorithm on the 153 filter sets used in the previous experiment. Fig. 2(b) shows that the mean SIR stays higher than 15 dB for down to 50 000 samples. V. CONCLUSION In this paper, we have introduced new fast fixed-point algorithms for BSS in the case of linear instantaneous and convolutive underdetermined mixtures. They are inspired from the well-known FastICA algorithm and from our recent C-FICA method only intended for (over)determined convolutive mixtures. The LI-DFICA and C-DFICA approaches proposed here are based on a new criterion, called differential nonnormalized kurtosis, that exploits the stationarity of some "noise" sources to achieve the partial separation of the sources of interest. We introduced differential sphering processes so as to use parameter-free fast fixed-point algorithms to achieve the optimization of this new criterion and differential correlation and Wiener filtering methods to recover the source contributions in each observation. As with the FastICA algorithm, a symmetric version of LI-DFICA is also proposed in the instantaneous case. In addition to the attractivity of the limited restrictions that we set about the sources compared to some other approaches for underdetermined mixtures, experimental results show the efficiency of our algorithms in comparison with the associated (over)determined FastICA and C-FICA algorithms. APPENDIX A GRADIENT OF THE DIFFERENTIAL KURTOSIS The nonnormalized kurtosis of a zero-mean signal is defined by (76) By taking its first-order derivative with respect to , we obtain Note that in the nondifferential FastICA approach, the gradient expression (77) can be simplified because the sphering process implies that and . The gradient expression is thus (78) In our approach, the differential gradient reads (79) Using the relations and , we can simplify expression (79) to obtain (80) APPENDIX B DIFFERENTIAL CORRELATION Here, we suppose that the extracted signal only contains one source of interest superimposed with noise sources so that (81) with . The scale factor associated with in (81) can be deduced from the differential power of constrained to be equal to 1 and from the unit differential powers of the nonstationary sources, i.e., which imply that . In the following, we suppose without loss of generality that (otherwise, we can redefine as its opposite). Besides, the th observation reads Let us now compute the differential correlation between and , defined by because of the independence of the zero-mean sources, because of the stationarity of the noise sources and taking into account (82). Therefore, yields the scale factor associated to the contribution of the th source in the th observation. APPENDIX C DIFFERENTIAL WIENER FILTERING Let us consider the difference between observation and a filtered version of the previously extracted signal with , i.e., where are the coefficients of a noncausal FIR filter. Due to the hypotheses of Section III-A, is a causal FIR mixture of all innovation processes, i.e., (47) yields (87) Moreover (88) where the bounds on need not be detailed here as the corresponding terms eventually vanish below in (92). Therefore, if is high enough so that and , (86)-(88) yield (89) where the weights depend on , and . These terms and the bounds on need not be detailed here for the same reason as previously. The differential power of is defined as (90) Taking into account that all innovation processes are zero-mean, mutually and temporally independent, and that Since we assumed all differential powers with to be strictly positive, (92) shows that the minimization of with respect to corresponds exactly to (93) By comparing this result to (89), we conclude that a criterion for obtaining a modified observation where the contributions of the th source have been removed consists in minimizing . We use Newton's method to optimize this criterion, which is denoted as follows and which may be expressed as Knowing that Newton's algorithm reaches a stationary point in one iteration for quadratic criteria and that our criterion is quadratic positive, we prove that the vector corresponds to the global minimum of . Note that this expression is the differential version of the standard Wiener filter defined in (58). APPENDIX D DEFINITIONS OF SIR AND SIR Here, we first define the criteria used in Section IV to measure the performance of our deflation-based convolutive BSS method for underdetermined mixtures (the definitions for its instantaneous version follow). In each test, we first apply the mixtures of all sources to our BSS system and estimate its parameters, i.e., and . Then, we freeze these parameters. Since we aim at evaluating the quality of the partial separation only achieved between the nonstationary sources, we then only transfer these sources through the mixing stage and the previously defined BSS system. Thus, we obtain a set of partial extracted signals , with . For each of them, we compute its colored versions , respectively, associated to each observation . These colored signals are denoted as follows. For each source and each signal , we then define the associated SIR as the ratio of the "signal" and "interference" power as follows: • the "signal" is the ideal value of when it extracts , which is equal to the contribution of in , that we denote ; • the "interference" is the deviation of from its ideal value, i.e., . This yields SIR (102) For each source and output , we only consider the single SIR corresponding to observation providing the signal which has the highest power. Then, for each source index , we only consider the maximum with respect to output index of the values SIR . Then, we derive the mean of these values on both time domains. This yields a single SIR for each of the sources of interest, defining the output performance of our system, which is denoted SIR hereafter. The mean over all sources of these SIR may then be derived. We perform this mean when computing the overall SIR of symmetric methods. The input SIR available from the observations is defined similarly as follows. First, we define the input SIR associated to each source as SIR (103) Then, we derive the global SIR as the mean of the previous contributions over all sources and both time domains. ACKNOWLEDGMENT The authors would like to thank J. Chappuis for her participation in the early stages of this investigation.	2014-10-01T00:00:00.000Z	2007-07-01T00:00:00.000	{ "year": 2008, "sha1": "289d072e11a3a112f2b1ff493ca35b4a5dd519ec", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0806.3195", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "b2ccea1c7fc1a01c972f3d32b25c8bdf54229024", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Mathematics", "Physics" ] }
10599728	pes2o/s2orc	v3-fos-license	Serial Cultivation of Human Scalp Hair Follicle Keratinocytes A mcthod is described for the serial cultivallon of adult human hair follicle keratmocytes. Plucked scalp hair follices, placed on bovine eye lens capsules as a grow1h sub�1ra1e. give rise to quickly expancling colonies within a few days. After trypsinizalion, thc cells arc replated wilh irradiated 3T3 cells as ·feeders'. Using this combinalion of techniques the keratinocyte� can be subcultured up lo four limes. In thi\ way about 107 keralinocytes can be generated from one single hair follicle. Moreover. the technique enables cryogenic storage of lhe cells, allowing for instance, convenienl lransportalion. Subculturcd hair follicle keratinocytes can be platcd on glass coverslips. This allows immunofluorescence studies. The keratin cytoskclcton, visualized using an antiserum again�t human keratin. Key word.v: Serial c11/t11re; Hair �eratinocytes: lmmunofluorescence; Cryogenic storage. (Received December 21, 1982.) Over the last 15 years, several methods for establishing culture:, of normal skin keralino cytes have been developed. In carly attempts, disaggregated epidermal keratinocytcs were grown succcssfully on a plastic substrate (I, 3, 4, 9. 20). However, large inocula of the cells were necessary and serial cultivat1on was not satisfactory. In 1975. Rheinwald & Green developed a tcchnique for the serial cultivation of human skin kcratinocytes. using lethally irradiated Swiss 3T3 cells as a feeder laycr ( 11 ). Concomitanily, much �maller inocula of keratinocytes werc found sufficienl to obtain growth. In reccnt years, wc have employed human scalp hair follicles. being an extremely convenient biopsy material. for the detection of a number of genetic disorders (16). In order to rendcr this biopsy material more generally applicable, wc tried to cultivate keratinocytes, obtained from follicles by trypsinization, in the 'feeder-cell' technique. However, this was successful only when a !arge number of hair follicles (more than 100) were used, which is obviously unsuitable for screening studies. Therefore, a different method was developed for the cultivation of keratinocytcs from one single scalp hair follicle: a frcshly plucked follicle is simply place<l on lhe cuhure substrate. a bovme eye lcns capsule, and within a few days a quickly expanding colony of epilhclial cells is formed (I 7). This technique probably represents the easiest way of establishing primary cultures of purely epithelial cells from human skin, since fibroblasts have never been observed in cultures of anagen follicles. Recently, we succeeded in the subcultivation of human hair follicle keratinocytes growing on Jens capsules by transplanting small fragments of these cultures onto new capsules (18). However. the presencc of the lem, capsule impeded immunofluorcscence studies on the cultured hair follicle keratinocytes. Moreover. further cultivation of the cells provcd impossible after cryogenic storage. In the present report we describe a method for the generation of large numbers of keratinocytes originating from single hair follicles. It requires a combination of our culture technique with bovine eye lens capsulcs for the primary culture. with the 'feeder-cell' tcchnique for subcultivation. Cryogenic storage appearcd possible. and immunofluores cence studies could be performed, as is shown by staining of the keratin cytoskeleton. Serial c11ltioo1ion Primary cultures of keratinocytes. originating from the outer root sheath of human scalp hair follicles. were e�tabli�hed a� described earl i er ( 17). In brief, frcshly pluckcd hair follicles were placed on bovine eye lens capsules mounted in culture dishes (Epicuh. cspecially developed for lhis purpose and now commercially available from Sanbio, Nistelrode, The Netherlands). MEM was supplemented with 15 % fetal bovine serum. 0.4 µg/ml hydrocorti�one and 4 µg/ml insulin. The cultures were placed in an mcubator wi1h humidified air for an initial period of 3 days and subsequently transferred 10 an atmosphere of 5 5t C0 2 in air. After 15 days in culture. stratitied colonies of keratinocytes are formed around each hair follicle. At that time the culture dishe, "ere dismounted and the capsules with the adhering colonie� were transferred to 0.2 'K lrypsin and 0.5 % EDTA in phosphate-buffered saline. Excess solution wa� carefully removed after I min and incubation was continued for 5 more min al 3rc. After the addition of culture medium the cells were gently suspended using a syringe and sedimented al 9()0 g. The cell pellet was resuspended in culture medium. and the quantity of the cells was determined using a hemocytometer. The contents of one Epicult dish (about 4x 10' cells) were inoculated into a 90-mm culture dish containing 10 6 Sw1ss 3T3 cells. according to thc technique of Rheinwald & Green (11). The Swis\ 3T3 cells (CCL-92) "ere obtained f r om the American Type Culture Collection. The cells were irradiatcd with a dose of 3000 r u,ing a cobalt source, as described by Kondo ct al. ( 10). The medium used for subcultivation of hair follicle kcratinocytes was Dulbec co·s Modified Eagle Medium supplernented with 20% fetal bovine ,erum (Boehringer Mannheirn), 0.4 µg/ml hydrocortisonc (Sigma), 4 µg/ml bovine insulin (Organon. The Netherlands). 10 9 M cholera toxin (Schwarz/Mann). 10 ng/ml ep,dermal growth factor (Collabora1ive Research) and 50 µg/ml gentamycin. The cultures were refed every 3-4 days and werc rurther subcultured every 2-3 weeks using the 'feeder-cell' technique. Cr y of(enic storage For storagc in liquid nitrogen the celh were released from the lens capsule as described above. After onc wa,h with culture medium the cell pellet w, ;s resuspcnded in I ml of fetal bovine serum (inactivated: 30 min at 56°C) containing 5 o/c dimethyl sulphoxidc (DMSO). The freeze medium was at 4°C. The cell suspension was transferred into freeze vials with a silicon ga�ket between cap and tube (Nunc. Denmark) and immediately moved to a -80°C freezer. Under these conditions the cooling rate was about 1°C per min. After 24 h the vials were transferred lo a liquid nitrogen tank. To recover the cells from frozen storagc the vial W'dS warmed to 37°C. The suspension was diluted in culture medium 10 reduce 1he concentrationof DMSO and ccn1rifuged at 900 g. The cell pellet wa, inoculated into a 90mm dish in 1he prcsence of irradiated 3T3 cells. De1em1i11a1io11 of tlre colo11)·-formi111? efficiency In order to determine the colony-forming efliciency (CFE) after freezing of the cells. two primary cultures of keratinocytes growing on lens capsules \\ere trypsinized as described. Half of the cells were immediately plated together with 10 6 irradiatcd 3T3 cells in a 90-mm dish. The other half of thc cells were quickly frozen in freeze medium at -80°C. After 3 h the fr eeze vial was transferred to a liquid nitrogen container and after 3 more hours the vial was thawed. The cells were recovered and plated as described above. At day 5 the colonies with more than 10 cells were counted using a phase• contrast microscope. lndirect imm1111of111oresce11ce microscopy An antiserum specilic for keratins of human stratum corneum was prepared as described by Sun & Green (13). and tested by double diffusion in agar ad modum Yen et al. ( 19). The antiserum W-dS partially purified by 50� ammonium sulphate precipitation of the immunoglobulins. For indirect immunotluorescence microscopy. hair follicle keratinocytes originating from one single primary culture were seeded together with 2x 10' 1rradia1ed 3T3 cells onto sterili7ed glass coverslip� in a 50-mm culture dish. After 5 days of cultivation. the coverslips were briefly rinscd with phosphate buffercd salinc (PBS) and fixed in a 4 'k formaldehyde wlution in PBS for 15 min at room tempera- Acta Derm Venereol (Stockh) 63 ture. The fixed cells were treated for 5 min with prechilled acetone (-20°C), and air-dried. 100 µI of the partially purified keratin antiserum (diluted I : 20 in PBS) was placed on the coverslips and incubated for 45 min at 37°C in a humidified atmosphere. The coverslips were thoroughly rinsed using an incubator-shaker (New Brunswick Scientific Co .. USA) for 20 min at room temperature. while the butTer was changed three limes. fluorescein-conjugatcd swine anti-rabbit lgG (Dako, Denmark) wa� applied at a dilution of I : 40. After incubation fora further 45 min al 37°C, thc covcrslips were rinsed in PBS for 20 min as above and mounted cell-side down in glycerol/PBS (I: I) on slides. Nail polish was used to prevent evaporation. The preparations were viewed in a Zeiss microscope using epifluorescence ilumination and IOx and 95x oil-immersion objectives. Pholographs were taken with Kodak tri-X film. Serial cultivation Primary cultures of hair follicle cells werc used for subcultivation after 2 weeks of culture. Older cultures became progressively more stratified and contained an increasing number of keratinized cells. After 2-3 days in subculture, groups of 2--4 keratinocytes surrounded by 3T3 cells were visible. These cel.ls formed quickly expanding colonies (Fig. I), pushing away the 3T3 cells and finally covering a !arge area of the culture dish. The presence of epidermal growth factor and cholera toxin in the culture medium has been shown to prolong considerably the culture lifetime and to enhance the rate of cell proliferation in cultures of epidermal keratinocytes (6, 12). Since we also observed a beneficial effect on the cultures of hair follicle cells growing on the lens capsule (18), these substances are new routinely included in our culture medium. The CFE of the keratinocytes at their first passage, determined as described in Materials and Methods, was 1.0±0.2 % (11=4). A maximum of four subcultivation steps could be performed, with gradually decreasing CFE. After 3 days in culture, the outgrowth from a single hair follicle placed on a !ens capsule contained 50 to 100 cells. In a total culture period of about 12 weeks. approx. l0 7 keratinocytes can be generated from one single scalp hair follicle. Compared with the externa] root sheath, which contains about 10000 cells (as judged from cell-counts after trypsinization), the increase is I 000-fold during the culture lifetime. Cells recovered from cryogenic storage and plated in the presence of irradiated 3T3 cells attached to the culture substrate just as quickly as non-frozen cells. There were no morphological differences between the colonies formed by these cells. Howcver. thc CFE of the cells after freezing was lower: 0.20±0.05% (11=4). The CFE did not decrease significantly upon storage of the keratinocytes for as long as 6 months (n=4). /mmunojluorescence microscopy The anti-keratin serum, prepared as described in Materials and Methods. produced a strong precipitin band against stratum corneum keratins as well as against extracts from plucked hair follicles and cultured cells in a double diffusion assay. according to Yen et al. (19). Cultured hair follicle keratinocytes, when grown on glass coverslips in the presence of irradiated 3T3 feeder-cells, stained strongly with the antiserum, while the 3T3 cells surrounding the hair follicle cell colonies remained unstained (Fig. 2). At high magnifica tion, a filamentous pattern was revealed in the tlattened part of some of the hair follicle keratinocytes (Fig. 3). This pattern is comparable to that of epidermal keratinocytes, as described earlier by Sun & Green (14). DISCUSSION The use of hair follicles as a biopsy material for biomedical research and diagnosis started only a decade ago. In recent years the hair follicle has been increasingly used as an enzymc source for molecular diagnosis ofin bom errors of metabolism (for review, see 16). Since the development of a method for the cultivation of hair follicle keratinocytes ( 17), this approach has found further applications. For example, in somc studies related to cancer. it is nece5sary to incubate for long periods of lime with carcinogens (2. 5, 21). This can only be done when cultured cells are used (7,8). We rcccntly dcscribed a method for subcultivation of human hair folliclc kcratinocytcs by mean� of tran�plantation of colony fragments onto new bovine eye tens capsules (18). This wa� an improvement ofthe culture mcthod de�cribed earlier (17), sincc il enabled us to cultivate morc cells derived from a single hair follicle. The present technique of co cultivation of irradiated 3T3 cells and hair follicle cells derived from primary cultures on bovine eye tens capsulcs allows serial cultivation of hair follicle keratinocytes. fhe advantagc of this rnethod is that the lens capsule is only essential for the primary culture and in consequence about I O times more cells can be generated than by transplantation. Alternatively. for biochemical studies thc transplantation technique is preferable, since the determination of biochemical parameters is not disturbed by the presence of othcr cell types, and since the cells have not been altered by proteolytic enzymes that have been shown to alter �ome of the biochemical parameters of cells in culturc (15). The techniquc presently described not only allows the generation of a !arge amounl of cells. but also enables lluorescence studies and cryogenic storage. As a result. transporta tion of the cells between laboratories is now possible. This i� especially important for the investigation of rclatively uncommon genetic diseases that manifest themselves in epithcli al cells.	2018-04-03T00:17:14.727Z	1983-07-01T00:00:00.000	{ "year": 1983, "sha1": "479a8d53e9bc0497312c2e3b143150696b5a615b", "oa_license": "CCBYNC", "oa_url": "https://medicaljournalssweden.se/actadv/article/download/7881/11357", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "3235a5b34f3b1a8bdef7e1450c5308dc99af0fcc", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
221779466	pes2o/s2orc	v3-fos-license	Analysis of Cardiac Metabolic Remodeling in Heart Failure Using Nuclear Medicine and Its Application: Japanese Society of Nuclear Cardiology Award Heart failure is associated with a significant change in the energy metabolism of the heart. We aimed to elucidate the altered energetics during the progression of heart failure. We used radioactive metabolic tracers to assess the substrate uptake. In a rat model of heart failure, the glucose uptake increased significantly at the stage of left ventricular hypertrophy, whereas the uptake of fatty acids decreased at the stage of heart failure, with decreased energy reserve during the transition of cardiac hypertrophy to failure. Metabolic modulator which enhances glucose oxidation ameliorated the decrease in cardiac function. We also validated the close correlation with mitochondrial membrane potentials and 99m technetium sestamibi ( 99m Tc-MIBI) in vivo and at the organ level. The retention of 99m Tc-MIBI signals was correlated with the severity of heart failure. Nuclear medicine is a powerful tool to understand the mechanism of cardiac remodeling in heart failure. H eart failure is associated with a significant change in the energy metabolism of the heart, and the altered energetics are hypothesized to play an important role in the progression of heart failure (1). The direct measurement of flux through various pathways of energy metabolism using radio-labeled substrates has served as a model system to elucidate the mechanism of energy metabolism in an organ and has significantly increased our knowledge of cardiac energy metabolism (2). Despite intense research efforts, the precise mechanism by which the alteration of cardiac energy metabolism is induced and causes progression of heart failure is not clear. One reason for this is that current knowledge is based on studies analyzing different parameters of metabolism using different animal models or patients with different clinical backgrounds (3). Thus, we simultaneously analyzed cardiac function, substrate uptake, cardiac energy reserve, gene and protein expression, as well as amounts of metabolites using metabolomics, in a rat model of heart failure that shows a distinct transition from compensated left ventricular hypertrophy to heart failure. Comprehensive analysis of the transition of cardiac hypertrophy to heart failure Dahl salt-sensitive rats fed a high-salt diet developed hypertension and left ventricular hypertrophy at 11 weeks of age and congestive heart failure at 17-19 weeks (4,5). At around 17 weeks of age, they showed signs of heart failure and decreased systolic function ( Figure 1). Dahl rats fed a low-salt diet were used as age-matched controls. The phosphocreatine to adenosine triphosphate (ATP) ratio started to decrease at the left ventricular hypertrophy stage, and significantly decreased at the heart failure stage ( Figure 1) (3). Glucose uptake increased and fatty acid uptake was preserved at the left ventricular hypertrophy stage, confirmed by auto-radiography protein expression (3). To test whether enhanced glucose metabolism is a protective mechanism in heart failure, we administered Dahl rats with dichloroacetate, which activates pyruvate dehydrogenase and carbohydrate oxidation. Dichloroacetate preserved contractile function and improved the survival of the rats. Through metabolome analysis, a pathway highlighted in the study of Dahl rats was the pentose phosphate pathway, which is contributes to maintaining reduced glutathione and redox homeostasis. Metabolic remodeling of the heart causes cardiac dysfunction and can be a new therapeutic target for heart failure, for example, glucose oxidation (3) and amino acid metabolism (7). Metabolic alteration from fatty acid to glucose in the failing heart is reportedly considered to be due to the fetal gene reactivation program (8). From the mechanistic viewpoints, the disruption of a sarcoglycan-sarcospan complex in vascular smooth muscle cell disturbs vascular function, which in turn initiates the development of cardiomyopathy and exacerbate myocardial ischemia due to intra-myocardial microvascular dysfunc-tion (9,10). In addition, pressure overload by transverse aortic constriction induces hypoxia-inducible factor-1 and angiogenesis (11). Sustained pressure overload inhibits the activity of hypoxia-inducible factor-1 through accumulation of p53 (11); however, the accumulation of hypoxia-inducible factor-1 and p53 was not observed in our Dahl rat model (3). Collectively, these mechanisms may be associated with metabolic alteration and mitochondrial dysfunction in the failing myocardium in each pathophysiological context. In clinical settings, 18 F-fluorodeoxyglucose is possible to differentiate ischemic and infarcted myocardium (12) or to visualize the granulomatous region of sarcoidosis (13) on the basis of the presence of altered glucose metabolism. Metabolic remodeling in the heart failure occurs not only in the heart, but also in the whole body (5,14). Analysis of radioactive metabolic tracers revealed that the liver incorporates greater amounts of glucose in rats with heart failure. In conjunction with other analyses, the paradoxical production of triglyceride synthesis is also observed and is associated with a Ann Nucl Cardiol 2020；6(1) ：91-94 -92 -Kato Metabolic Remodeling and Nuclear Cardiology proinflammatory response in liver (14). Using radioactive metabolic tracers, we also found that cardiac-specific overexpression of the sirtuin gene, which is associated with longevity, and phosphoglycerate mutase, a glycolytic enzyme, causes cardiac dysfunction with altered mitochondrial morphology (15) or reduced anti-stress resistance in mice (16). These studies illustrate the link between metabolism, aging (15,17,18), and anti-stress resistance in the heart. Mitochondrial dysfunction in heart failure The mitochondria have a central role in the production of ATP in cells. Many methods have been used to assess mitochondrial function using isolated mitochondria, intact cells, or in situ techniques. However, little is known based on in vivo studies or at the organ level. 99m Technetium sestamibi ( 99m Tc-MIBI), a lipophilic cation, is rapidly incorporated into myocardial cells by diffusion and mainly localizes to the mitochondria. We analyzed 99m Tc-MIBI signals in a perfused heart, excised heart tissue, and in vivo using Sprague-Dawley rats and carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler known to reduce the mitochondrial membrane potential. 99m Tc-MIBI signals decrease in rat hearts administered CCCP (Figure 2A) (19), and the ATP content, as measured by 31 P magnetic resonance spectroscopy, decreases simultaneously. The 99m Tc-MIBI signal per heart tissue weight is inversely correlated with the severity of heart failure in the Dahl rat model (19). On in vivo imaging, CCCP increases the clearance of 99m Tc-MIBI ( Figure 2B), showing that the washout rate is increased in rats administered CCCP (20). To gain insight into the mechanisms underlying mitochondrial dysfunction and exercise intolerance in heart failure, we analyzed 99m Tc-MIBI washout of the heart and leg muscles along with other clinical and cardiopulmonary exercise parameters. 99m Tc-MIBI washout of the heart is correlated with brain natriuretic peptide levels and the washout rate of the leg muscle (21). Peak oxygen consumption was negatively correlated with the 99m Tc-MIBI washout of the leg muscles (21). These studies indicated the potential linkage between mitochondrial function of the heart and leg muscles and brain natriuretic peptide levels in patients with heart failure, suggesting the mechanism of the beneficial effect of exercise in patients with heart failure. From the clinical viewpoints, the effects of inhibitors of sodium glucose cotransporter 2 highlighted the importance of cardiac and systemic metabolic remodeling in heart failure and diabetes mellitus (22). Conclusions Nuclear medicine has been and will continue to be a powerful tool to understand the metabolic remodeling of the heart both in clinical and basic contexts. Ann Nucl Cardiol 2020；6(1) ：91-94 -93 -Kato Metabolic Remodeling and Nuclear Cardiology Figure 2 99m Tc-MIBI signals and the heart. (A) Correlation between 99m Tc-MIBI signals and heart weight. 99m Tc-MIBI signal per gram of heart tissue was inversely correlated with heart weight. (B) Representative in vivo images of 99m Tc-MIBI distribution. Myocardial retention of 99m Tc-MIBI was markedly decreased after the CCCP injection (lower panels) compared to vehicle-administered rats (upper panels). White arrowheads indicate hearts. Analysis of in vivo images showed that the 99m Tc-MIBI washout rate was significantly increased in CCCP rats. CCCP; carbonyl cyanide m-chlorophenylhydrazone, 99m Tc-MIBI: Technetium-99m sestamibi, WR, washout rate. All bars indicate means and SEMs. *P<0.05 vs vehicle-administered rats.	2020-08-06T09:05:04.331Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "50018ed7f097890450310f02c204893e3c43ba33", "oa_license": null, "oa_url": "https://www.jstage.jst.go.jp/article/anc/6/1/6_20-00112/_pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "7fd55d3dfc6fef8877b2189b38154c467b510e78", "s2fieldsofstudy": [ "Medicine", "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
258865816	pes2o/s2orc	v3-fos-license	Entangled photons enabled ultrafast stimulated Raman spectroscopy for molecular dynamics Quantum entanglement has emerged as a great resource for studying the interactions between molecules and radiation. We propose a new scheme of stimulated Raman scattering with entangled photons. A quantum ultrafast Raman spectroscopy is developed for condensed-phase molecules, to monitor the exciton populations and coherences. Analytic results are obtained, showing an entanglement-enabled time-frequency scale not attainable by classical light. The Raman signal presents an unprecedented selectivity of molecular correlation functions, as a result of the Hong-Ou-Mandel interference. Our work suggests a new paradigm of using an unconventional interferometer as part of spectroscopy, with the potential to unveil advanced information about complex materials. Introduction.-With the advancements of quantum light sources, the study of spectroscopy and sensing draw much attention in a diversity of active research fields [1][2][3][4][5][6][7][8].Quantum states of light with different types of entanglement offer new freedom for the light-matter interactions, spectroscopy and precise measurement [9][10][11][12][13][14][15].Novel knobs can be developed thereby for controlling the atom and molecule motions at microscopic scale.A capability of controlling the multi-photon transitions, as a signature of nonlinear optical processes, was enabled by photon entanglement [16][17][18].Much attention has been drawn recently for the importance of entangled photons in various fields including quantum simulations [19] and further the marriage of molecular spectroscopy with quantum photonics. The multi-photon interactions with complex molecules were studied recently in a context of quantum-light spectroscopy.Several experiments indicated the extraordinary transitions with entangled two-photon absorption (ETPA)-the inhomogeneous line broadening can be circumvented for an efficient population of highly-excited states of molecules [1,16,[20][21][22][23][24][25][26][27][28][29][30][31][32].The multi-photon interaction has been studied much in atoms, it is however an open issue in molecules so far.This arises predominately from the couplings of electrons to more degrees of freedom, which bring up new challenges for the optical response.Elaborate experiments demonstrated the incredible power of entangled photons yielding the probe and control of electronic structures with unprecedented scales [33].Recent studies extended the ETPA to time-resolved regime, showing miraculous cancellation of molecular correlation functions not accessible by classical pulses [34].New control knobs by entangled photons may enable considerable suppression of background in the radiation.An improvement in signal-to-noise ratio can be thus expected.The entanglement-refined interactions with molecules may induce a nonlinearity prominently resonant with the excited-state relaxation as well as the many-particle couplings.These call for a thorough un-derstanding of the quantum-light interactions with complex molecules at ultrafast timescales. The Raman process, as a typical component of the multi-photon interactions, closely connects to the quantum-light fields [35,36].Coherent Raman spectroscopy including a variety of schemes provides a powerful tool for quantum physics and molecular characterization.Extensive studies have demonstrated the quantum advantage of entangled light in spectroscopy [37][38][39].The time-frequency entanglement of photons may enable a superresolved capability, free of the conjugation of temporal and spectral scales that posses the fundamental limit in Raman spectroscopy using classical light.A femtosecond CARS was proposed recently with entangled photon pairs, to monitor the ultrafast dyanamics of electronic coherence and the passage of conical intersections [40].Furthermore, recent progress reported a CARS with squeezed photons in a nonlinear interferometer [41].A quantum-enhanced measurement beyond the shot-noise limit was therefore performed.As a different scheme, the stimulated Raman scattering is sensitive to the molecular populations that are of fundamental interest and importance for the cooperative effects and multiexciton correlations.These can be monitored in a greater way by making use of entangled photons and nonlinear interferometry [42,43]. In this Letter, we propose an ultrafast stimulated Raman spectroscopy (USRS) using entangled photons.A microscopic theory is developed with molecular trimers.Here the molecules play an active role in interacting with optical signals, rather than the passive role of the beam splitters in quantum optics.Due to the entanglement, such a quantum USRS (Q-USRS) enables a superresolved nature of the spectrum with time-frequency scales not attainable by classical pulses.Moreover, as a result of the multi-photon quantum interference, the spectroscopic signals are presented.An unprecedented selectivity is thus shown by the signals, enabling a selective access to molecular correlation functions.This is a hard task for Quantum stimulated Raman scattering.-Weconsider a generic model of molecules interacting with entangled photons generated by nonlinear mediums in Fig. 1(a).The two entangled photons are shaped in a short pulse.The photons in s and i arms are jointly scattered by molecular excited states, inducing the stimulated Raman process.A coincidence counting of emission is measured, where no spectrometers are required. The Raman interaction between molecules and entangled photons is of the form where E s (t) and E i (t) play the roles of the respective pump and probe fields containing multiple frequency modes.α(t) = m>n α mn \|ψ m ⟩⟨ψ n \|(t) + h.c.defines the Raman polarizability operator and the elements α mn are given in Supplemental Material (SM) [44].Usually \|ψ m ⟩⟨ψ n \|(t) = \|ψ m ⟩⟨ψ n \|e iωmnt for closed systems but we will not adopt this assumption, so as to involve more general cases described by a reduced density matrix. Eq.( 1) resembles the beam-splitter interaction, indicating the two-photon interference that essentially interplays with the molecular excitations.Further results will elaborate the active role of the Hong-Ou-Mandel (HOM) effect in Raman spectroscopy for a monitoring of excitedstate dynamics. The Q-USRS is defined as the coincidence counting of the transmissions along s and i arms, and is given by where ψ(τ ) is the molecular wave function including full degrees of freedom.T s and T i are the central times of the pulses in s and i photons, respectively.∆T = T i − T s measures the time delay between s and i photons that is controllable via optical paths.This provides the arrival times of the two photons with delays relative to the resonant pump that creates the electronic excitations in molecules, shown in Fig. 1(b).The six-point field correlation functions are ).The two components with C I and C II in Eq.( 2) correspond to the loop diagrams in Fig. 2 which govern the multipoint Green's functions of Raman operators.It includes the components: pathway I for parametric process and pathway II for dissipative process [45]. Notably from Eq.( 2), C I ̸ = C II in general for quantum fields whereas C I = C II for classical fields.One can probably achieve the selectivity of the molecular correlation functions, not attainable by classical pulses. The density matrix ρ(τ ) = \|ψ(τ )⟩⟨ψ(τ )\| contains full information about the structures and dynamics of molecules.These are essentially imprinted into the Ra- man signal, so as to be read out by making use of the quantum-field correlations. Q-USRS with entangled photons.-Theentangled state of photons in Fig. 1 (3) is the two-photon wave function with a phase matching τ i where τ s (τ i ) is the time delay of s(i)-arm photons relative to the pump field A, due to the group velocity dispersion in the nonlinear mediums.A is a classical field with an effectively narrow bandwidth σ 0 , so that A(ω s − ω i − ω − ) → δ(ω s − ω i − ω − ) as σ 0 → 0. ω s − ω i indicates quantum correlated photon pairs rather than the anti-correlated nature directly from the down-conversion process. Notably, the entangled photon states cause a miraculous cancellation of the field correlation functions.In particular, C I ̸ = 0 but C II = 0.This is a typical twophoton interference, arising from the HOM effect [46]. As a result, the parametric component in Fig. 2 survives whereby the diagram II vanishes.A great selectivity of molecular Green's functions is thus expected, not accessible by the Raman spectroscopy using classical pulses.It should be aware of that the Raman interaction in Eq.( 1) largely differs from the 50:50 beam splitter in quantum optics.The destructive two-photon interference is therefore retained, giving C I ̸ = 0 rather than causing the full cancellation as in the original HOM scheme.The constructive two-photon interference groups two photons in one output mode, making C II = 0.These yield an aspect of conceptual importance for quantum-light spectroscopy, because the residue part characterizes the spectral lines that may provide a key monitoring of molecular structures and relaxation.Elaborate simulations of the Q-USRS incorporating the two-photon interference will be performed later on. S(ω e,e ′ e ′′ dtdτ e −iω e ′′ e ′ (t−τ ) ρ ee ′ (τ ) is the wave packet of the twin photons.The background has been dropped from Eq.(4a), in that no spectral lines are produced. Eq.(4a) indicates the role of the quantum-entangled photons whose unusual band properties may provide versatile tool for controlling the fast excited-state dynamics of molecules. We see clearly from Eq.( 5) the time-frequency-resolved nature of the entangled Q-USRS, beyond the spectral and temporal scales conjugated by classical pulses.More advanced information about molecules and environments would be therefore unveiled.These will be elaborated by simulating the Raman signal using certain molecular models. Microscopic theory.-Wewill adopt the molecular aggregate model into the Q-USRS.The molecular Hamiltonian is where Q n,s (t) is the coordinate of vibrations and f ns gives the exciton-vibration coupling strength.The model includes N photoactive molecules; σ + n is the raising operator of excitons at the nth molecule, in the limit of large onsite exciton-exciton coupling.ω n is the exciton energy and J is the hopping rate.A simple model for molecular trimer dyes can thus obtained by N = 3.Previous studies observed that the absorption/fluorescence spectrum of molecular aggregates show a dense distribution of vibrational states attached to electronic excitations, evident by the inhomogeneous line broadening characterized by a smooth spectral density of vibrations [48,49]. Averaging over the vibrations and radiative loss, i.e., ρ(t) = Tr B \|ψ(t)⟩⟨ψ(t)\|, the nonradiative relaxation is opened for the Frenkel excitons in the aggregates.We obtain the equation of motion ρ(t) = −i[H 0 , ρ(t)]+ Ŵρ(t) with the Lindbladian Ŵ = j>i Ŵji that contains the jumping operators between the eigenstates of H 0 [44].The density-matrix dynamics therefore provide a microscopic description for the Q-USRS. Simulation of Q-USRS.-Weused a Photosystem (PS) trimer model to simulate the USRS with entangled photon pairs combined with nonlinear interferometer scheme.The two-photon wave function in Eq.( 3) is taken to be ) assuming τ s = τ i = τ 0 along with before.In what follows, we assert that ω − can be tuned whereas fixing ω + .Fig. 3 shows the USRS signal with various photon statistics.The plots vary with ω − and arrival time T of the two photons with a zero optical delay (∆T = 0).Using the parameters from PS trimers, we consider N = 3 molecules and weak coupling J = 30meV that yields small energy splitting as illustrated in Fig. 1(b). Fig. 3(a) displays several peaks presenting dramatic different behaviors when varying the delay T (arrival time of entangled photons).In particular, at the Raman shift of ω − = −0.059,−0.189eV , two long stripe-shaped peaks can be observed promisingly at T > 400fs.The timeevolving dynamics ρ e1,e1 (T + τ0 2 ) is thus monitored, meaning a downhill transfer of exciton population towards \|e 1 ⟩.Likewise, the peak intensity at ω − = −0.13,0.059eV shows a rapid increase during 100fs and drops smoothly therein.This monitors the dynamics of exciton population at \|e 2 ⟩, i.e., ρ e2,e2 (T + τ0 2 ).The two peaks at ω − = 0.13, 0.189eV resolve the exciton dynamics at \|e 3 ⟩, whose population drops dramatically within 700fs.Nevertheless, a few side peaks can be seen within a shorter timescale, by a zoom-in.The oscillatory nature evidences the quantum coherence of excitons, as detailed next. Fig. 4(a) illustrates the fast dynamics of coherences coexisting with the exciton populations.The side peaks essentially monitor the coherences, provided that ρ ee ′ (e ̸ = e ′ ) are associated with the Raman resonances distinct from ρ ee , as given by Eq.( 5).For instance, the peaks at ω − = ±0.124eVresolve the excitonic coherence ρ e2,e3 and ρ e3,e2 , when observing an oscillation period of 25fs. Indeed, the exciton dynamics is subject to a temporal scale τ 0 (pulse duration of an entangled photon pair), whereas the line broadening of the Raman signal is given by σ 0 .These are evident by Eq.( 5) and are confirmed from the simulations.Fig. 3(b) and 3(c) plot the USRS without quantum entanglement.The result using uncorrelated twin photons is given in Fig. 3(b), where the each photon has a bandwidth σ0 = 1 2τ0 .Similar consideration applies to the classical USRS, whereby using two laser pulses with a bandwidth σ0 = 1 2τ0 to drive the SRS.Fig. 3(b,c) evidence fairly blurred spectral lines and dynamics if the pulses are short.And the classical USRS dilutes the selectivity of molecular correlation functions, disclosing an intrinsic limit for accessing the excited-state dynamics.This can be seen from the symmetric distribution of the Stokes and anti-Stokes lines in Fig. 3(c).Such a lack of pathway selectivity holds as well for the FSRS using combined narrow-and broad-band pulses. Multi-photon interference.-Tosee closely the quantumlight-enabled selectivity, we scan the optical delay ∆T = T i − T s so that the s and i photons have different arrival times.Fig. 4(b) shows a slice of the entangled Q-USRS at ω − = ω e3,e2 , T = 0.This is a typical HOM interference: a decaying envelop as the two photons get separated in time.The destructive interference is however retained, as a result of light-molecule interactions which yield a residue of the HOM dip at ∆T = 0.The dip intensity does not only reveal an overlap between wavepackets of twin photons, but also imprints the electronic structure and dynamics of molecules.The latter is extensively important for spectroscopy and sensing, in that the dip intensity indeed engraves the interference between the two parametric processes given in Fig. 2. The photon pair at outgoing ports interfere with its interchange s ↔ i essentially. Moreover, Fig.This makes sense once noting the frequency offset between the two wavepackets of the twin photons [47]. Conclusion.-In summary, (i) we develop a microscopic theory for the USRS with quantum-light fields, incorporating the photon-coincidence counting.(ii) A simple analytic expression is derived for the Q-USRS using entangled photons, yielding elaborate HOM interference which enables the selective access of molecular correlation functions.(iii) Unprecedented time-frequency-resolved property of the Q-USRS is shown explicitly, from the fact that the temporal and spectral scales are not conjugated due to the quantum correlations of photons.(iv) Fast exciton dynamics is visualized including the fluctuating energy-gap and populations in real-time domain.(v) No gratings are needed for spectrally-resolved lines. All these yield unprecedented scales for spectroscopy to be facilitated with quantum advantage, and would be sufficient for the tomography of density matrix of molecules. FIG. 1 : FIG. 1: (a) Schematic of entangled twin photons (correlated with each other rather than the anti-correlation) as an ultrafast probe for molecules, where the nonlinear mediums and the photon-coincidence counting measurement are presented; Small panel plots \|Φ(ω s , ω i )\| of the entangled twin photons.(b) Level scheme of molecular relaxation interacting with two entangled photons that induce the stimulated Raman scattering. FIG. 2 : FIG. 2: (Top) Feynman's loop diagrams for the stimulated Raman scattering with twin photons; (I) parametric process and (II) dissipative process.(Bottom) Two pathways of the two-photon interference in a fashion of the Hong-Ou-Mandel (HOM) scheme corresponding to the processes (I) and (II).Notice that the complicated light-molecule interactions lead to the two-photon interference not exactly the same as the original HOM using 50:50 beam splitters. Our work, as a new coherent Raman technique, may open a new frontier for studying the ultrafast processes in photochemistry, nano-plasmonics and semiconducting heterostructures.J.F. and Z.Z.gratefully acknowledge the support of the Early Career Scheme from Hong Kong Research Grants	2023-05-25T01:16:19.909Z	2023-05-24T00:00:00.000	{ "year": 2024, "sha1": "a3969f461d091d01fba71073fa0b973d905b9eb4", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "ArXiv", "pdf_hash": "a3969f461d091d01fba71073fa0b973d905b9eb4", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Medicine", "Physics" ] }
123660951	pes2o/s2orc	v3-fos-license	Dynamic spin-response function of the high-temperature Bi$_2$Sr$_2$CaCu$_2$O$_{8+\delta}$ superconductor form angle resolved photoemission spectra We introduce a formalism for calculating dynamic response functions using experimental single particle Green's functions derived from angle resolved photoemission spectroscopy (ARPES). As an illustration of this procedure we estimate the dynamic spin response of the cuprate superconductor Bi$_2$Sr$_2$CaCu$_2$O$_{8+\delta}$. We find good agreement with superconducting state neutron data, in particular the $(\pi,\pi)$ resonance with its unusual `reversed magnon' dispersion. We anticipate our formalism will also be of useful in interpreting results from other spectroscopies, such as optical and Raman responses. The linear response to an external probe as a function of momentum and frequency is of great importance in elucidating the properties of complex materials.Examples include various two-particle correlation functions involving spin, current and charge as measured by inelastic neutron scattering (INS), nuclear magnetic resonance (NMR), optical conductivity, and Raman scattering experiments.On the other hand, angle resolved photoemission spectroscopy (ARPES) [1] directly gives information about single-particle excitations of a system.The response function of a system can be expressed in terms of a two-particle correlation function of the observable to which the external probe couples.The goal of this paper is to develop an approach to use single-particle spectroscopy data to gain insight into two-particle correlation functions.In particular we focus here on using the Green's functions obtained from superconducting state ARPES data in the high T c cuprates to compute the dynamic spin susceptibility, which we then compare with INS data [2]. From a theoretical point of view, dynamic response functions are difficult to calculate in general and many different approximate formalisms exist in the literature.For instance, there are two rather different approaches for computing the dynamic spin response for the high T c cuprate superconductors.The first is based on the random phase approximation (RPA) [3] and related diagrammatic formulations [4].This approach not only assumes momentum is a good quantum number, but also that the spin and charge degrees of freedom are coupled.The second is based on spin ladders separated by one-dimensional domain walls known as stripes.In this formalism spatial inhomogeneity is important, and the charge sector is assumed to be secondary when calculating the spin response [5].Despite the quite different physics underlying these two schemes, the results for the calculated spin response function of the cuprates are similar -one of the current dilemmas facing the field of high T c superconductivity.It is thus important to go beyond a purely theoretical approach and directly employ information obtained from one experiment (ARPES) to make progress on interpreting the dynamic susceptibility measured by another (INS). We use a formalism based on a diagrammatic k-space approach which goes beyond RPA in that it uses fully dressed Green's function obtained from ARPES data on Bi 2 Sr 2 CaCu 2 O 8+δ (Bi2212).We compare the calculated superconducting state susceptibility with INS data.We obtain the (π, π) resonance seen in many cuprates [2], including Bi2212 [6], and also its unusual 'hourglass' shaped dispersion as observed in YBa 2 Cu 3 O 7−δ (YBCO) [7,8] and more recently in Bi2212 [9].We also find that the magnetic dispersion is sensitive to the momentum dependence of the effective interaction used to calculate the susceptibility. We use ARPES spectra from a near-optimal sample (T c =90K) of Bi2212, the data having been presented previously [10,11].While a resonance peak was observed in this material some time ago [6], a more detailed study with results similar to the much more extensive INS data for YBCO, has appeared only recently [9]. Quite generally, two-particle correlation functions can be written in terms of single-particle Green's functions and vertex parts [12].The lowest order term contributing to the spin susceptibility (the bare polarization bubble) in the superconducting state can be written as [13] where Im denotes the imaginary part of the normal and anomalous Green's functions G and F , and χ G 0 and χ F 0 denote the GG and F F contributions to χ 0 respectively.We next describe in detail how ImG is extracted from ARPES data and return later to the question of estimating the contribution of ImF (which is not directly measured).ARPES probes the occupied part of the spectral function leading to the intensity I(k, ω) ∝ n F (ω) ImG(k, ω), where n F (ω) is the Fermi function [14].In order to extract ImG from raw data we need to address several issues including data normalization, background subtraction, and removing the effects of the Fermi function.In addition we need to extend ImG to ω > 0 to calculate χ 0 . Starting from raw data, we first subtract the constant signal at ω > 0 (due to second order light).Next an 'unoccupied' state spectrum at a k far from k F is used as an energy-dependent background [15].The subtraction is performed by normalizing the background to each spectra at a given binding energy, ω c (320 meV for the data set in question), and then subtracting it [16].This effective spectral function represents the the renormalized band near the Fermi energy.Finally, we divide the data by a resolution [17] broadened Fermi function to obtain ImG(k, ω) for ω < 0. The next step is to determine the unoccupied part of the spectral function, ImG(k, ω) for ω > 0, which cannot be obtained directly from ARPES data.We obtain this by invoking particle-hole symmetry with respect to the Fermi surface: where k is directed along the normal to the Fermi surface.This assumption should be reasonable in the superconducting state of optimally doped cuprates over an energy range in excess of the gap, as evidenced by the approximate particle-hole symmetry seen in tunneling experiments [18].We have also checked that this assumption does not qualitatively affect our final results for χ 0 (by using ImG with p-h asymmetry put in by hand).We then normalize the obtained ImG so that the integral of the spectral function (−ImG/π) is equal to unity over the energy range of ±ω c .This minimizes the effect of dipole matrix elements.Now we may use the ImG derived from ARPES to calculate χ G 0 (we will discuss χ F 0 later).Finally, to perform the k-sum in Eq. ( 1), the ARPES data are interpolated to a regular grid and then reflected using square lattice group operations to fill the first Brillouin zone [11].We used a 100 × 100 grid.Fig. 1a shows the calculated Imχ G 0 at T=40K (superconducting state) as a function of the momentum transfer q along the zone diagonal.We note that Imχ G 0 is greatly suppressed at low energies due to the gap to particle-hole (p-h) excitations in the superconducting state and then increases quite sharply.The p-h gap is in general given by the sum of the superconducting gaps at two points on the Fermi surface separated by the wavevector q.The Q = (π, π) vector connects the hot spots (ǫ k = ǫ k+Q =0) which are not too far from the zone boundary in Bi2212, and thus the hot-spot gap is comparable to the one at the antinode.Consequentially at Q = (π, π), we see in Fig. 1a a large gap whose midpoint is around 80 meV, roughly twice the maximum d-wave superconducting gap of ≃ 40 meV at the antinode.We note that the threshold is quite broad (∼ 40 meV) as a result of the intrinsic broadening of ImG arising from self-energy effects as well as resolution.As q decreases from Q, one sees the p-h gap decrease due to the d-wave anisotropy of the gap, and then disappear at q n ≃ (0.76, 0.76)π.q n is the wavevector corresponding to node-node scattering with the d-wave gap vanishing at the nodes.For q < q n , the p-h gap reappears [3]. In Fig. 1b we show Reχ G 0 obtained from Eq. (1) [19].First concentrating on Q, we note the presence of a peak that corresponds to the gap midpoint of Fig. 1a as expected from Kramers-Kronig relations.This peak is broadest for q n where the p-h gap vanishes in the imaginary part. We now turn to χ F 0 .Since ImF is not available from experiment, we estimate the FF term as follows.We calculate the BCS χ G 0,BCS and χ F 0,BCS from Eq. ( 1) using the bare BCS Green's functions G ) with the experimentally measured dispersion [20] ǫ k and the measured ∆ k , which we find to be proportional to (cos k x − cos k y ) for this sample.We define the ratio of the real and imaginary parts, given by α R (q, Ω) = Reχ F 0,BCS /Reχ G 0,BCS and similarly for α I (q, Ω).We then assume that the missing contribution χ F 0 may be accounted for by Reχ We will discuss below the extent to which our final results are affected by this approximation. In order to carry out comparisons with INS data, or other probes such as NMR, the full spin susceptibility χ is needed.The most common approximation is to use the RPA form [3] where U is an effective interaction.In this paper, we will assume two limiting forms for this effective interaction, one where it is a constant (U 0 ), the other where it has the form U (q) = −U 0 (cos q x + cos q y )/2 corresponding to superexchange between near neighbor copper sites. The experimental results for χ 0 presented in Fig. 1 are similar to those obtained from BCS theory [3] especially at low energies where the incoherent spectral weight in the ARPES data is small.Within BCS theory, which uses bare Green's functions, one has a true gap in Imχ 0 at Q and a corresponding log divergence in Reχ 0 .These features still persist in Fig. 1 albeit broadened by selfenergy (and resolution) effects.As such, for some frequency smaller than the gap, one will obtain a pole in χ when 1 − U (q)Reχ 0 (q, Ω) = 0 provided Imχ 0 is small at the frequency of interest.This pole represents a collective mode, known as the spin resonance at Q, which is prominently observed in INS data for YBCO [2] and Bi2212 [6,9].Following this logic, we fix U 0 [21] by fitting the energy (44 meV) of the INS spin resonance at Q for optimal doped Bi2212 [6]. In order to compare the results of our approach with INS data we plot the imaginary part of the full susceptibility χ as calculated from ARPES data as discussed above for constant q cuts in Fig. 2 and constant Ω cuts in Fig. 3.The left panels assume a constant U , the right panels the near neighbor exchange form U (q).Let us first consider Fig. 2. For constant U (left panel), the resonance traces out a pronounced downwards dispersion which terminates at the node-node scattering vector q n , as seen in the INS data.For q < q n , a distinct second branch appears, which is broad and weak, that disperses upwards.The change of behavior at q n corresponds to the so-called silent band effect and second mode mentioned in connection with INS data of overdoped YBCO [22,23] and Bi2212 [9].But interestingly, the upper branch of the 'hourglass' is not apparent.In contrast, for U (q) (right panel), the mode is almost dispersionless near (π, π), then shows an upward dispersion for momenta q < 0.9(π, π).This difference is a direct consequence of the relatively weak momentum dependence of Reχ 0 (Fig. 1b) coupled to the decrease in U (q) away from Q = (π, π). The behavior of the dispersion observed in constant q scans can be contrasted with that from constant Ω scans (Fig. 3).For constant U , both types of scans yield a qualitatively similar mode dispersion (solid dots in Fig. 3a).However, for U (q), the dispersion obtained from the constant Ω cuts is hourglass-like, with an upward and downward branch that merge at (π, π), in good agreement with INS data in underdoped YBCO [8].We note that this downward branch is not visible in the constant q cuts in Fig. 2b.This difference is analogous to the different ARPES dispersions that one finds from energy distribution curves as compared to momentum distribution curves. We have also generated results involving only the GG contribution by setting α = 0.In order to compare with the results having both FF and GG contribution, we rescale U 0 to maintain the same resonance energy at Q.Only minor differences are found between this and the full calculation which includes both GG and FF contributions.This lack of sensitivity to the inclusion of FF terms is a consequence of the d-wave symmetry of the superconducting order parameter where χ G 0 and χ F 0 reinforce one another near (π, π).(In contrast, for an s-wave superconductor, χ G 0 and χ F 0 are opposite in sign, and there is no spin resonance.) Comparing our results with the INS data and earlier RPA calculations, we arrive at the following conclusions.First, the self-energy effects present in the ARPES-derived Green's function do not affect the low energy physics of spin excitations, such as the existence and sharpness of the (π, π) resonance, or the mode dispersion.Second, vertex corrections do not play a major role in the spin channel, except possibly in the overall scale of U .Third, the magnetic dispersion is sensitive to the q dependence of U .Finally, our results provide strong evidence for the interpretation of the resonance peak as a spin exciton [3]. To summarize, we computed the polarization bubble χ 0 using experimental Green's functions derived from ARPES spectra, and the full dynamic spin response obtained from a diagrammatic formalism assuming either a constant or a near neighbor exchange interaction.Although this analysis requires several approximations, we find surprisingly good agreement with inelastic neutron scattering data for high temperature cuprate superconductors.Our results demonstrate a close relation be-tween experiments probing the spin and single-particle excitations.Our formalism is quite general, and can be used as well to compute other response functions, such as the current-current response function measured by conductivity.	2016-02-12T08:34:30.430Z	2006-06-14T00:00:00.000	{ "year": 2006, "sha1": "ec96093f1a410dafdeeb2e2f9873e48a4cdc00da", "oa_license": null, "oa_url": "https://www.dora.lib4ri.ch/psi/islandora/object/psi:18249/datastream/PDF/Chatterjee-2007-Dynamic_spin-response_function_of_the-(published_version).pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "ec96093f1a410dafdeeb2e2f9873e48a4cdc00da", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Materials Science", "Physics" ] }
1486266	pes2o/s2orc	v3-fos-license	Characterization of the Gene Expression Profile of Human Hippocampus in Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis One of the main putative causes of therapy refractory epilepsy in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis is the overexpression of multidrug transporters (MDTs) at the blood-brain barrier (BBB). It steps up the removal of antiepileptic drugs (AEDs) out of the brain cells across the BBB resulting in a low concentration of AEDs within the target cells. Some of the mechanisms of AED resistance are most likely to be genetically determined. To obtain more information about the underlying pathophysiology of intractability in epilepsy, we compared the global gene expression profile of human hippocampus and hippocampal-derived microvascular endothelial cells from MTLE with HS patients and controls. At the level of MDT, a significant up-regulation was found for ABCB1 (P-gp), ABCB2, ABCB3, and ABCB4, which was mainly related to endothelial cells. The data on the MDT were validated and extended by quantitative RT-PCR. Surprisingly, inflammatory factors such as interleukins (IL-1α, IL-1β, IL-6, and IL-18) and cytokines (TNF-α and TGF-β1) were found to be up-regulated in hippocampal parenchyma. The overexpression of P-gp, IL-1β, and IL-6 was also confirmed by immunohistochemistry (IHC). Our results suggest that complex expression changes of ABC-transporters may play a decisive role in pharmacoresistance in MTLE. Further studies on the new and unexpected overexpression of inflammatory cytokines may unlock hitherto undiscovered pathways of the underlying pathophysiology of human MTLE. Introduction Mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis (HS) is the most common type of epilepsy in adults [1]. HS consists of gliosis, neuronal loss and cell dispersion. As many as 75% of the patients with MTLE are considered to be resistant to drug therapy with AEDs. Pharmacoresistance is a common problem in neurological and psychiatric disorders which leads to a big distress among patients and family members. The long-term followup after selective amygdalohippocampectomy (sAHE) in the presence of severe HS showed a greater than 80% of patients becoming seizure free [2]. The causes and mechanisms underlying drug resistance in MTLE are still badly understood, which hampers the development of new drugs and thus the availability of better treatment options [3][4][5][6]. The main factors which might contribute to the development of medically intractable MTLE are aetiology of epilepsy, the progression of seizure activity under drug therapy, changes in the targets of AEDs, abnormalities in 2 Epilepsy Research and Treatment the neuronal networks owing to damage in the epileptogenic focus during epileptogenesis, and changes in the druguptake across the BBB [7]. Genetic factors may explain why patients with the same type of epilepsy respond differently to the same drug therapy. In recent years, several putative mechanisms underlying drug resistance in epilepsy have been identified. One theory that has received considerable attention is the removal of AEDs from the epileptogenic tissue through overexpression of multidrug transporters at the BBB. This theory is biologically feasible and can partly explain drug-resistance. Although MTLE is usually not regarded as a neuroimmunological disease, proinflammatory cytokines and chemokines may play a role in seizure onset [8,9]. It has also been reported in experimental models of limbic seizures that there is a rapid and transient increase of IL-1β, IL-6, and TNF-α mRNA in the hippocampus, and that intrahippocampal injection of IL-1β has a detrimental effect on seizures [10]. Furthermore, a polymorphism in the IL-1β gene has been associated with MTLE-HS compared to patients without sclerosis and nonepileptic controls [11,12]. Febrile seizures, particularly complex febrile seizures and status epilepticus during early childhood, have been associated with hippocampal sclerosis and overexpression of IL-1β [13]. The relevance of sclerotic hippocampus in seizure maintenance and therapy refractoriness has led to us to investigate the mRNA expression profile of the sclerotic hippocampus in order to obtain a comprehensive view of this particular pathology at the molecular level. Accordingly, we used a DNA microarray approach. The main aim of this study was to identify those genes which may be involved in the pathogenesis of MTLE-HS. A separate comparison was performed for mRNAs extracted from the entire tissue and from MVECs prepared from the resected tissue. In the interpretation and discussion of the data, we focused on the multidrug-transporter overexpression theory and on the genes which were found to be among the most overexpressed in sclerotic hippocampus. Surgical Specimens. Clinical specimens were obtained from ten caucasian patients with chronic pharmaco-resistant MTLE, who underwent surgical treatment at the Department of Neurosurgery, University Hospital Zurich (Table 1). Surgical removal of the hippocampus was clinically indicated in every case. The specimen was obtained by selective amygdalohippocampectomy (sAHE). With this surgical approach, parts of the amygdala, the hippocampus, and the anterior portion of the parahippocampal gyrus (Gph) are selectively removed. All tissues were diagnosed by two pathologists at the Department of Neuropathology, University Hospital Zurich. The hippocampus and the Gph were examined and separately rated for the presence and severity of hippocampal sclerosis (HS) based on the extent of gliosis and neuronal loss (mild, marked, and severe). All hippocampal regions revealed HS with various degrees of gliosis and neuronal loss. All analyses were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Canton of Zurich. Informed written consent was obtained from all patients. Control human brain hippocampus total RNA were commercially purchased by Ambion (Ambion, Inc. Texas, USA). The autopsy controls (n = 3; 45, 5 ± 10 years old; 2 female, 1 male; Caucasians) had no history of brain-related disease and suffered sudden death without associated brain damage. Autopsy was rapidly performed with a short postmortem delay. Autopsy hippocampi were dissected and immediately collected in RNA-Later tubes and frozen until processing. Totally RNA was isolated using the modified version of Ambion's RNA KIT for RNA-Isolation, and RNA was stored in 1mM sodium citrate (pH 6.4) at −70 • . Endothelial Cells (MVECs). MVECs were isolated as described by [14,15]. Briefly, ependym, large vessels and pia membrane were dissected away with the help of a microscope, and the remaining tissue minced and homogenized with a 10 mLsyringe in 10 mL MCDB 131 medium (Gibco, Invitrogen, Basel, Switzerland), containing 2% FBS (PAA Laboratories, Linz, Austria), 16 U/mL heparin (Sigma, Fluka Chemie, AG Buchs, Switzerland), 20 μL BBE, 5 μL hEGF, 5 μL hydrocortisone, 5 μL FGF (all from Cell System Inc. Kirkland. WA), 50 μL ECGS (Serva, Heidelberg, Germany), 500 μL gentamicin (Sigma). The tissue was dissociated in a glass flask in dispase II solution (Roche, Rotkreuz, Switzerland) at a final concentration of 0.096 U/mL and the solution was placed in a water bath at 37 • C and stirred for approximately 1 hour. The homogenate was centrifuged (1,000 ×g, 10 min, 4 • C) and the supernatant was removed. The pellet was dissolved in 2 mL MCDB 131 medium, containing 20% dextran, (Sigma) and gently homogenized with a fire-polished pipette and centrifuged (1,700 ×g, 10 min, 4 • C). The top layer containing most of the myelin was discarded. The pellet was resuspended in MCDB 131, containing 2% FBS medium, layered on 2 mL 13% dextran, and again centrifuged (1,700 ×g, 10 min, 4 • C) to remove the remaining myelin. The pellet was solubilized in 1 mg/mL collagenase/dispase (Roche) and 0.05 mg/mL DNase I (Sigma) and stirred for 1 hour. The cells were pelleted (380 ×g, 10 min, 4 • C) and washed with MCDB 131 containing 2% FBS medium, and the pellet was finally placed in 1 mL QIAzol lysis reagent (Qiagen, Basel, Switzerland) frozen and kept at -70 • C until further processing. Processing of Hippocampal Tissues. Immediately after surgery hippocampal specimen from epileptic patients were stored in RNAlater stabilization reagents (Qiagen) in order to prevent degradation of RNA. The samples were stored at 4 • C overnight and subsequently frozen and kept at -70 • C until further processing. Isolation and Analysis of RNA. Total RNA from isolated MVEC was extracted by using QIAzol lysis reagent according to the manufacturer's protocol. The isolated MVECs were placed in 1 mL QIAzol reagent, frozen and kept at -70 • C. After thawing, 0.2 mL dichloromethane was added and the samples were centrifuged ( separation. The RNA from the upper aqueous phase was precipitated by mixing with 0.5 mL isopropyl alcohol. After 10 min incubation the samples were centrifuged (12,000 ×g, 10 min, 4 • C) and the RNA pellet was washed with 1 mL 75% ethanol. At the end of the procedure, the RNA was briefly dried and then dissolved in RNase-free water. The hippocampal biopsies were thawed, placed in 1 mL QIAzol reagent for RNA isolation, and processed according to the protocol RNeasy Lipid Tissue Mini Handbook (Qiagen). The quality of the isolated RNA was determined by NanoDrop ND 1000 (NanoDrop Technologies, Delaware, USA) and a Bionalyser 2100 (Agilent Technologies, Basel, Switzerland). Only those samples with a 260 nm/280 nm ratio between 1.8-2.1 and a 28S/18S ratio within 1.5-2 were processed further and investigated. Control human hippocampal RNA was commercially purchased (Ambion Ltd., Huntingdon, UK). cRNA Preparation. 3 μg and 100 ng of total RNA per sample were reverse-transcribed into double-stranded cDNA with one-cycle and two cycle cDNA synthesis kit (Affymetrix Inc., P/N 900431, Santa Clara, CA), respectively. The doublestranded cDNA was purified using a Sample Cleanup Module (Affymetrix Inc.). The purified double-stranded cDNA was in vitro transcribed (IVT) in the presence of biotinlabeled nucleotide using an IVT Labeling Kit (Affymetrix Inc.) and the biotinylated cRNA was purified using a Sample Cleanup Module (Affymetrix Inc., P/N 900371). The quality and quantity of biotin-labeled cRNA were determined using NanoDrop ND 1000 and Bioanalyzer 2100. Microarray Data Analysis. Raw data processing was performed using the Affymetrix Genespring software (Silicon Genetics, California, USA). After hybridization and scanning, probe cell intensities were calculated and summarized for the respective probe sets by means of the MAS5 algorithm [16]. To compare the expression values of the genes from chip to chip, global scaling was performed, which resulted in the normalization of the trimmed mean of each chip to target intensity (TGT value) of 500 as detailed in the statistical algorithms description document of Affymetrix. Quality control measures were considered before performing the statistical analysis. These included adequate scaling factors (between 1 and 3 for all samples) and appropriate numbers of present calls calculated by application of an algorithm. The efficiency of the labelling reaction and the hybridization performance was controlled with the following parameters: present calls and optimal 3 /5 hybridization ratios (around 1) for the housekeeping genes (GAPDH and ACO7), for the poly A spike in controls and the prokaryotic control (BIOB, BIOC, CREX, BIODN). Analysis was performed by setting controls sample data set to zero and filtering and utilizing only genes with transcripts flagged "present" or "marginal". Gene transcripts with a difference of 2-fold or greater "induction" or "reduction" when compared to controls were subsequently annotated and grouped according to their molecular and cellular function. Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total RNA was extracted from hippocampal biopsies and MVEC using QIAzol reagent (Qiagen) according to the manufacturer's protocol. Quantification of ABC transporters was performed by a TaqMan Low Density Array, which is based on an Applied Biosystems 7900HT Micro Fluid Card. The method allows simultaneous analysis of 47 human ABC transporters and the reference gene 18S rRNA in two replicates of four different samples per run [17]. Results The GeneChip expression analysis was performed from control hippocampi (n = 3), hippocampal MTLE biopsies (n = 8), and isolated MVECs (n = 2) thereof. A total of 27,000 out of 54,000 mRNA transcripts of known or predicted function were found to be present in all samples. The expressed genes were normalized and compared with control. Gene transcripts with >2-fold induction or reduction were analyzed. The comparison of the gene expression profiles of AHEs, MVECs, and controls is shown in the multidimensional hierarchical cluster analysis (Figure 1). This analysis revealed remarkable differences between the three groups. When AHEs were compared with controls, a total of 1,253 genes were found to be significantly upregulated and 637 genes down-regulated. Among the many up-regulated genes we found also GFAP (7.6-fold) as a bona fide marker for astrogliosis. The comparison between MVEC Epilepsy Research and Treatment and controls revealed 7,862 up-regulated and 9,504 downregulated genes. These much higher numbers compared to AHE reflect enrichment of endothelial mRNA upon eliminations of glial genes from the highly purified MVEC population. Various multidrug transporters of the ATP-binding cassette protein, subfamilies A (ABCB) and B (ABCC), were found to be overexpressed in AHEs and MVECs compared to the control group. A significant up-regulation of ABCB1/Pgp, ABCB2, and ABCB3 was found in MVECs of epileptic hippocampi (Table 2). Among the ABCC transporters, only ABCC4 was significantly up-regulated in both AHEs and MVECs (Table 2). These data were validated by quantitative RT-PCR analysis, as summarized in Table 3. The trend which was found by micro array analysis became statistically significant by RT-PCR for P-gp, MRP-2, and MRP4 (Table 3). As we had to deal with a large number of differentially expressed genes we evaluated those genes which were found to be highly overexpressed in epileptic patients compared to controls. Surprisingly, immunological factors such as cytokines were significantly up-regulated in AHEs (n = 8) compared to controls (n = 3). This includes proinflammatory interleukins (IL-1α, IL-1β, IL-6, and IL-18), and cytokines (TNF-α and TGFβ-1) as shown in Figure 2. The immunohistochemistry staining of human hippocampal section for IL-1β revealed a strong expression, mainly localized at the cerebrovascular unit from epileptic hippocampus (Figures 3(c) and 3(d)), and staining for IL-6 showed positive expressing cells to be evenly distributed in vessels, neurons, and glial cells of epileptic hippocampus (Figure 3 The morphology of cultured MVECs derived from epilepsy patients had a coblestone morphology with some spindleform-like, elongated cells ( Figure 5(a)) which was also seen in control MVEC cultures (data not shown) and their purity was demonstrated by expression of the endothelial marker CD105 (Figures 5(b) and 5(c)). Interestingly cultured MVECs isolated from MTLE patients revealed by flow cytometry an enhanced P-gp expression in comparison to controls (Figures 5(b) and 5(c)). In contrast, no difference was found in their level of MRP1 expression. These data are consistent with the findings on the mRNA level. Discussion In our study resected hippocampi and hippocampal MVECs, forming the BBB from epileptic patients, were evaluated for their global gene expression profile. Our analysis was based on the "multitransporter up-regulation hypothesis". We also singled out those genes that were found highly up-regulated. In examining the gene expression profile of the multidrug transporters, we included all transporters that have been described in the literature: 13 ABCC and 12 ABCB transporters. Some of them have been associated with medically intractable epilepsy: ABCB1/P-gp. [18][19][20][21][22]: MRP1 and MRP2 [18][19][20]: MRP3 and MRP5 [19]. Most of these findings are based on IHC. However, Dombrowski et al. performed a gene expression analysis of MVEC isolated from human epileptogenic hippocampi and discovered an up-regulation of P-gp, MRP2, −3 and −5, which is partly consistent with our findings. In addition, we observed an up-regulation of ABCB2, ABCB3, ABCB4, and MRP4. Until now these transporters have not been identified as being overexpressed in epileptogenic hippocampi, suggesting that of the list of gene products contributing to the "transporter hypothesis" may need to be enlarged. With regard to the localization of these transporters, our results show an up-regulation, mainly in endothelial cells. Several studies have addressed the question of where these transporters are exactly localized; however, the answers are still highly controversial (luminal versus basolateral or even both). At the protein level, IHC of hippocampal sections revealed that P-gp and MRP1-5 were largely confined to the endothelial cells of the hippocampus. The overexpression of these genes was validated by quantitative real-time RT-PCR, and the results were compared to that of the gene array. No significant differences were found by gene array when the averaged values of P-gp of individual epileptic patients and controls were compared. However, P-gp values obtained by RT-PCR revealed a statistically significant difference between them. The same situation was also observed by comparing the MRP4 values. This finding suggests that the RT-PCR method is more sensitive; further support for this is given by the threshold cycles (CT-values). When comparing MRP1, MRP2, and MRP3, no significant differences were obtained between the two methods. In contrast, a statistical significance for MRP5 was found by gene array analysis, but not by the RT-PCR analysis. Unexpectedly, we observed a strong up-regulation of various cytokines (IL-1α, IL-1β, IL-6, IL-18, TNF-α, and TGF-β1) in the hippocampus of epilepsy patients. The fact that MTLE-HS has not been considered to be an immunological disorder makes this finding even more interesting. Only few papers addressed a possible link between immunology and epilepsy, using different approaches. Two studies have been published on the gene expression profile of hippocampus in MTLE [23,24]. In one of the studies, the mRNA expression was compared in the hippocampi of three patients with sclerosis and three without, using a small array representing 588 genes. This study revealed 9 overexpressed genes and 12 underexpressed genes. Among these genes are those associated with gliosis (GFAP), cell death/survival, and neuronal plasticity, but none associated with inflammation [23]. In the second study, five sclerotic hippocampi from MTLE patients were analysed, using the Affymetrix GeneChip U133A [24]. Unfortunately, this study does not report on the full expression pattern in these specimens, but only the expression pattern that was also present in the hippocampi from rats having pilocarpineinduced seizures. It is important to mention that the lack of a suitable control ("healthy" hippocampus) makes it difficult to interpret these results. With respect to the link between MTLE and immunology, one of the most remarkable findings is the role of IL-1β in the development of febrile seizures in animal models as well as the assumption that a polymorphism of IL-1β at position −511 may favour a high production of IL-1β in patients with MTLE-HS, possibly a major trigger for the development of HS [12]. In another study, the role of cytotoxic T cells and auto-antibodies against neurons in the brain in Rassmussen encephalitis was examined suggesting an immunotherapy as a treatment approach [25]. In a recent article, possible pathways underlying neuronal death in HS have been postulated, including the participation of cytokines and chemokines, leading to a disturbance of the glutamatergic system, and subsequently to the persistence of seizures by chronic neuronal overexcitation [10,[26][27][28]. Among the different factors potentially involved in the molecular pathway underlying glutamate release from astrocytes, chemokines, chemokine receptors, IL-1β, and prostaglandin E2 have been found to be highly up-regulated in both epileptogenic hippocampus and MVEC. In experimental models and in clinical cases of epilepsy, proinflammatory and anti-inflammatory cytokines have been described in brain and plasma after seizure induction. Experimental findings demonstrated that proinflammatory cytokines IL-1β, IL-6, and TNF-α in microglia and astrocytes are increased and followed by a cascade of downstream inflammatory events which may recruit cells of the adaptive immune system [29,30]. In addition to inflammatory disease, recent evidence shows the activation of innate immune system and associated inflammatory reactions in epilepsy may modulate some of the molecular and structural changes occurring during and after seizure activity [30]. Moreover, experimental strategies aiming to block CNS or systemic inflammatory pathways reduce status epilepticus duration and seizure frequency [31]. Nevertheless, little is known about the possible underlying mechanisms which might be responsible for the fact that inflammatory factors are strongly overexpressed [32]. How inflammatory reactions intrinsic to the brain compared with those mediated by peripheral immune cells can be contributed to the epileptogenesis is poorly understood [33]. BBB failure might be a link between these two mechanisms. In summary, whether these findings contribute to the persistence of seizures despite treatment with AEDs, or whether this event must be regarded as an epiphenomenon, is currently an open issue. It remains elusive, however, if such changes are causes or consequences of the disease. Since the underlying pathophysiology of MTLE-HS remains an issue of debate, more research is needed with the common goal of finding new targets and therapeutic strategies. The findings that inflammatory mediators contribute to the onset and recurrence of seizures in experimental models, as well as the presence of inflammatory molecules in human epileptogenic tissue, do highlight the possibility of targeting specific inflammation-related pathways to control seizures that are otherwise resistant to the available AEDs. Drugs that block IL-1β actions have entered clinical trials as potential therapeutics for autoimmune and inflammatory pathologies and may also have therapeutic potential in refractory epilepsies associated with proinflammatory processes in the brain [34].	2016-05-12T22:15:10.714Z	2011-03-06T00:00:00.000	{ "year": 2011, "sha1": "c5e741ad97c6233c7e9b40c33a23cfa8cae608d4", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/archive/2011/758407.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "c5e741ad97c6233c7e9b40c33a23cfa8cae608d4", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
266057968	pes2o/s2orc	v3-fos-license	FUNCTIONAL AND PSYCHOSOCIAL OUTCOMES ON PATIENTS WITH MODERATE-TO-SEVERE COVID-19 DISEASES: A POST-HOSPITALIZATION FOLLOW-UP STUDY Intro COVID-19 survivors suffer from variable limitations affecting their quality of life. We examined the functional and psychosocial outcomes among COVID-19 patients with moderate-to-severe diseases by three months post-hospitalization. Methods This retrospective cohort study included 510 COVID-19 patients admitted to Kepala Batas Hospital with moderate-to-severe diseases, requiring oxygen therapy during hospitalization (Malaysia COVID-19 severity category ≥5; WHO scale ≥5), between January and August 2021. We followed up with telephone surveillances by 90 days post-discharge from the hospital, assessing their performance in activities of daily living and psychosocial implications. Relevant clinical data were extracted from medical records. We compared patients with low (<10L/ min) versus higher (≥10L/min) oxygen requirements on the patient-reported outcome variables. Findings Among 441 survivors (86.5%), half (n=223, 50.6%) were male, with a relatively young population with a mean age of 50.2 (13.73) years. Only 17.9% were partially vaccinated and 5.7% had complete vaccination before hospitalization. Nearly 70% were supplemented with nasal prong or face mask oxygenation (<10L/ min), 26.1% received high flow oxygenation and 4.1% were mechanically ventilated. By 90-day follow-up, >90% had their functionality returned to baseline before hospitalization. Only 1.6% required home oxygen supplementation. Compared with their baseline functionality, 4.8% were unable to perform basic household chores, 4.1% required assistance in mobilization and 2.5% became fully dependent on caretakers. Among 254 patients returning to work, 98% worked in the same institution but 18.9% required job scope adjustments. About 7.7% experienced post-covid stigma at home and/or work, 3,9% suffered from depression 5.7% became self-isolated and 0.9% had suicidal ideation or attempts. Functional and psychosocial outcomes were similar between patients with low and higher oxygen requirements (all p>0.05). Conclusion Despite fair recovery outcomes reported by survivors with moderate-to-severe disease, a small proportion suffered from significant functional limitations and psychosocial adversity. Post-hospitalization care is essential to screen-detect post-COVID complications and provide timely interventions. Intro: A few communities collect date palm sap throughout the year to ferment and consume. This study's objective was to characterize Pteropus bats' sap feeding behavior around the year to identify the potential for sap contamination with bat excreta. Methods: We used infrared cameras to observe bats' feeding behavior for 28 tree-nights per month for 22 months from March 2013 to December 2014. We placed the cameras at 4 sap-producing date palm trees focused on the sap-producing surface and collection pot from 5:00 PM to 6:00 AM for seven consecutive nights. We extracted the number and duration of bat visits and duration of contact with date palm sap from the images. Findings: We recorded a total of 26,870 bat visits (5% Pteropus, 90% non-Pteropus and 5% unidentified) from 616 observation treenights. Median duration of each visit was higher for Pteropus bats than non-Pteropus bats (8 versus 0.03 minutes, P < 0.001). Median duration of contact with date palm sap was higher for Pteropus bats (0.67 versus 0.03 minutes, P < 0.001) for each visit. The average number of Pteropus bat visits per night was the highest during spring (17) followed by winter (14), post-monsoon (6), and monsoon (3). Conclusion: Even when date palm sap is harvested year-round, Pteropus bats visit the date palm trees more frequently during the spring and winter, perhaps due to lack of other available food. Feeding behavior could be one reason why the risk of Nipah infection to people has been concentrated in the winter season, even when fermented sap is consumed year-round. Sap harvesters should regularly use skirts to prevent bats from contaminating the date palm sap to prevent Nipah virus and other bat associated zoonoses. Intro: COVID-19 survivors suffer from variable limitations affecting their quality of life. We examined the functional and psychosocial outcomes among COVID-19 patients with moderate-tosevere diseases by three months post-hospitalization. Methods: This retrospective cohort study included 510 COVID-19 patients admitted to Kepala Batas Hospital with moderate-tosevere diseases, requiring oxygen therapy during hospitalization (Malaysia COVID-19 severity category ≥5; WHO scale ≥5), between January and August 2021. We followed up with telephone surveillances by 90 days post-discharge from the hospital, assessing their performance in activities of daily living and psychosocial implications. Relevant clinical data were extracted from medical records. We compared patients with low ( < 10L/ min) versus higher ( ≥10L/min) oxygen requirements on the patient-reported outcome variables. Findings: Among 441 survivors (86.5%), half (n = 223, 50.6%) were male, with a relatively young population with a mean age of 50.2 (13.73) years. Only 17.9% were partially vaccinated and 5.7% had complete vaccination before hospitalization. Nearly 70% were supplemented with nasal prong or face mask oxygenation ( < 10L/ min), 26.1% received high flow oxygenation and 4.1% were mechanically ventilated. By 90-day follow-up, > 90% had their functionality returned to baseline before hospitalization. Only 1.6% required home oxygen supplementation. Compared with their baseline functionality, 4.8% were unable to perform basic household chores, 4.1% required assistance in mobilization and 2.5% became fully dependent on caretakers. Among 254 patients returning to work, 98% worked in the same institution but 18.9% required job scope adjustments. About 7.7% experienced post-covid stigma at home and/or work, 3,9% suffered from depression 5.7% became self-isolated and 0.9% had suicidal ideation or attempts. Functional and psychosocial outcomes were similar between patients with low and higher oxygen requirements (all p > 0.05). Conclusion: Despite fair recovery outcomes reported by survivors with moderate-to-severe disease, a small proportion suffered from significant functional limitations and psychosocial adversity. Post-hospitalization care is essential to screen-detect post-COVID complications and provide timely interventions. Methods: The SARS-CoV-2 RNA detection by real time RT-PCR is a molecular in vitro diagnostic test that aids in the detection and diagnosis of SARS-CoV-2 in nasopharyngeal and oropharyngeal specimens. This test is based on nucleic acid extraction and amplification technology and uses oligonucleotide primers and duallabeled hydrolysis probes. RNA is isolated and purified from specimens using the Abbott m20 0 0sp. This technology uses magnetic particles to capture and purify the RNA. The bound RNA is eluted and transferred to a 96 deep-well plate and is ready for amplification. The master mix is prepared manually and is added to a PCR plate together with the extracted RNA. The RNA is reverse transcribed to cDNA and subsequently amplified in the Abbott m20 0 0rt. In this process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension step of the PCR cycle, the 5' nuclease activity of Taq polymerase degrades the probe, causing the reporter dye molecules to be cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle on the Abbott m20 0 0rt instrument. Findings: The clinical evaluation was performed by testing patient samples in a blinded fashion. The performance of SARS-CoV-2 Assay was established using 60 clinical specimens. The positive and negative percent agreements were analyzed by comparing the SARS-CoV-2 Assay results to Seegene's AllplexTM 2019-nCoV which showed 100% concordance. Conclusion: This assay demonstrated accuracy and reproducibility for the detection of SARS-CoV-2. Intro: Surveillance of antimicrobial resistance (AMR), use and consumption of antimicrobials are critical elements to inform policy and practice as well as monitoring the effectiveness of implementation of national Action Plans on AMR. The burden of AMR can be determined through quality microbiology laboratories which are connected through a well-organized national surveillance system. Designing, building and deploying an efficient surveillance network remains a challenge in most resource constrained countries. Lack of robust information technology (IT) infrastructure and technical capacity and an appropriately designed Laboratory Information Management System LIMS) are perceived to be major barriers to comprehensive AMR surveillance. Methods: A review of the early implementation of the National AMR surveillance system as per the National AMR Surveillance Strategy was conducted by multiple stakeholders over several consultative meetings. Gaps in the implementation process were identified and recommendations made for improvement in systems deployed to support National AMR Surveillance functions. Findings: Even when functioning microbiology laboratories are established, systems that can transmit and aggregate results as shown in the illustration below, are required. Several software such as Labware, WHONET, Bliss have been deployed in some hospitals but often lack interoperability and integration with the national AMR surveillance system, and may not combine all required surveillance data elements. Major barriers to improving the quality of surveillance include: costs of software and hardware, system support and maintenance, and shortage of IT experts. Conclusion: There is need to fully understand the AMR surveillance systems in place, appropriateness for intended use, barriers to implementation of AMR surveillance system. Identifying easy to use platforms combining clinical data, pharmacy data, and microbiology data is a challenge in settings such as Kenya. Designing a surveillance system that integrates required IT infrastructure, LIMs, personnel, data governance to support effort s to monitor AMR and antibiotic use is key to an efficient surveillance system. https://doi.org/10.1016/j.ijid.2023.04.373 Aga Khan University, Medicine, Karachi, Pakistan Intro: Dexamethasone, a corticosteroid, was recently demonstrated to be the only medication capable of reducing mortality in severe COVID disease in the UK's Recovery Trial. There is a need to compare different steroids because it is well recognised that different corticosteroids have varied pharmacodynamic properties. The aim of our study was to compare outcomes in severe or critical	2023-05-18T05:06:16.882Z	2023-05-01T00:00:00.000	{ "year": 2023, "sha1": "60f7a6aba42b0dd06a2a238a628f01ab3d5ade28", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.ijid.2023.04.371", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "60f7a6aba42b0dd06a2a238a628f01ab3d5ade28", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
237506426	pes2o/s2orc	v3-fos-license	Decreased Sleep and Subjective Well-Being as Independent Predictors of Injury in Female Collegiate Volleyball Players Background: The relationship among sleep duration, subjective well-being, and injury risk in athletes is poorly defined. Purpose: To evaluate the independent effects of sleep duration, sleep quality, and subjective well-being on in-season injuries in collegiate female volleyball athletes. Study Design: Cohort study; Level of evidence, 2. Methods: During a 9-month competitive season, 17 female National Collegiate Athletic Association (NCAA) Division I volleyball players reported mood, fatigue, stress, soreness, sleep duration (hours), and sleep quality every morning. Well-being measures were recorded from 0 (worst) to 5 (best), and all time-loss injuries were recorded by the team athletic trainer. Separate mixed-effects logistic regression models were used to evaluate the effects of sleep and subjective well-being on in-season injury. Each well-being variable was also included in a separate mixed-effects logistic regression model with sleep duration as a covariate. Results: A total of 54 injuries were recorded during the study period. Compared with days without an injury, mood, fatigue, stress, soreness, sleep quality, and sleep duration were significantly worse the day before an injury occurred. In the separate prediction models, in-season injury was significantly predicted by fatigue (odds ratio [OR], 0.56 [95% CI, 0.36-0.86]; P = .008), mood (OR, 0.52 [95% CI, 0.35-0.78]; P = .002), stress (OR, 0.63 [95% CI, 0.42-0.94]; P = .023), soreness (OR, 0.54 [95% CI, 0.38-0.79]; P = .001), sleep quality (OR, 0.49 [95% CI, 0.34-0.7]; P < .001), and sleep duration (OR, 0.69 [95% CI, 0.55-0.87]; P = .001). In the multivariable models, sleep duration remained a significant independent predictor in each of the subsequent multivariable models (OR, 0.72-0.74; P < .05 for all), as did mood (OR, 0.55 [95% CI, 0.36-0.83); P = .005) and soreness (OR, 0.57 [95% CI, 0.39-0.83]; P = .003), while fatigue (OR, 0.65 [95% CI, 0.42-1]; P = .054) and stress (OR, 0.68 [95% CI, 0.45-1]; P = .061) no longer reached statistical significance. Conclusion: Increased sleep duration, mood, and decreased soreness were independently associated with a reduced risk of in-season injury in this cohort of female NCAA volleyball players. The benefits of sport participation on physical and mental health are well-established, 27 but injuries continue to be an inherent risk of participation in athletics. When athletes sustain injuries resulting in time lost from participating in their sport, it can lead to significant negative outcomes, such as lower quality of life, incidence of depression, lower level of play after return to sport, and a threat to athletic success. 1,9,21,22 Injured college athletes exhibit significantly higher emotional distress for at least 2 months after injury, 14 and a significant portion of injured young athletes report symptoms of posttraumatic stress disorder after an anterior cruciate ligament rupture. 20 Although there is a growing understanding of the potentially long-term negative impacts of injury on athletes' well-being, factors unrelated to training that affect the likelihood of injury are not well-understood. Volleyball is a popular sport, particularly among female collegiate athletes. In the 2018-2019 competitive season, there were 1112 National Collegiate Athletic Association (NCAA) women's volleyball teams, with a total of 17,780 athletes participating. 19 While volleyball is considered a noncontact sport, injuries are relatively common, largely ankle and wrist sprains and concussions, and female collegiate volleyball players sustain more injuries per athlete exposure (AE; individual training sessions and competitions) than male volleyball players. 2 In data originating from the NCAA Injury Surveillance Program, a nonprofit research organization analyzing data from women's volleyball injuries from the 2013-2014 and 2014-2015 academic years, it was found that female athletes sustained 7.07 injuries per 1000 AEs . 2 Competitive college and high school athletes have reported depression symptoms 1 week up to 1 month after injury, and women are particularly at risk. 1 Collegiate student-athletes balance their time between athletic, academic, and social commitments, and sleep often suffers as a result. There is accumulating evidence that sleep duration and quality are associated with improved performance and competitive success in athletes and that a lack of sleep can be inhibitory of endurance performance and reaction time. 35 Sleep is also acknowledged among elite athletes as an important recovery tool. 5 Longer sleep duration counters the negative effects of high training loads on well-being in female adolescent soccer players, and sleep and training load had independent effects on fatigue, mood, and stress in athletes. 31 While sleep can have a positive effect on athletes' performance and recovery, aspects of sports participation can have a negative impact on sleep. In collegiate soccer players, greater levels of fatigue, tension, depression, and anger are associated with global sleep dysfunction. 3 Subjective measures of well-being (mood, fatigue, stress, and soreness) and sleep quality have been shown to be predictors of injury in various populations. 24,28,29,33 In athletes, impairments in subjective wellbeing and sleep quality contribute to increased risk of injury, 9,13,32 and increased sleep duration is independently associated with a reduced risk of in-season injury in male collegiate division I basketball players. 34 Taken together, these findings suggest that lower sleep duration and well-being both increase the risk of injury in athletes. However, few studies have attempted to study the interaction of sleep and well-being on injury risk, and these limited data should be replicated in female athletes. Therefore, the purpose of this study was to evaluate the independent effects of sleep and well-being on in-season injuries in collegiate female volleyball athletes. We hypothesized that both decreased sleep duration and subjective well-being would be independent predictors of in-season injuries in female collegiate volleyball players. Study Design This study used a prospective cohort design, and the participants in this study were 17 female NCAA Division I volleyball athletes who provided information regarding self-reported training load, sleep, and subjective wellbeing every day during a 9-month study period, including the fall championship and spring competitive seasons. All procedures were approved by an institutional review board. Because the data were collected as part of the standard athlete-monitoring practices of the team involved and analyzed retrospectively, informed consent was not deemed necessary for inclusion in this retrospective analysis. Height and mass were measured at the start of each testing session with a stadiometer and a standard electronic scale, respectively. No data were included from the 4-week winter break period between the fall and spring seasons. Each morning during the study period, before any volleyball events, athletes were asked to provide ratings of fatigue, mood, soreness, stress, and sleep quality on a 0 (worst) to 5 (best) Likert scale, with descriptive text prompts 31 as well as prior night sleep duration in hours, using online software (Metrifit). The evaluation of sleep quality through self-report has been suggested as an appropriate measure in team sport athletes, even when compared with objective measures, such as wrist actigraphy. 8 Finally, self-reported sleep duration is a commonly used, low-cost metric, which has been found to be highly correlated with objective measures of sleep duration in athletes. 7 Daytime naps were not included as part of sleep duration. Throughout the study period, injuries that resulted in time loss were recorded by the team athletic trainer and reported as both injuries per day and injuries per week. The date of injury onset was determined by the athletic trainer as the first date the injury was recorded. Both first-time and repeat injuries were included if they were felt to represent new injuries based on resolution of symptoms and return to full participation between time-loss injuries. Because the athletic trainers worked directly with the student-athletes during treatment of injuries, blinding was not possible. Compliance with the completion of daily training load and well-being ratings was encouraged periodically throughout the study period by coaching staff. Statistical Analysis Data were initially evaluated for normality using descriptive statistics and histogram analysis. Prior night sleep and subjective well-being measures were compared between days with and without an injury using least squares means from mixed-effects linear regression models, including individual repeated measures as a random effect. A mixedeffects logistic regression model was developed to predict injury, including increments of sleep duration (<6 hours, 6-6.9 hours, 7-7.9 hours, and 8 hours) 35 as a fixed effect and individual repeated measures as a random effect. To evaluate the association between injury and prior night sleep duration and subjective well-being measure from the morning of the same day (mood, fatigue, soreness, and stress), separate mixed-effects logistic regression models were used to evaluate their association with in-season injury by including the variable as a fixed effect and individual repeated measures as a random effect. This tested the relative ability of each variable reported in the morning to predict the likelihood of injury later that same day, while adjusting for the repeated measures from each individual. To determine the independence of sleep and well-being as predictors of injury, separate mixed-effects logistic regression models were developed to predict injury with each well-being variable (mood, fatigue, soreness, and stress) and sleep duration included as fixed effects and individual repeated measures as a random effect. Statistical significance was determined a priori at the .05 level, and all tests were 2-tailed. All statistical analyses were performed in R (The R Project for Statistical Computing). 23 RESULTS Anthropometric data for the participants are presented in Table 1. A total of 54 injuries were identified on 45 days in 14 of the 17 athletes. The distributions of injuries throughout the study period by day and week are shown in Figure 1. Three players left the team in the middle of the year because of graduation and end of eligibility, resulting in some missing data from the second half of the year. Combined with other randomly missed data entries by players, the overall missing information was 13% for the year. If the data of the 3 players' who left the team midyear were not considered missing after they were no longer on the team after the middle of the year, the missing values would be 6.8%. The mean sleep duration during the study period was 7.9 ± 1.2 hours, and 70.6% of the athletes had a mean of <8 hours of sleep per night. The mean nightly sleep duration and quality are shown in Figure 2. Compared with days without a reported injury, days in which an injury occurred were found to have significantly lower (worse) mood, fatigue, stress, soreness, sleep quality, and sleep duration the night before the injury (Figure 3). Sleep duration and quality were found to be significantly related to each of the subjective well-being measures the following morning ( Table 2). The relative risk of injury following nights with varying amounts of sleep the prior night are shown in Figure 4. Daily fatigue, mood, stress, soreness, sleep quality, and sleep hours were all found to be significant predictors of injury in the separate mixed-effects logistic regression analysis; however, after inclusion with sleep duration in the multivariable models, sleep duration, mood, and soreness remained significant, independent predictors of injury (Table 3). DISCUSSION The primary finding of this study was that both sleep and subjective measures of well-being are independent predictors of injury in female NCAA Division I volleyball athletes. Specifically, mood and soreness remained independent predictors of in-season injury, even after adjusting for sleep duration the night before. Worse morning stress and fatigue also seemed to be associated with an increased risk of injury later that day, but these relationships did not meet our statistical significance level, perhaps because of the relatively small sample size. Our findings are consistent with previous studies in elite athletes that found that decreases in sleep volume contribute to a higher risk of injury. 18,29,34 Specifically, we sought to reproduce previously published findings related to well-being and injury risk regarding collegiate men's basketball athletes. With respect to sleep duration, our results are very similar, suggesting that these findings may be generalizable to other populations of collegiate athletes. In contrast to our findings, however, a recent report in male Division I football players found that sleep duration was not correlated with injury incidence during a football season. 6 That study used time loss as a surrogate for injury incidence and reported low compliance with sleep duration data collection, which may have undermined their ability to identify a relationship between sleep and injury risk. In the current study, athletes reported sleep metrics and subjective well-being metrics every day for an entire school year, allowing us to develop a more complete data collection and analysis with insight into the relationship among sleep, subjective wellbeing, and injury risk. While the relationship between sleep and subjective well-being has been previously documented, 4,10,31 the relationship between subjective well-being and injury risk in athletes is incompletely understood. In the present study, we found that all of our measures of well-being were predictive of in-season injury. Because sleep duration and well-being may be related, we included both sleep duration and well-being in our multivariable models to identify the independent effects of each well-being measure. Our findings that well-being and injury risk are related also are in line with findings from our previous study in collegiate male basketball players, where it was found that subjective well-being variables were significant predictors of injury. 34 Our findings differ slightly, however, in that while that study found that soreness in male athletes was the only independent subjective well-being metric predictive of injury after adjusting for sleep duration, we found that both soreness and mood were independent predictors of injury in female athletes in the multivariable models. Nonetheless, the finding that worse subjective well-being is correlated with higher incidence of injury is consistent with prior research exploring this relationship. 11,26 In a retrospective study, Galambos et al 11 asked athletes to report injuries and well-being in the preceding 12 months, where it was found that higher rates of anger, confusion, depression, fatigue, tension, and life stress were all significantly higher in athletes who sustained injuries than those who reported no injuries. Smith et al 26 found that while physical factors did not significantly predict injuries in male hockey players, psychosocial factors, including low vigor and high fatigue, significantly predicted athlete injuries. Predictors of injury may be sex-and sport-specific. Although prior research has continually identified the value of subjective well-being as a predictor of injuries in athletes, little research has attempted to specifically evaluate this in female athletes. Female athletes have different rates of injury from male athletes as well as higher rates of Sleep duration is shown in hours, while sleep quality is reported from 0 (worst) to 5 (best). depressive symptoms following athletic injury than male athletes. 17 In a group of adolescent female soccer players, lower mood and higher acute training were associated with increased injury risk. 32 Although it is widely reported that athletes do not get the quantity and quality of sleep that is b Separate mixed-effects logistic regression models to predict injury with morning well-being or prior night sleep measure as a fixed effect and individual as a random effect. c Separate mixed-effects logistic regression models to predict injury with morning well-being measure and prior night sleep duration as fixed effects and individual as a random effect. recommended, 15 we found that athletes who slept less than 7 hours the prior night had a significantly higher risk of injury than those who slept more than 7 hours. This suggests a minimum sleep duration of 7 hours to minimize injury risk in collegiate athletes, which is in line with other recommendations in the literature. 25 However, prior research in youth athletes has suggested an 8-hour minimum cutoff to minimize injury risk 18,30 ; moreover, the most recent consensus statement from the American Academy of Sleep Medicine and Sleep Research Society recommends a minimum of 7 hours of sleep and that sleeping over 9 hours may be most appropriate for young adults. 36 Our finding that sleep duration, mood, and soreness are independent predictors of injury in female collegiate athletes suggests that a more holistic approach to injury prediction may be warranted in this population. Mental health depends on affective, subjective states, including emotions, stress responses, impulses, and mood, all of which affect how a person thinks, feels, and behaves. 12 Unlike objective measures of physical health and fitness, subjective measures may be equally, if not more, relevant to injury risk in athletes. The traditional focus of sports team training has been focused on daily training, conditioning, nutrition, and coaching, but there is recent evidence that a shift in sports team culture to include sleep as a critical component of peak performance has resulted in both improved sleep and athletic performance among college athletes. 16 Consistent with prior evidence in high-level athletes, our data suggest that daily reporting of sleep, soreness, stress, mood, and fatigue may help identify collegiate female volleyball athletes at risk of injury and facilitate interventions to reduce the risk of injury. In addition, it may allow coaches and health care providers to intervene on behalf of their athletes to mitigate negative psychosocial symptoms and improve mental health. Given the significant impact that injury can have on future health outcomes in athletes, developing robust methods to identify athletes at greater risk of injury is imperative. Limitations This study has several limitations. As with virtually all longitudinal studies, we did have missing data. This was felt to be missing at random within the data set, however, and imputation of missing sleep and well-being data did not result in a substantive change in the results of our analyses. Naps and travel for competitive events were not included in the sleep-duration data collection, and thus, their impact on the results is unknown. We did not include additional risk factors for injury, such as anatomic differences, body composition, fitness level, or prior injury, which could potentially confound our results. Furthermore, our findings do not confirm the cause-and-effect relationship between sleep and injury risk in athletes, and further study that explores the relationship among overtraining, sleep, and injury is warranted. While we sought to identify relationships within a specific sport and sex, a larger sample size in future studies would allow for the inclusion of additional risk factors and their interactions as well as separate prediction models for different injury mechanisms. The mechanisms by which sleep, mood, and soreness play a factor in injury risk is beyond the scope of this study; however, they could all have a negative effect on reaction time, cognitive function, and confusion, which could lead to greater injury risk. Additional research is necessary in other populations of athletes to determine whether these relationships are similar and more generalized or if sport-specific recommendations can be made. CONCLUSION This study demonstrated that increased sleep duration and improved subjective well-being are associated with a decreased risk of injury among collegiate female volleyball athletes. Although we also found that several subjective well-being measures were predictors of injury risk, mood and soreness remained independent predictors of injury after adjusting for the effects of sleep duration. This seems to suggest that among female collegiate volleyball athletes, both sleep duration and subjective well-being exert separate effects on injury risk. Given the known benefits of sleep, however, efforts to promote proper sleep may improve well-being and decrease the risk of in-season injury. Alongside the existing literature, these results suggest that proactive monitoring of sleep and well-being on a daily basis may help identify individuals at risk for injury and facilitate interventions to reduce risk.	2021-09-15T05:21:38.240Z	2021-09-01T00:00:00.000	{ "year": 2021, "sha1": "857336f4d74b7b6b65cc911e5c30a0266e73f734", "oa_license": "CCBYNCND", "oa_url": "https://journals.sagepub.com/doi/pdf/10.1177/23259671211029285", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "857336f4d74b7b6b65cc911e5c30a0266e73f734", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
259810131	pes2o/s2orc	v3-fos-license	Role of the village head in handling inheritance disputes outside the court in the customary inheritance law of the Osing Tribe (Blambangan) (Study in Kemiren Village, Glagah District, Banyuwangi Regency) id INTRODUCTION Development in the field of law with the existing legal system is also encouraged to continue, so that it does not lead to a situation where physical development moves quickly and on the other hand development in the field of law moves slowly. Every country has its own legal system that is different from one another. The positive law system in Indonesia applies three legal systems namely, customary law, Western law and Islamic law. Customary law is one of the important sources of formal law for the development of national law towards legal unification which will be implemented through the making of statutory regulations. One of the elements forming national law is customary law. The background that makes the Customary Inheritance Law different from other legal systems is the thought of the Indonesian nation which prioritizes kinship and brotherhood so as to create harmony and harmony and peace in life. The similarities between the three legal systems are that there are both heirs who pass on their assets, both have heirs as subjects entitled to inheritance, and both have inherited assets as objects to be distributed. Customary Inheritance Law divides the inheritance to several parties either before the heir dies, or can also distribute the inheritance after the heir dies. The heirs referred to in this case are all people who will receive the distribution of inheritance, both as heirs, namely people who have the right to inherit and people who are not heirs but receive inheritance. Likewise, in the context of fostering the National Inheritance Law, there are elements of Customary Inheritance Law. Therefore, materials on customary inheritance law need to be explored by conducting research on existing literature and field research to obtain and find out whether the various systems and principles of customary inheritance law found throughout the archipelago can be drawn into conformity and reach common ground. customary law has developed under the influence of several factors, namely the horizontal process factor, this factor can be caused by economic pressure or to break away from the backwardness of the communal community, towards the development of a freer individual identity, the vertical process factor is a symptom that destroys the boundaries of "social stratification" "(social stratification), the process factor of modernization and emancipation is a factor that coincides with economic, political, scientific, and technology. Customary law is a living law, because it embodies the real legal feelings of its people. Mochtar Kusumaatmadja stated that the function of law is as a development tool. According to him, good law is law that is in accordance with the living law in society, which of course is also appropriate and is a reflection of the values prevailing in society. Customary Inheritance Law contains rules governing the process of forwarding and transferring goods -property and intangible goods from a generation of humans to their descendants. This often causes disputes within a family which is caused either by the inheritance factor or by the heirs, as well as the influence of different customs, so that the procedures for distributing inheritance are different. The Indonesian nation in its mind is based on the principle of kinship, which prioritizes living in harmony and peace over material and self-serving traits. There is a phenomenon in our society that there has been a tendency for families to be more concerned with material possessions by neglecting and destroying harmony in life and kinship or brotherhood, ignoring the legal principles in the Indonesian Customary Inheritance Law, so this cause discord. The principles of customary inheritance law are: Principles of Godhead and self-control; The principle of equal rights and collective rights; The principle of harmony and kinship; The principle of deliberation and consensus; The principle of justice and parimirma. The division that is felt to be unfair because it uses the "sepikul segendong" distribution system or the distribution with a ratio of two to one for men causes problems in the distribution of inheritance. On a small scale, customary law usually applies in villages that still follow the customary rules that are still The highest leader in customary law is the village elder or village head. The village head as a leader who is respected and respected in a village community plays a major role in maintaining the continuity and existence of the customs of the local community he leads. The measure of justice used to decide on the distribution of inheritance for all heirs is also not the same in all regions, depending on what inheritance system is used in that area, and its application by the village head. METHOD In compiling this research, the authors used a research method approach. The approach used in this research is the sociological juridical method, which is a method intended to determine the relationship between juridical factors and sociological factors from the problems to be analyzed. Location of the research to be carried out in this research is in Kemiren village, Taman Sari sub-district with the consideration that Kemiren village is a village in Banyuwangi Regency which is used as a cultural tourism village and is a traditional village of the Osing tribe with residents who still adhere to and carry out customary law provisions. Kemiren Village is a village where an inheritance dispute has occurred, involving the Village Head as a mediator in its settlement. the types of data sources obtained are secondary and primary data sources and the population used are all elements that are related and have an interest in relation to Customary Inheritance Law in the village of Kemiren, in this case the Village Head, Village officials (such as Kaur Kesra), Kemiren community leaders such as Kyai. The determination of the sample was carried out based on a non-random technique, namely purposive sampling, in which the authors chose subjects from members of the population, namely representative parties, including: Village head; Village Administration Devices; like Kaur Kesra; Indigenous community leaders, such as Kyai. The analytical method used in this study is a qualitative descriptive method, namely analyzing data that has been obtained from respondents orally and in writing, as well as their real actions. From the data collected, it is then presented in order to obtain an overview of the problem being discussed, then looks for a solution and draws conclusions according to the problems in the research. RESULTS AND DISCUSSION The results of this study began with a detailed introduction to the location, such as the area of Kemiren village which is approximately 177,052 m2. Kemiren village has 2 hamlets, namely Kedaleman hamlet and Krajan hamlet. Kedaleman Hamlet has 4 RW and 15 RT with a total of 488 families, with a male population of 629 people and a female population of 689 people with a total of 1,318 residents in Kedaleman Hamlet. The population of Kemiren village from the table above is more female than male, namely 52. Disputes regarding land boundaries owned by the community that could not be resolved by both parties, with the assistance of the village head, were finally resolved peacefully by producing evidence of a land boundary agreement signed by both parties. In 2005, there were 2 disputes regarding inheritance disputes that were requested for settlement assistance from the Village Head during 2005. Description of the Kemiren Village Inheritance System Harmony and mutual respect are still held firmly in the life of the Kemiren village community. All kinds of disputes as much as possible can be resolved peacefully and amicably. Likewise the problem of inheritance disputes, as much as possible can be resolved in the family deliberation. The model for dividing inheritance in Kemiren village until now has been varied, there are times when an equal distribution is given to male heirs and female heirs. The use of the "sepikul-sesuwunan" system is the same as in Javanese inheritance, namely giving a larger share to male heirs and giving half of the male share to female heirs is also found in the life system of the Osing Kemiren community. If the division is carried out according to the provisions of "sepikul-sesuwunan", then the decision received by each heir sincerely and with full respect for the heir. Harmony and a sense of brotherhood are prioritized in the distribution of inherited assets so as to avoid disputes as much as possible. Disputes cannot be resolved by deliberation between families and brought before the Village Head. The report submitted by one of the parties, namely Sutris, was then followed up by the Village Head amicably. The Village Head then assists in formal/official dispute resolution because the family deliberation does not reach an agreement between the two parties. Efforts by the Village Head in resolving disputes Settlement in peace is also intended to eliminate feelings of resentment due to disputes that arise. Disputes in terms of inheritance in general have several alternative solutions with the stages of being resolved among the heirs themselves by holding a meeting or deliberation between the parties concerned. the intervention of the elders or close relatives and family members who have influence as intermediaries. The village head's actions are an obligation and a form of responsibility of the village head for the maintenance of order and peace in the village community. So far, based on the information from the Head of Kemiren Village, Mr. Bambang Sugiharto, the problem of disputes over inherited assets brought to the Kemiren village hall can always be resolved peacefully in a family manner. The Village Head generally intervenes in the resolution of inheritance disputes between heirs which are limited in nature when reports are received by the Village Head. Look for the family tree of the parties; Collect information regarding the origin of the disputed assets; Initiating deliberative meetings; Proposing alternative solutions to problems; Provide the necessary suggestions. Inhibiting Factors and Supporting Factors for the Village Head's Efforts to Resolve Inheritance Disputes The Head of Kemiren Village, in his efforts to provide services to his community, faces various obstacles which often make it longer to resolve disputes. The inhibiting factors that make it difficult to resolve disputes and are faced by the Village Head in handling inheritance disputes in the village of Kemiren are as follows: It is difficult to know the position of the inherited assets; Constraints regarding limited number of witnesses; The human factor; Transfer of ownership rights to land without being recorded. It is difficult to know the position of the inheritance. To determine whether the inheritance is the original property or the joint property of the husband and wife which they produced during their marriage, the constraints regarding the witnesses required to make it clear that the inheritance case is minimal. This situation makes it difficult to prove that the disputing parties are entitled or not to receive the disputed inheritance because most of the witnesses are old or have died. Constraints regarding the witnesses needed to make clear inheritance cases were minimal. This situation makes it difficult to prove that the disputing parties are entitled or not to receive the disputed inheritance because most of the witnesses are old or have died. The transfer of ownership rights to property in the form of land has become a habit of the village community until now without being accompanied by registration. A case that is not settled through a court is a peace trial.51 This kind of peace does not only apply to rural communities, but also to advanced communities such as cities. This shows that the principle of kinship and harmony in people's lives is still maintained to create harmony and peace in life. 51 This kind of peace does not only apply to rural communities, but also to advanced  ISSN: 2085-7233 (Print), xxxx-xxxx (Online) Requisitoire : Law Enforcement, Vol. 14, No. 01, July 2022: 8-13 communities, such as in cities. This shows that the principle of kinship and harmony in people's lives is still maintained to create harmony and peace in life. 51 This kind of peace does not only apply to rural communities, but also to advanced communities, such as in cities. This shows that the principle of kinship and harmony in people's lives is still maintained to create harmony and peace in life. CONCLUSION From the discussion that has been described, it is consistent with the results of the study that has been done, at the end of this paper it can be stated that the Head of Kemiren Village in resolving inheritance disputes made efforts to reconcile it by seeking family trees from the parties, gathering information about the origins of the disputed assets, initiating deliberation meetings, proposing alternatives solving problems, providing necessary suggestions. Settlement of inheritance disputes by the Village Head often encounters obstacles due to factors that make it difficult to know the position of inherited assets, constraints regarding limited witnesses, the human factor, transfers of ownership rights to land that are not accompanied by records.Factors that make it easier for the Kemiren Village Head to resolve inheritance disputes are that the Village Head has very strong influence and the attitude of the village community that views inheritance disputes as a disgrace, deliberations are conducted in a spirit of kinship, disputes in the District Court which are considered more complicated, cost a lot and took a long time.	2023-07-12T15:15:46.349Z	2022-07-30T00:00:00.000	{ "year": 2022, "sha1": "d493cbbacfaa543238e84b4a5f184a851f040368", "oa_license": "CCBYNC", "oa_url": "https://ejournal.cria.or.id/index.php/relae/article/download/72/52", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "b237568647c33689cad866165c2c6a1a8b7f5f36", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [] }
141604954	pes2o/s2orc	v3-fos-license	Using Professional Certification Criteria to Assess Occupational Safety Curricula in Degree Programs Investigating Accreditation This paper demonstrates a novel assessment method developed to determine if the curriculum from two separate safety degree programs provided sufficient opportunity for students to obtain the knowledge required for professional practice in occupational safety. The method relies on the Board of Certified Safety Professionals (BCSP) examination blueprints. In the graduate program case study, over 88% of the BCSP criteria were met through an explicit means and up to 64% through assignments or better. Aggregating criteria into respective subject areas showed that the curriculum covered anywhere from 58% to 100% of the items within each BCSP topic. In the undergraduate case study, over 96% of the BCSP criteria through an explicit means, and 82.8% of knowledge items were assessed in assignments, exams or better. Aggregating criteria into respective subject areas showed that the curriculum covered anywhere from 75% to 100% of the items within each BCSP topic. Once briefed on the results, all faculty/instructors agreed that the approach helped identify strengths and weaknesses in their current curriculum. Most importantly, presentation of results acted as a catalyst for curricular discussions amongst the faculty that resulted in improvement priorities and a better understanding of student learning potential in course assignments. Introduction Since 2010, the American Society of Safety Engineers (ASSE), in cooperation with the Board of Certified Safety Professionals (BCSP), has increased their promotion of ABET-ASAC accreditation of occupation safety, safety and health, and environmental safety and health degree programs. Some advantages of increasing the number of accredited safety degree programs include, but are not limited to: more current curricula and curricula improved through data analysis, required faculty development, evidence for more labs and lab equipment, better educated graduates, and the graduate safety profession (GSP) credential granted to graduates. However, the ABET-ASAC accreditation requires a considerable financial investment by the host-university and time and resources from the degree program faculty. Unfortunately, due to the depressed U.S. economy and reduced state budgets, universities are being required to reduce costs, postpone hiring, and simply "get by" with what they currently have. Due to the higher unemployment, college graduates are having difficulty gaining employment in their chosen field and national news outlets are questioning the value of a 4-year baccalaureate or terminal master's degree. Although the occupational safety and health field employment outlook is rather promising (NIOSH 2011), degree programs across the country to reflect on the quality, value, and efficiency of their degrees in the face of budget issues. Safety degree programs across the U.S. are taking efforts to maintain the quality of instruction while reducing costs included efforts to more efficiently utilize instructional resources (e.g., cross listing of courses, sharing electives between programs, restructuring curriculums to reduce dependence on outside instructors) and develop consistency in admissions standards and program prerequisites across graduate programs. Whether considering curriculum-and program-level changes or committing to the accreditation process, faculty members in safety degree programs are forced to ask themselves: Are we in a position to invest our limited time and the universities' limited resources to pursue program accreditation? Background Faced with increasing pressure to demonstrate degree value and justify the investment into pursuing degree program accreditation, department faculty from two separate safety degree programs (at two separate times) decided that an objective, externally based set of criteria was needed to validate the curriculum and provide evidence to support or argue against proposed curricular changes. The first program was a one-totwo year terminal master degree in environmental safety and health, located at a university in northern Minnesota. The other program offered both undergraduate and graduate degrees in occupational safety, located at a university in southern Wisconsin. The one commonality in these two program case studies was a single faculty member who worked at conducted the evaluations at both sites but at different times (approximately two years apart). ABET accreditation seemed a logical first step, especially given its critical role in the Graduate Safety Professional certification developed by the Board of Certified Safety Professionals for students in BCSP-approved graduate safety programs. But, as noted in the next section, ABET criteria for safety programs are also quite vague (ABET, 2008) and provide little specific guidance on curriculum content. Instead, the faculty needed to find some other set of criteria if they were to conduct an in-depth curriculum assessment. 34 T. W. Loushine and R. G. Feyen It should be noted that this assessment was not focused on an outcomes assessment. Although the curriculum assessment results would eventually be used to help assess how well its students were learning the subject matter being presented in the curriculum, the faculty was more interested in the fundamental questions of curriculum assessment mentioned above. After all, a key assumption underlying outcomes assessment is that a good outcome measure indicates effective learning that, in turn, positively correlates with the professional quality and competence of a program's graduates. However, what if a student learns a topic well, but the topic is irrelevant to practice? Or, what if a topic relevant to practice is only mentioned in the curriculum-or worse, not presented at all? Without proper curriculum assessment, outcomes assessment may reliably measure student learning, but runs the risk of being an invalid tool for assessing their professional quality and competency. In terms of assessing student learning in a higher education setting, the general assessment cycle appears to follow these steps (e.g., Allen, 2004;Diamond 1998;Maki 2004): 1. Establish new or review existing learning goals for the program 2. Provide opportunities to achieve these goals 3. Assess if students are learning from these opportunities to meet the goals 4. Use information from assessments to adjust program (and thus competency) 5. Repeat As inferred from above, the researchers were interested primarily in step 2 of this sequence -yet the literature search yielded little specific information on how to select and assess these "opportunities." Rather, student learner outcomes were emphasized heavily while curricular content assessment received far less attention. In this context, learning outcomes can be considered the foundation for driving programmatic changes, but at least for STEM-based programs, most outcomes are adapted directly from ABET criteria for accreditation and are accordingly vague (e.g., "an ability to communicate effectively"). Worth noting as well, although the authors would general use the terms 'outcomes' and 'objectives' interchangeably, ABET clearly defines the terms for assessment (ABET, 2008): Program educational objectives [emphasis added] are broad statements that describe the career and professional accomplishments that the program is preparing graduates to achieve. Program outcomes [emphasis added] are narrower statements that describe what students are expected to know and be able to do by the time of graduation. These www.hlrcjournal.com Open Access Using professional certification criteria to assess occupational safety curricula… 35 relate to the skills, knowledge, and behaviors that students acquire in their matriculation through the program. (p. 1) Once again, the "skills, knowledge, and behaviors" are not defined in any way. In fact, it is the authors' understanding that most accreditation and certification boards leave it to program faculty to decide what skills, knowledge, and behaviors should be included in their program in order to meet their stated outcomes. The typical argument behind this is similar to the one expressed by the National Council of Examiners for Engineering and Surveying (NCEES) when discussing the use of their Fundamentals of Engineering licensure exam for outcomes assessment, "[t]he exam should not, for example, be used to determine the curricular content of any program. Its purpose is to test competency for licensure; it is not intended to force programs to be similar" (Barrett et al., 2010, p.2). Specific to this program, the Educational Standards Committee of the American Society of Safety Engineers (ASSE) had worked with ABET in the mid-2000s to specify specific program criteria required to be in place if a program wanted ABET accreditation. Subsequently, this committee published guidelines for the broad topics to be included in a safety curriculum, but with a caveat similar to that expressed by the NCEES (ASSE, 2011): The committee did not want to provide a long list of required courses or topics areas that were common in previous safety curriculum criteria by the [Board of Certified Safety Professionals] and ABET. The committee believes strongly that programs should be provided flexibility […]. So, how do programs decide what skills, knowledge, and behaviors are needed? Anecdotally, most don't assess their curricula at that level of detail. But, in programs that conduct detailed evaluations, curriculum mapping appears to be the most common tool used to make this decision (e.g., Soulsby, 2006). This method requires identifying what students do in their courses and what the faculty expects them to learn (the skills, knowledge and behaviors) and then clarifying the relationship between the two, or "mapping the curriculum." This process reveals if a student's learning opportunities are linked or consistent with faculty expectations. Inconsistencies suggest places for curriculum improvement that bridge the gap between the two and, in turn, increase the likelihood of meeting program objectives. However, to identify the skills, knowledge, and behaviors needed by a student, common practice is to glean information from a program's stakeholders (e.g., faculty, administration, alumni, employers, funding agencies, peer programs, and professional societies), which can easily suffer from the same issue described earlier: everybody seems to provide a different opinion about what (they believe) students need to know when they graduate. Under the advice of each case study's department faculty, the researchers went back to the ASSE Educational Standards Committee caveat mentioned prior. Although the ABET criteria had not proven to be helpful, the "long list" attributed to the Board of T. W. Loushine and R. G. Feyen Certified Safety Professionals (BCSP) turned out to be a promising lead. The BCSP has a primary mission similar to the NCEES: assess the professional competency of safety professionals via the Associate Safety Professional (ASP) and Certified Safety Professional (CSP) exams. Notably, certification and licensure agencies utilize recognized methodologies (e.g., ISO/IEC 17024 -Conformity Assessment) to ensure that their examinations test people on the activities, knowledge, and skills required in their profession. The key step in this process involves a job analysis of current practitioners. However, unlike the NCEES and many other licensure and certification agencies, the BCSP was very transparent in its exam development process, publishing highly-detailed "blueprints" outlining the skills and knowledge expected of a safety professional and from which the ASP and CSP exams were developed. The exam blueprints were derived from a three-stage job analysis study of current safety professionals, targeting 1500 survey subjects, with respect to the skills and knowledge needed to perform the safety job in a professional, competent manner (BCSP, 2008b(BCSP, , 2008c. BCSP then categorized the resulting 249 knowledge items as either "foundation" (relevant to the ASP exam) or "advanced" (relevant to the CSP exam) and listed the 249 knowledge (and an additional 298 skill items) under a hierarchy of domains (e.g., risk management) and tasks (e.g., design effective methods to reduce or eliminate risk). Relevant to this initiative, the BCSP also undertook a generalized curriculum mapping effort, linking the skills and knowledge items with 15 "subject matter" topics typically taught in a safety program (BCSP, 2008d) -but provided no guidance on how to adapt this generalized curriculum map to a specific program. However, in a separate publication, one of the individuals involved in the original job analysis study did provide some guidance. Brauer (2005) not only described the job analysis survey and, in turn, suggested several ideas for using its results to assess a safety curriculum. With these two sources of information in hand, the program faculty now had an objectively derived set of skills, knowledge, and behaviors, as well as some ideas on how to assess the curriculum. All in all, the research efforts of BCSP created a comprehensive list of subject matter categories, (foundation and advanced) knowledge items, and skill items to use as a reference for the development of certification exams. The same certification exams that deem whether a person is a "certified safety professional" (CSP) or not. And it is this basic delineation that provided the basis for evaluating degree program curriculum based on evidence of course coverage and/or assignments. Study Objectives The overall goal of this study was to create and test a novel curriculum assessment method (based on 2004 BCSP Blueprints). Two types of safety degree programs (graduate and undergraduate), at different universities in the Upper-Midwest, were used in this study. The specific study objectives are: 1. What percent or count of knowledge items is covered in course lectures? www.hlrcjournal.com Open Access Using professional certification criteria to assess occupational safety curricula… 37 2. What percent or count of knowledge items is covered in course assignments? 3. Which knowledge items are covered most frequently? Or not covered at all? 4. Which topic areas need attention for faculty discussion of curricular changes? Methods Two Case Studies -Two Unique Safety Degree Programs in the Upper-Midwest The assessment tool derived from BCSP's 2008 Blueprints was first applied to a terminal master's degree program in 2009. The master's program had three core-faculty and four part-time or adjunct instructors, and generated approximately 10 graduates per year, all of whom earned full-time employment in safety after their capstone internship project. Based on lessons learned from the master's degree program assessment, an updated version of the assessment technique was applied to an undergraduate degree program in 2011 (improvements/changes noted below). The baccalaureate program had six core-faculty and six part-time or adjunct instructors, and generated approximately 40 graduates per year, most of whom gained full-time employment in safety after their capstone internship. In the graduate program assessment, both recent graduates and faculty were involved in the selection of topics and knowledge items rated within their fourteen courses (ten required courses and four elective courses). In the undergraduate program assessment, only faculty were involved in the selection of topics/knowledge items rated within their 22 courses (11 required courses and 11 elective courses). In the undergraduate study, faculty were also asked these questions: 1. Do you dedicate a lecture (or portion of a lecture) to a discussion on ETHICS? 2. Do you assign at least one (individually graded) writing assignment (paper) of 10 pages or more? 3. Do you assign (on average) 20 or more pages of reading per week? 4. Do you assign group or team projects (either graded as a team or individually)? 5. Do you assign student presentations (either graded as a team or individually)? 6. Do you assign technical problem solving assignments (i.e. math-based problems)? Preparation for course assessment interviews. In both case studies, the capstone internship was not included in the assessment, because each student's experience in that course was different. In addition, due to the sheer number of skill items and the difficulties in teaching and assessing skills in a traditional academic setting (most skill development occurs during actual practice, such as in an internship or after graduation, although lab experiences mitigate this to some degree), the faculty decided to exclude the 298 skill items identified by the BCSP from the assessment and focus exclusively on the 249 knowledge items. T. W. Loushine and R. G. Feyen Faculty recognized that evaluating each course against the full 249-item list would take too much time and effort, contributing to half-hearted responses or unwillingness to participate throughout the entire project. Therefore, the first step was to narrow down the list by deciding which knowledge items from the BCSP blueprints were relevant to each course, so only those relevant items would be rated for coverage and/or application in an assignment. The faculty also recognized, however, that the course instructors and students may differ in whether or not they perceive a knowledge item to be relevant for a course. In this context, a knowledge item's perceived relevance to a course could fall into one of three categories: "definite" (all respondents reported the item as relevant to the course), "likely" (at least half, but not all, of the respondents reported the item as relevant), and "possible" (less than half, but at least one, of the respondents reported the item as relevant). This suggests that, if all respondents report an item as relevant ("definitely"), a reasonable course expectation is that the item would be covered in some depth; but, if only one respondent lists the item as relevant ("possible"), minimal coverage of the item would be sufficient or even unnecessary. Accordingly, the perceived relevance of each knowledge item played a key role in the aggregate data analysis and in selecting recommendations for improving course coverage of a specific subject area or knowledge item. Creation of Data Collection Instruments The resulting methodology required two separate rounds of data collection, each with its own data collection instruments: one for selecting topic areas or knowledge items to be rated within each course and another for the customized interview with instructors to rate knowledge items based on evidence of coverage and/or student learning (i.e. exams, projects, papers, etc.). The first round of data collection consisted of a cover letter describing the study and instructions for users to select the knowledge items (sorted by subject area) relevant to the specified courses based on the original presentation of the BCSP blueprints. The cover letter and Excel spreadsheet (containing the course description in one tab and all the BCSP knowledge items in a second tab) were sent to a group of students who recently completed the courses, the course instructor, and were also completed by the student research assistant (whom also recently completed all the courses). The second round of data collection consisted of an interview with each course instructor using a customized datasheet based on the results of the first round of data collection. The customized interview datasheet had three sections: knowledge items with "definite" relevance to a course, knowledge items "likely" to be relevant to the course, and knowledge items "possibly" relevant to a course. Within each section, items were grouped by subject area and each item was preceded by a text box in which the item's coverage rating could be entered. www.hlrcjournal.com Open Access Using professional certification criteria to assess occupational safety curricula… 39 Data collection. The first round of data collection was driven by personalized email messages, with the cover letter language in the body of the e-mail and a selfadministered survey instrument as an Excel attachment. After the initial submission, several reminder e-mails were sent to all respondents encouraging participation (this was primarily for the student volunteers as all faculty had already agreed to participate and did so immediately). After a week, the student research assistant tallied the responses, and created customized interview spreadsheets for each course with the relevant knowledge items, grouped by subject area. In the second round of data collection, the student research assistant helped the instructors rate the course coverage of each knowledge item listed for the course, regardless of relevance level. This entailed face-to-face interviews, conducted individually with each course instructor who had been instructed to be inquisitive and acquire anecdotal evidence supporting each knowledge item rating. Based on course instructor evidence of coverage and/or assignment (exam, project, paper, etc.), each knowledge item was rated on a scale of 0 through 5 (in the graduate case) or 0 through 7 (in the undergraduate case). The ratings were entered into a single master spreadsheet, with courses across the column headings by item priority area (four columns per course, three priority and "no rating") and the 249 knowledge items horizontally (by subject area) by row. Thoroughly covered in lecture. Projects, presentations, quizzes, tests or other tangible products were utilized to assess knowledge item. 4 Discussed extensively in lecture. Material related to the item was included in homework assignments or quizzes to assess the level of knowledge acquired. 3 Item was covered in the course and included in notes, slides, handouts, activities, etc. However, students were neither tested nor asked to demonstrate their understanding of the item. 2 The knowledge item may not have been covered or discussed in lecture, but was included in assigned reading material. 1 Although possibly relevant, the item was not covered in any way in the course. 0 The knowledge item is not relevant to this course. Data analysis. After considerable trial and error, the following data analysis approach was selected as optimal for extracting reliable results from the master spreadsheet. The first step was to calculate the total number of course ratings given to each knowledge item, and then sorting the ratings by their perceived relevance (rating). Within each level of relevance, the ratings equal to 3 or higher (per knowledge item) were tallied. From the resulting table of rating frequencies, the total percent of knowledge items by coverage rating and relevance level were calculated to provide an estimate for the quality of knowledge item coverage across the program curriculum. The percentages for each subject matter area were then calculated (percent coverage and ratings of knowledge items within subject area). The second step consisted of transferring the data from each subject area worksheet into a new table to evaluate the aggregated ratings for the associated knowledge items and identify subject area coverage by course. The calculation of total counts and percentages was similar to the methods used on the entire data set. For each subject area, a bar chart illustrated the percent of knowledge items covered adequately (rating of 3 or greater) and inadequately (rating of 2 or less). Because items not receiving any ratings ("not rated") were excluded from this analysis, the sum of the adequate and inadequate percentages within a course fell between zero and one-hundred percent. This allowed the knowledge items with low ratings to be easily identified visually and noted for later consideration. In addition to the bar charts, two matrices were created containing each course's percentage of adequate coverage and inadequate coverage for each knowledge item. Within each matrix, courses were listed on the horizontal (top) and the subject areas were listed on the vertical (left). The matrices were then populated with the respective coverage ratings and color-coded to indicate areas receiving either above or below a specified threshold. This helped identify which courses either covered a topic well or should be considered for material additions or structural changes to improve subject area coverage and/or type of assignment to potentially assess (in the future) student learning. The following sections describe in more detail how the resulting data was interpreted and pilot-tested for curricular improvements. Case Study 1: Graduate Program -Individual Item Coverage Within Courses Each individual course was rated based on items selected by faculty/alumni: 60 to 124 knowledge items out of a total of 249 items (Figure 1). Results of the entire curriculum assessment indicated that an individual course might cover anywhere from 5 to 26 "definitely" relevant topics, another 9 to 51 topics "likely" to be relevant to the course, and anywhere from 10 to 89 additional topics "possibly" relevant to the course. Using professional certification criteria to assess occupational safety curricula… 41 Graduate Program -Individual Item Coverage Across the Curriculum Of the total 249 knowledge items, only three items (business management software, Poisson distributions, and agricultural/food supply safety) were not identified for course rating in any of the 14 courses evaluated. On the other hand, 11 items were covered at some level in at least 10 of the 14 classes (including education and training methods, several types of administrative hazard controls, facility safety principles, and hazard identification); no items appeared in all 14 courses. For example, 13 courses covered some aspect of "Administrative controls: Written plans, procedures, and work practices." Results of coverage rating and analysis indicated this topic was considered definitely relevant to two courses, and those two courses covered the topic through a written assignment (coverage rating = 5), whereas the topic was considered likely relevant to six other courses and possibly relevant to five others. Of the six courses for which the topic was likely to be relevant, two were reported as having covered the topic with a paper assignment. Of the remaining five courses for which the item was possibly relevant, one course was reported as having covered the topic to the highest rating-but, exemplifying the pattern reported the end of the prior section, the instructor reported this while the students did not consider the topic as relevant. Overall, further analysis (Table 3) suggests that the program delivered up to 88% of the BCSP knowledge items through an explicit means, such as lecture or other in-class activity, indicated by a quality of coverage rating of 3 or higher. Two-thirds of all items (64%) were thoroughly covered in the courses (rating = 5), indicating that these topics T. W. Loushine and R. G. Feyen were not only covered in class, but also through projects, tests, and other methods to help students demonstrate learning. Graduate Program -Items That Need Faculty Attention Aggregating the results into the respective subject matter areas showed that, within the each of the 15 subject matter areas, anywhere from 58% to 100% of the relevant knowledge items were explicitly covered in a course (i.e., quality of coverage rating equal to 3 or better). For example, the curriculum explicitly covered all the knowledge items in four subject areas (ergonomics, measurement/monitoring, organizational/behavioral sciences, and risk assessment/ management) but failed to adequately cover between 20-42% of the items in another four subject areas (business management principles, general sciences, EHS management and auditing systems, and security sciences; Table 4). Each of these latter subject areas was investigated further by Using professional certification criteria to assess occupational safety curricula… 43 identifying coverage (or lack of it) within individual courses for each knowledge item within a subject area. At this point, the analysis could also have explored knowledge items with coverage limited to lecture or assigned reading (rated 3). Although these items are covered, no evidence of student learning exists for them, so it is in the best interest of faculty to discuss these items (Table 5). Each individual course was rated based on knowledge items within subject areas selected by the most recent instructor for that course: 3 to 15 subject areas or 11 to 186 knowledge items out of a total of 244 items (Figure 2). The lower number of knowledge items per course was the results of choosing subject areas and not individual knowledge items for rating a course. This made the pre-selection process quicker and easier, and reduced some confusion during the interview and in analysis. Another difference from case study 1 is that the rating scale went from 0 to 7 (instead of 0-5). Although the scale is not a truly validated scale, a rating of 6 or 7 would be considered consistently better than a 4 or 5 because when a student produces a paper or written project, the instructor can assess several forms of student learning. The fluctuation in number of items rated per course was the result of course instructor perception and the unique nature of certain elective courses (such as Safety 472 -Advanced Ergonomics). Undergraduate Program -Individual item coverage across the curriculum. Another improvement over the graduate case study was that all items were rated several times due to instructors choosing topic areas instead of individual knowledge items. Indepth analysis (Table 6) suggests that the program delivered up to 96.3% of the BCSP knowledge items through an explicit means, such as lecture or other in-class activity, indicated by a quality of coverage rating of 3 or higher. Over four-fifths of all items (84.8%) were thoroughly covered in the courses (rating = 4 to 7), indicating that these T. W. Loushine and R. G. Feyen topics were not only covered in class, but also through projects, tests, and other methods to help students demonstrate learning. *It was discovered during interviews that some items appeared to be very similar, so they were combined. Undergraduate Program -Items that need faculty attention. Aggregating the results into the respective subject matter areas showed that, within the each of the 15 subject matter areas, anywhere from 75% to 100% of the relevant knowledge items were explicitly covered in a course (i.e., quality of coverage rating equal to 3 or better). The results were sorted down to individual knowledge item ratings to identify which items were rated below 3 (not covered in a lecture or reading; Table 7) or those that received no higher rating than 3 (only covered in lecture; Table 8). The topic areas under 100% and the individually identified knowledge items were identified and prioritized for discussion with faculty about what could be done to improve coverage and/or develop course assignments to evaluate student learning. www.hlrcjournal.com Open Access Using professional certification criteria to assess occupational safety curricula… 47 Discussion As the results suggest, both degree programs demonstrated that their curriculum covered most of the BCSP knowledge items, with fairly well-defined areas for improvement. The analysis revealed and prioritized subject areas and knowledge items for faculty discussion about improving evidence of student learning. The discussion about individual course content and how student learning is assessed through assignments actually began in the interview process, because instructors had to provide evidence and explanation to support their rating of knowledge items. The overall results show "overlap" and "gaps" in the overall degree program that can be further analyzed and discussed by faculty to determine the best means to improve curriculum to conserve resources and time. In the case of the graduate program, the internal auditing procedure revealed a possible tool for outcomes assessment, even though that was not the focus of the assessment. Consider the occasional disconnect reported between the instructor and the student respondents in terms of knowledge item relevance. Two possible reasons may explain this: one, perhaps an instructor may have planned to present on an item, yet for whatever reason, did not do so during the most recent semester. Or perhaps, students didn't learn the item sufficiently to understand its relevance to the course. Either way, instructors could assess the effectiveness of knowledge item coverage by comparing their perceptions of item relevance with student perceptions. Instructors could also use the information over multiple semesters to explore how the use of different topics or modes of presentation might fill gaps between their intentions and the students' actual exposure to the material. With regards to the curriculum assessment and the information gleaned from the results, the program faculty felt strongly that this approach using professional certification criteria is promising, but could still use some fine-tuning. The biggest disadvantage is the time and effort required. On average, the surveys and interviews took about one to two hours per course, and each participant had to take their task seriously to provide accurate information. One other major difficulty encountered was that the terminology used in the BCSP documentation (e.g., blueprints, curriculum mapping, etc.) was sometimes unclear and redundant. BCSP personnel were helpful, but often were not able to provide sufficient clarification on what was meant by a specific skill or knowledge item. Nonetheless, the consensus definitions may represent a concept differing from the knowledge item originally captured in the original BCSP job analysis study. Despite these challenges, the approach clearly helped the program answer the questions raised earlier in terms of whether or not program graduates are exposed to the material they should know in order to practice as EHS professionals. This curriculum assessment methodology provided answers at several levels by providing baseline Using professional certification criteria to assess occupational safety curricula… 49 measurements of knowledge item coverage both within individual courses and in the overall program. Within courses, as hinted earlier, the level of item coverage-particularly for items identified as definitely or likely to be relevant to the course-can be easily examined and modified if necessary. At the program level, the approach allows faculty to more effectively explore the impact of both temporary and permanent changes to the program curriculum-particularly in light of recent economic constraints. Using this approach provides program faculty with an opportunity to objectively evaluate the impact of adding or dropping classes from the curriculum, changing the frequency of course offerings, and whether a class should be required or can be offered as an elective. Further, in terms of program objectives, the faculty was able to seriously consider examining knowledge item coverage in terms of "foundation" and 'advanced" items to distinguish how well the program prepares graduates not only for professional practice, but either the ASP or the CSP certification exam. Perhaps more importantly, the BCSP foundation provides a significant degree of objectivity to curriculum assessment. Rather than rely on feedback from numerous stakeholders in the program, each with different agendas and conflicting opinions, the knowledge items used in this approach are derived from a profession-wide job analysis study conducted in compliance with an accepted international standard (ISO/IEC 17024) and utilizing data collected in three stages, that targeted 1500 survey subjects from practicing EHS professionals (BCSP, 2008a). Regardless of academic institution, the vast majority of faculty would not be unable to perform a study of this depth for their program. This externally validated set of skills and knowledge items removes the discussion about the value or necessity of certain topics (e.g., between college administration and program faculty) from the realm of opinion. Because BCSP has been open with publishing the skills and knowledge items sets derived from their job analysis studies, this approach can be readily adapted to any EHS program. In a move that should simplify this assessment methodology and help address the concern regarding interpretation of terminology within the knowledge items, the BCSP has since simplified its blueprints, reducing the number of domains and more clearly detailing the knowledge and skills areas within those domains (BCSP, 2009). Interestingly, the approach could also be adapted to other degree programs with a certification or licensing body such as in the engineering disciplines. For example, the NCEES regularly conducts PAK (professional activities and knowledge) studies for the various engineering disciplines as part of its exam development process (e.g., NCEES, 2011). However, NCEES does not make these studies readily available and, should an engineering program wish to utilize this approach, they would have to obtain access from NCEES to the latest PAK data for their discipline. T. W. Loushine and R. G. Feyen Conclusions Overall, reflection on this curriculum assessment process identified opportunities for improvement, including streamlining the data collection and analysis process and clarifying the meaning of individual BSCP knowledge and skill items. But, ultimately, by using the certification agency's job analysis data to indicate the knowledge needed by graduates of a safety program and developing a combined rankings and ratings methodology to assess coverage of this knowledge, the faculty was able to satisfactorily answer the initial question: Are we in a position to invest our limited time and the universities limited resources to pursue program accreditation? The authors believe that for both case studies, although changes need to be made and a decent amount of effort is required, that pursuit of ABET-ASAC accreditation be warranted. Other EHS-related programs could employ this new approach, and perhaps even other disciplines, to answer the same question and make fine-tuning improvements to the methods. Limitations The use of the BCSP blueprints assumed that subject areas and items encompassed the breadth of knowledge required of students graduating from an accredited degree program. This may not be the case, but an argument can be made that curricula that covers near 100% of material planned to be on a professional certification exam means that students of that degree program are at an advantage to passing the exam. And that those students, if they kept good lecture notes and/or created detailed course binders, would not have to invest in costly study materials or attend study review sessions. The rating scales used to assess how well a knowledge item was either covered in a course or potential to assess student learning was not validated. From a logical standpoint, the greater the time and intensity used to study or develop written projects, the more likely a student would both understand (and possibly apply) and remember that information. However, this is not true 100% of the time, and again, is only a theoretical assumption for these case studies. All in all, the faculty received an inexpensive review of their program curricula with recommendations on what they can do to improve topic coverage in courses and identify critical assignments in courses that demonstrate student learning outcomes, which are a key metric in ABET-ASAC accreditation reviews.	2018-12-05T06:40:47.013Z	2013-05-13T00:00:00.000	{ "year": 2013, "sha1": "84bb580be7c1f1df774f7c6abb25a92b0cec2c87", "oa_license": "CCBY", "oa_url": "https://doi.org/10.18870/hlrc.v3i2.113", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "c49e5431f94dab3ad11c788a5777e83a490846d8", "s2fieldsofstudy": [ "Education" ], "extfieldsofstudy": [ "Psychology" ] }
54535347	pes2o/s2orc	v3-fos-license	Seed germination studies of tree species: Radermarchera xylocarpa and Dolicandrone falcata Seeds of Radermarchera xylocarpa and Dolicandrone falcata were soaked overnight in water and germinated in different substrates. In both the plants, the highest percent of germination was recorded in cocopeat (Radermarchera xylocarpa:62%; Dolicandrone. falcata: 41%) followed by MS basal medium (Radermarchera xylocarpa: 46%; Dolicandrone falcata:20%), cocopeat: sand (Radermarchera xylocarpa:30%; Dolicandrone. falcata: 8%) and least being in filter paper (Radermarchera xylocarpa: 23%; Dolicandrone falcata: 0%). Prior to germination in optimised substrate, seeds were soaked overnight in distilled water (control) and in gibberllic acid (GA3) (5 & 10 μM). In both the plants GA3 treated seeds failed to increase percent germination and the growth of seedlings. In control the seedling growth was better with respect to all the parameter for both the species. Seedlings which were grown in cocopeat, rapidly developed into healthy plants after transfer to field Introduction The Indian Flora is rich in medicinal plants and over 7000 different plant species present in the different ecosystems are used for various medicinal purposes (Rajendra and Souza, 1999).To meet the growing demand of herbal/medicinal industries, these plant resources especially the tree species are exploited in the country and are rapidly being extinct from the wild. Under natural conditions, the seeds of some tree species have low percent of germination and are difficult to grow through conventional methods.Because of that their large scale plantations becomes limited.Such seeds must be pretreated to hasten germination and seedling establishment (Maku et al., 2014).Recent advances in agricultural research has suggested that the improvement in crop productivity and quality can be further enhanced by incorporating new technologies in the traditional breeding programs (Mhatre and Rao, 1998).In tree species there are several reports which mention methods for enhancing the seed germination as well as growth of seedlings (Azad et al., 2011;Kandya, 1990;Khan et al., 2001;Annapurna et al., 2005;Maku et al., 2014;Trivedi andJoshi, 2012 and2014). Radermarchera xylocarpa and Dolicandrone falcata are tree species belonging to family Bignoniaceae, which are an important component of semi-moist forests.But due to poor natural regeneration, harvesting them from the wild has placed these plants under threat category. Radermarchera xylocarpa (Roxb.)Schum is a critically endangered middle sized deciduous tree belonging to family Bignoniaceae, growing up to 5-10 m tall.Capsules are 1-3 ft.long slightly curved, rough with numerous winged seeds.The oil from the wood is used in cutaneous affections and resin extracted from it, is used in skin diseases as well as an antiseptic (Kirtikar and Basu, 1975).Dolichandrone falcata (Wall. ex DC) Seem is a small deciduous tree belonging to family Bignoniaceae, growing up to 20-50 ft.tall which is now placed under vulnerable category.Capsules are flat much falcately curved, glaborous bearing seeds which are broad, rectangular and winged.Traditionally the juice of leaves is used for treatment of diabetes and decoction of the fruit is used medicinally (Kirtikar and Basu, 1975). The propagation for both the plants is mainly through seeds (Cooke, 1908).These trees are important component of semimoist forest, but are poor in natural regeneration (Sabnis and Amin, 1992).Hence propagation of these members is essen-Seed germination studies of tree 51(1) 2016 tial and therefore the present work on seed germination studies of R. xylocarpa and D. falcata was taken up with an aim to improve the rate of germination utilizing different substrates.It was carried out with two objectives.Initially the planting substrate was standardised for developing large number of seedlings, and then different growth parameters were studied in the optimised substrate. Materials and methods Seeds of R. xylocarpa (Plate 1.a) were collected from Shoolpaneshwar and D. falcata (Plate 1.e) were collected from Dharampur forest.To carry out seed germination studies of these species, the substrates utilized were, cocopeat, cocopeat:sand(1:1), sand, filter paper and MS Basal medium. 150 gms of cocopeat (dry weight) was soaked in distilled water (4 times of dry weight) overnight.Except sand, individual cocopeat, a mixture of cocopeat and sand (1:1), petriplates with filter paper and MS basal medium were autoclaved at 15 psi for 25 min. Seed germination in three different planting substrates under aseptic conditions Seeds were soaked overnight in distilled water (control), treated with 0.1% HgCl 2 for 2 minutes and were washed 3 times with sterile water.All the manipulations for germinating the seeds were carried out in the Laminar air flow cabinet for the following substrates: Cocopeat and Cocopeat:Sand The seeds were germinated singly in each well of the root trainer containing a specific type of substrate.Bavistin (200mg/l) was added on to the substrate and the root trainers were kept in culture room at 25 ± 2 o C. MS Basal Medium 5 seeds were kept per flask containing basal medium and these were kept in culture room at 25 ± 2 o C. Filter paper 5-10 ml sterile water was added in each petridish with filter paper.Thereafter 5 seeds were inoculated in ten petridishes and were sealed with parafilm.All the petridish were kept in culture room at 25 ± 2 o C. In vivo seed germination in sand Sand (2.2Kg) was filled in polybags and single seeds (overnight soaked in distilled water) were planted in each bag.Fifty polybags were used for the experimental studies and daily watered to keep the substrate moist. Seed germination and growth in cocopeat Seeds were soaked in GA 3 (5µM and 10µM) solution overnight and were germinated in cocopeat substrate filled in root trainers.These root trainers were kept in culture room at 25 ± 2 o C.After seedling emergence, 5 seedlings from each treatment as well as control (seeds soaked in distilled water) were randomly selected and observations for percent germination and growth parameters like seedling length (shoot and root length), fresh weight, dry weight and collar diameter were recorded after 20 days in R. xylocarpa and in D. falcata. Results and discussion Results showed that out of the different planting substrates that were used to study the seed germination by soaking the seeds in distilled water, cocopeat proved to be a better substrate compared to all others in both the species R. xylocarpa and D. falcata (Fig. 1).It resulted into highest percent of germination (62 %) (Fig. 1) in R. xylocarpa (Plate 1.b) while it was 41.4 % in D. falcata (Fig. 1, Plate 1.f) followed by MS medium with 46% and 20%, which was higher as compared to cocopeat: sand.While in the other two substrates ie.sand and filter paper, lowest percent of germination was recorded.Earlier studies on seed germination in different planting substrates in Bignoniaceae members, O. indicum (Trivedi and Joshi, 2012) and S. suaveolens (Trivedi and Joshi, 2014), also reports cocopeat to be optimum for highest percentage of seed germination. Seed germination is regarded as a series of steps which occur prior to the emergence of radical from the seed coat (Mayerand andShain, 1974, Rahman et al., 2007).Since cocopeat and MS medium proved to be beneficial for seed germination, these substrates were evaluated weekly for seedling emergence in both the species and it was noted that, from first week to the third week, number of seedlings increased every week in both the substrates (Fig. 2).In R. xylocarpa it increased from 20 to 44% whereas in D. falcata it was from 7 to 29% in cocopeat substrate.In MS medium the percentage increased from 15 to 23 % in Radermarchera and 3 to 10 % in Dolicandrone.It was found that the growth of the seedlings in MS medium was normal but when transferred to in vivo conditions, they failed to survive.Cocopeat proved to be better substrate than MS Basal medium in terms of growth for both the plants. The effect of gibberllic acid (GA 3 ) on germination and growth of seedlings in both the species showed that GA 3 failed to improve percent germination as it was less than the control (Fig. 3).Usually gibberellic acid is used to promote seed germination (Joshi et al. 2010;Yamaguchi and Kamiva, 2001), but in the present studies it proved to be inhibitory.Similar results are reported in P. brevifolia (Wall et al., 2010).Seeds soaked in distilled water proved to be better than the GA 3 treated seeds and similar findings are observed in Tetrapleura tetraptera (Maku et al., 2014).The assessment of growth parameters for seedlings of Radermarchera (Plate 1.c) in control showed that the length (3.3 cm), biomass (FW: 0.14g; DW: 0.01g) was better than GA 3 (5 & 10 µM) except for collar diameter (0.7cm) which was higher in 5 µM of GA 3 (Table I). Hence from the above results it could be inferred that GA 3 pretreatment was not effective for germination and growth in both the species.The distilled water soaking was helpful for overall growth of seedlings in the two species.Similar obser vations were recorded in Tetrapleura tetraptera in which least performance in seedling growth characteristics was observed in GA 3 treated seedlings compared to control (Maku et al., 2014). As cocopeat substrate generated highest number of seedlings in both species, these seedlings were transferred to pots containing soil and were kept in garden for their further growth.It was observed that the seedlings of both species developed into a very healthy plants within a month showing vigorous growth and attained a considerable height within three months (Plate 1. d, h) Improved growth of seedlings using cocopeat has been reported in number of other species like Fig. 3 . Fig. 3. Effects of Gibberllic acid on seed germination in cocopeat of R. xylocarpa and D. falcata	2018-12-03T17:20:44.418Z	2016-03-28T00:00:00.000	{ "year": 2016, "sha1": "0bc38200e598f4f9683a97d3d6227d48e1a175a5", "oa_license": "CCBYNC", "oa_url": "https://www.banglajol.info/index.php/BJSIR/article/download/27056/18058", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "0bc38200e598f4f9683a97d3d6227d48e1a175a5", "s2fieldsofstudy": [ "Environmental Science", "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Biology" ] }
236966604	pes2o/s2orc	v3-fos-license	Accumulation of Regulatory T Cells in Triple Negative Breast Cancer Can Boost Immune Disruption Background and Aim The present study was conducted to evaluate the number of Tregs in triple negative breast cancer (TNBC), in normal breast parenchyma and in the peripheral blood of these patients and controls, in addition to their correlations with the clinico-pathologic features and the outcomes of TNBC. Methods Thirty adult treatment-naïve women with non-metastatic TNBC were recruited. In addition, 20 ages matched healthy females participated as a control group. Peripheral blood samples were collected from all participants in tubes containing heparin, fresh tumor tissues were also obtained from all patients undergoing surgery, and 20 normal breast tissue samples were obtained from the same patients’ areas adjacent to the safety margins; all these samples were taken for flow cytometric detection of Tregs. Results The mean percentages of CD4+CD25+highT cells and Tregs were higher in TNBC peripheral blood than healthy controls and in malignant tissue than normal tissue. Moreover, the frequencies of tumor-infiltrating CD4+T cells and Tregs were exceeding those in the peripheral blood of cancer patients. Only tumor-infiltrating Tregs have shown increasing levels with the increase in the tumor size and were significantly higher in patients with local recurrences than those without recurrence. In addition, Tregs showed significant inverse relation with DFS and direct relation with the level of the peripheral Tregs. Conclusion The findings of the current study support the possibility that TNBC microenvironment conveys specific characteristics on Tregs distinguishing them from those in normal breast tissue or Tregs in peripheral blood, improving the capabilities of tumor-infiltrating Tregs to enhance tumor growth, local recurrence and reduce the DFS. Introduction Significant attention has been given to regulatory T cells (Tregs) expressing the transcription factor fork head box protein P3 (Foxp3). 1 Tregs have a fundamental role in sustaining immunological tolerance and controlling autoimmunity. 2 They act by controlling the activation and differentiation of CD4 + Th cells and CD8 + cytotoxic T cells in response to environmental, autogenous, or tumor associated antigens. Studies have confirmed that Tregs have opposing actions in cancer immunity leading to immune evasion of cancer cells and implying a functional impact on tumor progression and metastasis. [3][4][5] Interestingly, the clinical relevance of tumor-infiltrating Tregs has been found ambiguous. For instance, a high Tregs density in hepatocellular carcinoma is predictive of poor prognosis, in line with the hypothesis that Tregs enhance tumor progression through tumor-specific T cell suppression. On the other hand, improved clinical outcome in other tumors as colorectal carcinoma is associated with a high Tregs density. These contrasting results indicate that the role of Tregs in tumor development may vary substantially according to the affected site. 6 Similarly, Tregs were suggested to be correlated with good outcome of breast cancer in one study, 7 while other studies revealed that Tregs were associated with poor outcome of breast cancer. 8,9 Triple negative breast cancer (TNBC) is a type of breast tumors that do not express estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) on the surface. 10 Patients with this TNBC have enhanced risk of metastasis and relapse, and cannot utilize targeted therapy. 11 Increased tumor-infiltrating-lymphocytes (TILs) in TNBC support the high immunogenic nature of this subtype of breast cancer. [12][13][14] It is not evident whether the Tregs found intra-tumoral are comparable to those in normal tissues or in the peripheral blood. The tumor microenvironment might imprint distinctive transcriptional and functional characteristics upon Tregs. 15,16 Recently, peripheral blood Tregs represented the main source of intra-tumoral Tregs in human breast cancers and that their response to cytokine signaling indicates intra-tumoral immunosuppressive possibility and predicts clinical outcome. 17 So far, the prognostic value of Tregs in breast cancer is still controversial, and further studies are needed to fully understand its significance. [18][19][20][21][22] The present study was conducted to evaluate the number of Tregs in TNBC, in normal breast parenchyma and in the peripheral blood of these patients and controls, in addition to their correlations with the clinico-pathologic features and the outcomes of TNBC. Patients and Methods Thirty adult treatment-naïve women undergoing surgical treatment of non-metastatic TNBC in the South Egypt Cancer Institute and Clinical Oncology Department, Assiut University Hospital were enrolled. The clinical and histopathological characteristics of the patients are shown in Table 1. In addition, 20 age matched healthy females participated as a control group. Ethical Statement The Committee of Medical Ethics of faculty of medicine, Assiut University reviewed and accepted the research proposal (IRB no. 17300416) and the study was done in compliance with the ethical guidelines of the Declaration of Helsinki 1975. Informed consent was obtained from all research participants before sharing in the study. Surgical Procedures After confirmation of breast cancer by needle biopsy, patients were offered preoperative assessment by multisclice cut scans of chest and abdomen, bone scan, and tumor markers including CA15-3, and CEA in order to exclude metastatic cases, and then patients underwent either modified radical mastectomy (MRM), or breast conservative surgery (BCS). Adjuvant Therapy After surgery, pathologic assessment of tumor type, size, grade, lymph node (LN) status was done, followed by immunophenotyping to ensure negativity of estrogen and progesterone receptors, and HER2neu. All patients received adjuvant chemotherapy according to standardized guidelines, patients with BCS, > T2 lesions, positive LN, positive surgical margins, and perineural invasion were treated with 3 DCRT with doses ranging from 40 to 66 Gy over 15-33 fractions. Follow Up TNBC women in our study were followed up monthly by clinical examinations for the first 2 years, then every 3-6 months for additional 3 years, then yearly later on, MSCT chest and abdomen and tumor marker were done every 3 months in the first 2 years, then every 6 months in the next 3 years, and then yearly later on, bone scan was done when indicated (bone pain, rising ALP, rising serum calcium, etc.), these follows ups continued until disease recurrence or death of patients to determine their disease free survival (DFS). Sample Processing Peripheral blood samples were collected from all participants in tubes containing heparin. Fresh tumor tissues were also obtained from all patients undergoing surgery for primary breast cancer immediately after surgery. In addition, 30 apparently normal breast tissue samples were obtained from the same patients from areas adjacent to the safety margins (20 tissue samples were areas devoid of any abnormalities; whether inflammatory or benign lesions and subsequently included for comparison, while the other remaining 10 sample tissues were found to have abnormalities subsequently, were excluded). The tumor tissues were mechanically fragmented to prepare single-cell suspension. The cell suspensions were filtered through cell strainers (100 μM). The contaminating red blood cells were removed by incubation with lysing solution for 5 minutes at 4°C, and the resultant suspension was washed twice with phosphate buffered saline (PBS). Flow Cytometric Detection of Regulatory T Cells in Peripheral Blood and Breast Tissue Fluoroisothiocyanate (FITC)-conjugated-Foxp3 (clone PCH101, eBioscience, Invitrogen, Thermofisher, US), phycoerythrin (PE) conjugated-CD25 (clone 2A3, Becton Dickinson (BD) Bioscience, CA, USA) and peridinium-chlorophyll-protein (Per-CP)-conjugated-CD4 (clone SK3, BD Bioscience, CA, USA) were used to detect Tregs. For assessment of Tregs, 1×10 6 cells of the breast tissue sample in 100 µL of PBS in one tube and 50 µL of blood sample in another tube were incubated with 5 µL of CD4, CD25 for 15 minutes at 4°C in the dark. Following incubation, red blood cells lysis, washing with PBS then addition of fixation solution to fix the cells and incubation for 10 minutes were done. After that, the cells were washed with PBS, and permeabilization solution (IntraSure TM kit, BD CA, USA) and 5 µL of Foxp3 were added and incubated for 20 minutes. The cells were then resuspended in PBS and analyzed by FACSCalibur flow cytometry with Cell Quest software (Becton Dickinson Biosciences, USA). An isotypematched IgG negative control was used for each sample. Forward and side scatter histogram was used to define the lymphocytes population. Then CD4 + cells were gated. Total CD4 + CD25 +low , CD4 + CD25 +High and CD4 + CD25 +High Foxp3 + T cells were evaluated as percentages of CD4 + cells in both blood and tumor tissue as shown in Figures 1 and 2, respectively. Statistical Analysis Numerical data was expressed as mean, median and standard deviation or standard error mean. Qualitative data were presented as number and percentage. The independent sample t-test and One-way ANOVA were used to assess the statistical differences between groups. Paired-t-test was applied to compare the percentages of cells between tumor tissue and peripheral blood. Pearson correlation was used to evaluate the strength of linear association between variables. The disease-free survival (DFS) was calculated using Kaplan-Meier curve and the receiver operating characteristic (ROC) curve was employed to estimate the accuracy of tumor-infiltrating Tregs in prediction of DFS (≥3 years). All analysis was performed by SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). Characteristics of the Patients' Group The patients' ages ranged from 28 to 77 years with a mean of 50.4±10.4. The tumor size in most of the patients was T2 (63.3%). Most of the patients were either N0 (46.7%) Assessment of Tregs Levels in Peripheral Blood and Breast Cancer Tissue of Patients with TNBC in Comparison with the Control Group As shown in Table 2, while the level of total CD4 + T cells in the peripheral blood was significantly lower in patients than healthy controls, their level in the malignant breast cancer tissue was higher than that in the normal tissue. The mean Relations Between Tumor-Infiltrating and Peripheral Blood Tregs and the Clinicopathologic Characteristics of Patients with TNBC CD4 + CD25 + subsets and Tregs have shown no significant associations with most of the tested clinicopathologic characteristics in both breast tissue and peripheral blood. Only tumor-infiltrating Tregs have shown increasing levels with the increase in the tumor size (p<0.0001) and were significantly higher in patients with local recurrences than those without recurrence (p =0.001), (Tables 3 and 4, Figure 3). Discussion TNBC is an aggressive type of breast cancer characterized by poor prognosis and lack of targeted therapy. 23 TNBC has higher immunogenicity and tends to have higher Tregs infiltration than other subtypes. 22,24,25 Inconsistent findings on the influence and prognostic value of Tregs in TNBC has been reported in previous reports. [18][19][20][21][22] In this study, the mean percentages of Tregs were higher in the peripheral blood of TNBC patients than healthy controls and in tumor tissue than normal breast parenchyma. Consistent with our findings, Wang and Huang reported significantly increased serum levels of CD4+CD25+Foxp3 + Tregs in patients with breast cancer compared with healthy individuals. 26 In addition, Plitas et al. 16 found increased Tregs in breast cancer tissue as compared to normal breast parenchyma and peripheral blood. Furthermore, breast tumor cells utilize immune regulatory cells such as Treg and different immunosuppressive pathways involving CD39, PD-1 and CTLA-4 molecules in creating disturbed immune environment for them to survive. 27 The increased tumor-infiltrating Tregs could be explained by expression of homing of receptors on Tregs that directs the migration of distinct populations to certain tissues 28 and regional extension of pre-existing tissue resident Tregs. 16 In addition, powerful stimulation of T cell receptors is needed for Treg cell activation, proliferation and inhibitory function. 29 Additionally, chemokine signaling, and cell migration were found to be the main single group of genes enriched in tumor-infiltrating Tregs. 16 The tumor-infiltrating Tregs showed significant direct relation with the level of Tregs in the peripheral blood. Similarly, Cai et al. 30 reported that the level of CD25 +Foxp3+ Tregs in circulating CD4+T cells was positively correlated with the level ofCD25+Foxp3+Tregs in CD4 +tumor-infiltrating lymphocytes in TNBC. In contrast to tissue infiltrating Tregs, peripheral blood CD 4+CD 25+Foxp3+ Tregs had no association with any of the clinico-pathological features of TNBC. These findings support the notion that tumor resident Tregs have distinct features that differ from Tregs in peripheral blood. 16 On the other hand, the proportions of circulating Tregs were found to be associated with an increased occurrence of breast cancer. 26 The association between the Tregs and the clinicopathological features of TNBC suggested that increasing 6025 tumor-infiltrating Tregs was associated with increased tumor size and local recurrence as well as decreased disease-free survival. Increased frequency of tumor-infiltrating Tregs was observed in the more aggressive BC subset; TNBC and was associated with higher-grade lesions among all studied breast cancer subsets. 16 Liu et al. 31 observed increased CD4 + CD25 + Foxp3 + Tregs infiltration in breast cancer tissues and that was associated with high histologic grade, negative estrogen and progesterone receptors status, and overexpression of human epidermal growth factor receptor type 2, along with diminished overall as well as progression-free survivals. On the other hand, Yeong et al. 21 reported that high number of tumor-infiltrating CD4 + CD25 + Foxp3 + Tregs in TNBC patients was linked to a higher tumor grade, lymph node status and better prognosis. Increasing the numbers of tumor-infiltrating Tregs may augment local immunosuppressive abilities, suppressing the anti-tumor immunity, thus enhancing tumor growth and invasion, 32 in addition; early breast cancer has an inflammatory milieu characterized by mDC, Treg, and cancer stem cells (CSC) infiltration. The frequencies of Treg, CSC and CD8/Treg ratio were associated with disease progression including lymph node metastasis. 33 Moreover, a previous review 34 proposed that Tregs have cytotoxic capability that may directly kill effector T cell, which may explain the association between Foxp3+ Tregs infiltration and poor recurrence free survival of breast cancer patients. 35 The study findings support the notion that Tregs can directly contribute to tumor progression rather than they The current study had a number of limitations, the small number of patients was a crucial limitation that was responsible for absence of several statistical relations, and heterogeneity of patients as the study recruited early and locally advanced diseases. Conclusion The findings of the current study support the possibility that TNBC microenvironment conveys specific characteristics on Tregs distinguishing them from those in normal breast tissue or Tregs in peripheral blood, improving the capabilities of tumor-infiltrating Tregs to enhance tumor growth, local recurrence and reduce the DFS. They also suggest the therapeutic value of targeting the function of tumor-infiltrating Tregs in TNBC. Data Sharing Statement All analyzed data are included within the article. Funding There is no funding to report. Disclosure All authors reported no conflicts of interest for this work. Publish your work in this journal Cancer Management and Research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.	2021-08-11T05:25:45.844Z	2021-08-01T00:00:00.000	{ "year": 2021, "sha1": "f564fe2d33b6d189ab66e9500a01a2ea3fc63724", "oa_license": "CCBYNC", "oa_url": "https://www.dovepress.com/getfile.php?fileID=72274", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f564fe2d33b6d189ab66e9500a01a2ea3fc63724", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
250490647	pes2o/s2orc	v3-fos-license	Metabolite profiling of abalone (Haliotis iris) energy metabolism: a Chatham Islands case study Introduction The Chatham Islands has some of the most prized black-footed abalone (Haliotis iris) beds in New Zealand. This well-managed fishery includes restrictions on catch and size limits, selective fishing methods, and shellfish management. However, recent declines in biomass and growth parameters have prompted omics research to characterise the biological responses of abalone, potentially contributing towards animal management strategies. Objectives The aim of this study was to characterise the metabolite profiles of slow and fast growing, juvenile and adult abalone, relating to metabolites supporting energy metabolism. Methods A gas chromatography–mass spectrometry metabolite profiling, applying methyl chloroformate alkylation, was performed on juvenile and adult abalone samples collected from Point Durham and Wharekauri sites, Chatham Islands, New Zealand. Results The results obtained from haemolymph and muscle samples indicated that abalone from the fast-growing area, Wharekauri, fuelled metabolic functions via carbohydrate sources, providing energy for fatty acid and amino acid synthesis. Conversely, higher amino acid levels were largely utilised to promote growth in this population. The metabolism of juvenile abalone favoured anabolism, where metabolites were diverted from glycolysis and the tricarboxylic acid cycle, and used for the production of nucleotides, amino acids and fatty acids. Conclusions This research provides unique physiological insights towards abalone populations supporting the use of metabolomics as a tool to investigate metabolic processes related to growth. This work sets the stage for future work aimed at developing biomarkers for growth and health monitoring to support a growing and more sustainably abalone fishery. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s11306-022-01907-6. Introduction The New Zealand black-footed abalone (Haliotis iris) is endemic to the coastal waters of New Zealand, encompassing cultural, ecological and economic importance (Will et al., 2015). In 2019, abalone exports were worth $50 million, including shell, by-products and nutraceutical sales. The commercial abalone fishery is managed in accordance with total allowable commercial catch systems. Of the managed areas, the Chatham Islands contribute more than a quarter to the national 720 tonnes of commercially harvested wild abalone (PMCSA, 2021). In recent years, a decline in abalone biomass has been observed in the Chatham Islands, resulting in new research and managing approaches to ensure the fishery is sustainable for future generations. Translocation and reseeding programmes are examples of such initiatives, where new spawning banks are established, or where abalone with slower growth are moved to areas with higher recorded growth rates (Fisheries`New`Zealand, 2019a). Furthermore, the implementation of a minimum harvest size of 125 mm helps to increase the spawning biomass and allows areas with abundant abalone growth, previously depleted, to recover (Fisheries`New`Zealand, 2019b). Characteristically juvenile abalone prefer habitats sheltered from wave exposure, but when they transition to adults, they are often found in areas of high wave exposure and dense macroalgal cover (Shaffer & Rovellini, 2020). As abalone of different life stages have different habitat preferences, investigations into abalone performance need to be size-and habitat-specific to assist management decisions. In addition, environmental factors, such as food availability also affect abalone growth and health and need to be considered for physiological assessments . To fill gaps in our knowledge regarding H. iris growth and performance, insights into abalone physiology and the effects of various biotic and abiotic factors on the organism's growth and health are essential (Venter et al., 2018a). This information is also crucial to improve our understanding of the physiological mechanisms that underpin abalone population variations, susceptibility and resilience to environmental change to ensure a sustainable future for wild stocks and the fishery. Various tools and techniques exist to measure physiological parameters of molluscs. These are often focused at biological responses at the molecular, cellular, biochemical, physiological, or behavioural level (Waller & Cope, 2019), typically measured using omics approaches . Generally, omics-based approaches identify sets of gene products such as transcripts, proteins and metabolites, within a biological sample (rather than single products at a time) . Of these, metabolomics is at the endpoint of the omics cascade, and the closest to the cell's functional phenotype, which is dictated by both the genome and environment, making metabolites more likely to contribute to the functional state of cells and serve as a direct signature of biochemical activity (Venter et al., 2018a). The aim of this research was to characterise the metabolite profiles of juvenile and adult abalone collected from two sites at the Chatham Islands with different animal growth rates. Specifically, we study the endo-and exometabolome of abalone adductor muscle and haemolymph samples, respectively, using a gas chromatography-mass spectrometry (GC-MS) metabolomics approach, and profile metabolites contributing to energy metabolism adding new knowledge to a larger project investigating H. iris physiology. Abalone sampling Abalone were collected under special permit (720, client number 9791209) issued by Fisheries New Zealand, from two catch zones (as defined by the Ministry of Fisheries statistical area maps) around the Chatham Islands: 1) Zone 437-Awatotara Creek-Awa Raku (locally referred to as Point Durham: Lat -44.009608, Long -176.687622); 2) Zone 415-Te Awamuti-Okahu Creek (locally referred to as Wharekauri: ). Point Durham is classified as a low recovery area, which is not fished often due to the slow growth of abalone in this area, while Wharekauri is classified as a high fished (abalone fast growth) area. Juvenile (n = 20) and adult (n = 20) abalone were collected by divers from Point Durham and Wharekauri sites. Haemolymph were taken from the pedal sinus and transferred into cryovials followed by snap-frozen using liquid nitrogen. Animals were weighed ± 0.01 g, and the shell lengths and heights were measured ± 0.10 mm. Following shucking, a sample of the adductor muscle was taken from the ventral surface towards the central point, where the muscle attaches to the shell, and snap-frozen. All samples were shipped to the laboratory on dry ice and stored at − 80 °C until metabolomics analyses. Extracted metabolites were derivatised by methyl chloroformate (MCF) alkylation and analysed via GC-MS . Quality control (QC) samples were included in every batch by preparing a pooled mixture of haemolymph or muscle tissue (Broadhurst et al., 2018). Additionally, a derivatised sample blank containing the internal standard, an in-house prepared derivatised amino acid mix and a nonderivatised alkane mix were also injected and analysed for QC purposes (Young et al., 2019). The MCF derivatives were analysed with an Agilent GC7890B and autosampler coupled to a MSD5977A, with a quadrupole mass selective detector (EI) operated at 70 eV, using a ZB-1701 GC capillary column (30 m × 250 μm id × 0.15 μm with 5 m stationary phase) and helium as carrier gas (flow of 1 mL min −1 ). Samples (1 μL) were injected under pulsed splitless mode with the injector temperature at 260 °C, following a temperature program as reported by Alfaro et al. (2021). Identification of compounds was carried out using mass spectra acquired in scan mode from 38 to 550 amu, with detection threshold of 100 ion counts (view supplementary material for detailed description of methods used) (Smart et al., 2010). Data processing Raw spectra were processed using AMDIS (v2.66) software. Metabolite identifications and peak integrations were conducted using Chemstation software and customised R-XCMS based scripts (Aggio et al., 2011), based on an inhouse mass spectral library of MCF derivatised commercial standards. Compound identifications were based on matches (≥ 70%) to both the MS spectrum of the derivatised metabolite and its respective retention times. Identified compounds can be assigned a level 1 identification confidence level (Schymanski et al., 2014). Unknown features are not shown. The data matrices of peak intensities were pre-processed for quality control purposes prior to statistical analyses using the web-based tool MetaboAnalyst 5.0 (Pang et al., 2021). Data were normalised to the peak intensity of the internal standard and to sample biomass in the case of muscle tissue . Statistical analysis QC samples analysed amongst the batches were assessed in terms of coefficient of variance percentages and data were evaluated for within batch effects. The data were generalised log (glog) transformed and two-way analysis of variance (ANOVA) was used to determine the influence of collection site and animal life stage (between subject, p < 0.05). The metabolite response was further analysed and visualized in a blocked manner, with principal component analysis (PCA) (Xu & Goodacre, 2012), and by constructing individual metabolite maps of metabolite findings linked to animal collection site and life stage. Grouping of experimental groups on the PCA scores plots were highlighted with the use of 95% confidence ellipses around each group. Data analyses for morphological parameters were conducted with SPSS® software, version 23.0, using student t-tests to identify differences between collection site and life stage (p < 0.05). Results and discussion Marine invertebrate populations generally span habitats with a range of physical and biological characteristics (Sebens, 2002), which can significantly influence their metabolism, growth and health. This link between the environment and growth was investigated in this study by focusing on metabolic differences between slow growing abalone, collected from Durham, and fast-growing abalone, collected from Wharekauri (Fig. 1a). Both adult and juvenile abalone were studied in an attempt to reveal key metabolic activity that could explain increased growth in the Wharekauri abalone. Understanding how the environment influences the metabolism and growth of abalone in fished populations is central to effective fisheries management (Naylor et al., 2006). Metabolic studies have previously been performed on juvenile or adult H. midae (Venter et al., 2019), H. iris (Grandiosa et al., 2018), H. fulgens (Tripp-Valdez et al., 2019) and H. discus hannai (Xu et al., 2020), yet metabolomics characterisation of a population to guide stock and fisheries management or translocation practises remains an untouched subject. The morphological parameters (Fig. 1c), showed an average wet weight of 290.55 ± 57; 322.05 ± 68 g, shell length of 110.42 ± 7; 117.60 ± 8 mm and shell height of 140.90 ± 4; 136.70 ± 3 mm (average ± standard error) for abalone collected from Durham and Wharekauri sampling sites, respectively. No differences were found in morphometric measures between animals collected from Durham and Fig. 1 Overview of metabolomics results as a Venn diagram (a); PCA plots (b) and morphometric (weight, length and height) measures (c) from two abalone collection sites (Durham and Wharekauri) and abalone of two life stages (adults and juveniles) Wharekauri. Juvenile abalone had an average wet weight of 119.15 ± 11 g, shell length of 90.91 ± 2; mm and shell height of 129.30 ± 1 mm. While adult abalone had an average wet weight of 493.45 ± 19 g, shell length of 137.11 ± 3; mm and shell height of 148.30 ± 2 mm (average ± standard error). Adult abalone were significantly heavier and longer than juvenile abalone (p < 0.05). Based on two-way ANOVA, the largest number of metabolites were affected by animal life stage, yielding a total of 45 metabolites. Abalone collection site as experimental factor resulted in 35 significant different metabolites, as shown in the Venn diagram (Fig. 1a). The covariance of these metabolites was visualised with PCA in a multiblock manner, to highlight their combined discriminatory power (Xu & Goodacre, 2012) (Fig. 1b). A moderate overlap was seen in the scores of the different collection sites (across life stages), showcasing the lower impact of this experimental factor on the metabolism of adult and juvenile abalone from both sites. The relevant metabolic variation due to collection site is mainly captured by PC1, which explains 45.3% of the variance. PC2 explains 12.6% of the variation in the data, which is mostly unrelated to collection site, adding to a larger variance in the metabolism of the abalone from Durham. Complementary loadings data are provided in the supplementary material (Table S1 and Figure S1). Similarly, the PCA comparison of life stages (from both sites) gave clear distinctions between juvenile and adult abalone, with PC1 of the plot explaining most (37%) of this variance. PC2 explains 19.9% of the variation in the data, largely unrelated to life stage. Complementary loadings data are provided in the supplementary material (Table S2 and Figure S2). The metabolites that differed significantly between collection sites are shown in Table 1 as metabolite response of Wharekauri (compared to Durham) abalone (p < 0.05). Table 2 shows the metabolites that differed significantly in the juvenile abalone when compared to the adults, irrespective of the collection site. Metabolites in bold in Tables 1 and 2 indicate significance within the site or stage groups. A matching online Kyoto Encyclopedia of Genes and Genomes (KEGG) database ID (Kanehisa et al., 2021), metabolite classification, tissue finding, and the metabolite response is found in both tables. These findings are further illustrated in the metabolic maps of Figs. 2, 3 and 4. Central carbon metabolism is mostly conserved Based on video footage collected from the sampling sites, Durham was more sheltered, with less exposure to wave action, while Wharekauri was more exposed. Not only does this influence immediate nutrient and waste levels but also oxygen which could influence energy metabolism considerably. In a previous study on H. iris, abalone from wave exposed and sheltered sites showed large differences in the glycolytic pyruvate reductase enzyme, tauropine dehydrogenase, and glycogen content (Wells et al., 1998), hinting to perturbed carbohydrate metabolism. In this study, the concentration of several metabolites was higher in the animals from the more exposed area (Wharekauri), however, intermediates of the central carbon metabolism [glycolysis pathway and tricarboxylic acid (TCA) cycleillustrated in Fig. 2] reflected few changes which suggests unperturbed energy production from carbohydrates. This suggests that sufficient algal sources were obtained at both sites for metabolic functioning, as previously seen in H. laevigata (Bansemer et al., 2016). Central carbon metabolism results from the current study in Fig. 2, show prominent differences between adult and juvenile abalone. It can be hypothesised that juvenile abalone are in a steady growth phase, with anabolic reactions considerably upregulated. Once cell proliferation takes priority, as in the case of growing juveniles, metabolic demands change, enabling dietary nutrients to support cell growth, and manage redox challenges associated with anabolic processes. Typically, during anabolic processes, metabolites are diverted from the central carbon metabolism, to be used in the production of nucleotides, amino acids and fatty acids (Zhu & Thompson, 2019). Most of these anabolic pathways have a high energy demand which necessitates upregulation of the central carbon metabolism and oxidative phosphorylation. The TCA cycle in particular keeps functioning due to anaplerotic reactions involving branched chained amino acids (leucine, isoleucine and valine), glutamine, alanine, lactate and tyrosine (all affected in this study), which replenishes the TCA cycle to ensure production of essential energy (Inigo et al., 2021). Interestingly, in the current study, the metabolites supporting anaplerotic reactions were mostly lower in juvenile abalone, supporting the idea that these metabolites are utilised by the TCA cycle for energy purposes, as well as other pathways discussed below. Focusing on Fig. 2, intermediates in first half of the TCA cycle (citrate, aconitate, itaconate, and isocitrate) were significantly elevated in juvenile abalone (both sites) compared to adults. 2-Ketoglutaric acid and (systemic) succinate were significantly lower in the juvenile animals. An increase in citrate for example serves as an important signal for the inhibition of catabolic reactions, favouring anabolic reactions (Frezza, 2017), supporting the hypothesis that juvenile abalone are diverting energy towards growth in the current study. Lower succinate levels could be an indication of an upregulated aspartate-succinate pathway important in the synthesis of nicotinamide adenine dinucleotide (NAD + ) and flavin adenine dinucleotide (FAD) to support energy production in the presence of stressors (Venter et al., 2018a). Lipogenesis in Wharekauri abalone apparently upregulated The concentrations of several saturated and unsaturated fatty acids, illustrated in Fig. 3, were elevated in the muscle of abalone collected at the Wharekauri site (both adults and juveniles). Since these animals are considered faster growers, it is possible that fatty acid synthesis from glucose via de novo lipogenesis is upregulated in these animals, as cells accumulate lipids under conditions such as growth (Giese, 1966). Additionally, the diet consumed by the abalone at Wharekauri can serve as a rich source of fatty acids (lipids), attributing to the higher levels of muscle 9E-heptadecenoate, dihomo-gamma-linolenate, DPA, myristate, (Xu et al., 2004) and H. tuberculate (Hernández et al., 2013). Malonate and gamma-linolenate were significantly lower in the haemolymph of Wharekauri abalone (Fig. 3). Malonate is typically used as a precursor for de novo synthesis of fatty acids (Guan & Nikolau, 2016), but can also act as a competitive inhibitor of succinate dehydrogenase, limiting mitochondrial respiration (Bowman & Wolfgang, 2019). In addition, gamma-linolenate plays a role as an polyunsaturated fatty acid, vital towards the production of eicosanoids (signalling molecules) (Sergeant et al., 2016). From this context, these haemolymph metabolites support the utilisation thereof by muscle metabolites, where fatty acids are incorporated into cells via a process that is not energy dependent but requires additional proteins (amino acids), which does require energy for fatty acid transport (Laposata, 1995). The concentration of most fatty acids differed markedly between the adult and juvenile abalone (seen in Fig. 3). Increased citrate levels are associated with elevated lipogenesis (Lee et al., 2018), which correspond with the elevated long chain fatty acids detected in the muscle tissue of juvenile animals. Upregulated lipogenesis and storage of dietary lipids not only ensure a supply of triacylglycerols for later use, but also phospholipids needed for cell division (Schönfeld & Wojtczak, 2016;Tang et al., 2018). Juvenile, H. asinia, showed weight gains following feeding experiments with supplemented essential fatty acids (Bautista-Teruel et al., 2011), while research on juvenile H. discus hannai, supported the accumulation of fatty acids in soft tissue, due to algal diets (Pan et al., 2018). This study supports the function of fatty acids in juvenile H. iris, as an anabolic source, that assists with organismal growth. Additionally, Wharekauri abalone retains amino acids (and nitrogen) better The concentrations of most amino acids and many of their associated intermediates were elevated in the muscle of Wharekauri abalone compared to the Durham animals (Fig. 4). Higher concentrations of amino acids can be linked to anabolic activities, promoting growth, as previously reported in H. midae (Venter et al., 2018c). Generally amino acids are degraded by different pathways that feed into energy production pathways, yet in scenarios where sufficient carbohydrate sources are present for energy production, amino acids can fulfil other functions like osmoregulation (Venter et al., 2018a), production of proteins and neuropeptides (Sharker et al., 2020) and shell formation (Marie et al., 2010). When excessive glycolytic activity is experienced, associated intermediates are diverted to other pathways assisting with the production of non-essential amino acids and lipids, typically required for cell growth (Zhu & Thompson, 2019). Apart from upregulated protein synthesis, the carbon and nitrogen derived from these affected amino acids can also support nucleotide production. Elevated methionine and S-adenosylmethionine (SAM) concentrations could also be linked to down-regulated gene methylation (Roznere et al., 2017), which could contribute to the faster growth rate in the Wharekauri abalone. Lower concentrations of serine, threonine, SAM and methionine were detected in juvenile haemolymph collected at both sites (Fig. 4). Since these metabolites are used by the folate-and methionine cycle for the regulation of gene expression, energy balance and the synthesis of biomacromolecules (i.e., nucleotides) (Pan et al., 2021), it could lead to the same conclusion that gene silencing is possibly down-regulated. As seen in Fig. 4, the branched chain amino acids were markedly elevated in the muscle of the Wharekauri abalone (especially the adults) which highlight their ability to retain essential amino acids for anabolic processes or as alternative fuel to replenish TCA cycle intermediates when in catabolic state. Likewise, the aromatic amino acids (phenylalanine and tyrosine) with their associated intermediates were also elevated in the Wharekauri group, as seen in fast growing H. midae (Venter et al., 2018c). Additional amino acids and metabolites affected in this study can be attributed towards an antioxidant function. A study on H. midae reported that juvenile abalone are more resistant to oxidative stress because they have higher levels of antioxidant enzymes (Vosloo et al., 2013). Juvenile H. iris, from this study, displayed lower concentrations of metabolites relating to the glutathione metabolic pathway (glutamine from glutamate, and ornithine) and oxidative stress mechanisms (serine, cystathionine, methionine and GABA), showcasing that these animals had no requirement to scavenge reactive oxygen species or respond metabolically to stressors (Delorme et al., 2021). Indeed, urea cycle-related intermediates were also lower in juvenile abalone in both sites which highlights the retention of nitrogen. The urea cycle also functions as a collection point for nitrogenous waste (Azizan et al., 2021), especially during stressed conditions as previously demonstrated in abalone (Venter et al., 2018b). Glycine, one of the severely elevated metabolites detected in the juveniles (Fig. 4), contributes to the biosynthesis of heme, purines, creatine, glutathione and uric acid, which would require upregulated de novo glycine synthesis (Venter et al., 2018c). Furthermore, during anaerobic conditions glycine is utilised to produce strombine, releasing NAD + for use in the glycolysis pathway. Higher strombine was also found in the current study, which links to the increased glycine levels. The production of strombine can be considered as a valuable carbon chain store (like pyruvate and glycine) for use in the abovementioned pathways and TCA cycle. It is arguably also a better storage form than lactate, with better buffer capacity (Venter et al., 2018a). In contrast to strombine, lactate along with alanine, were significantly lower in the haemolymph samples from juvenile abalone tested. Lactate is a well-known metabolite of anaerobic metabolism, often reported as increased in abalone subjected to stress . Elevations in alanine have also been attributed to abalone as a mechanism to buffer H + ions during stress and to regulate intracellular osmotic pressure. Moreover, the reduction hereof in juvenile abalone reflects the transport function of haemolymph where metabolites central to the glycolysis pathway and TCA cycle are absorbed from the haemolymph by tissues (muscle) where they can be utilised (Venter et al., 2018b). Fishery considerations This research provides unique physiological insights towards abalone populations of New Zealand and globally. In the Chatham Islands, Wharekauri is classified as a high abalone catch area, where animals typically achieve the required fishing size in a shorter time period, arguably due to their surrounding conditions and the interaction with their environment. A similar trend was reported in greenlip abalone, where better quality habitat positively influenced growth patterns (Dixon & Day, 2004). Abalone populations are classified as stunted (slow growers), where individuals grow slowly and/or achieve a smaller maximum size in comparison with other populations, and has been observed in greenlip abalone (H. laevigate) (Hart et al., 2013), and black-footed abalone (H. iris) (Naylor & Andrew, 2004). Additionally, protected marine areas are being sought out to aid the restoration of white, pink (H. corrugate) and green abalone (H. fulgens) (Rogers-Bennett et al., 2002). This brings to light the need to characterise "fast-growing/ non-stunted" areas to provide biomarkers relating to the biological response of abalone to their environment, for potential reseeding or translocating initiatives. The current research provides insight into Chatham Islands abalone sites, where the translocation of abalone from other populations to Wharekauri can be achieved, as long as abalone metabolic energy is invested into anabolic activities and not maintenance or recovering from stress, resulting in limited energy for growth (Venter et al., 2018c). Freshwater mussel research proved that characterisation of metabolite levels can provide a framework for management decisions and the development of tools to improve conservation techniques (Roznere et al., 2017). Yet, various environmental variables will also need to be considered, as demonstrated in caged mussels, where the effect of seasonal patterns on condition and tissue biochemistry have been highlighted as other considerations for translocation (Gray & Kreeger, 2014), something which is unknown for H. iris at this stage. Additional research on H. iris, identified faster growing animals in areas were the mean monthly maximum sea surface was lower, while the opposite was also true, where slow growth was linked with higher temperatures (Naylor et al., 2006). From the current investigation no temperature data was collected, creating an important factor to take into account in future studies and translocation strategies. Metabolomics results of juvenile abalone indicated that anabolic metabolism is dominant, supporting steady growth mechanisms, suggesting that juvenile abalone can be translocated around the Chatham Islands. In saying that, biomarker analyses will be essential to monitor animal physiology and the metabolic response to environmental conditions. For example, the survival of juvenile H. roei was compromised by high wave energy and temperature environment (Strain et al., 2019). Restoration work on juvenile H. kamtschatkana revealed that monitoring of genetic diversity of hatcheryproduced and habitat quality are required to ensure survival (Read et al., 2012). The use of metabolomics to identify candidate biomarkers in abalone following exposure to stressors have been well-documented, yet validation of these candidate biomarkers remains largely unexplored . Conclusions From the metabolomics results, it is clear that fast growing abalone from Wharekauri obtained sufficient carbohydrate sources from the environment to support energy production functions. Additionally, the metabolite profiles showed increased fatty acid synthesis essential for structural functions and higher amino acids to support protein synthesis. In juvenile abalone, energy produced via glycolysis and the TCA cycle was utilised to support anabolic reactions and the production of amino acids and fatty acids, corresponding to nucleotide production for cell growth. The lack of increased metabolites previously associated with abalone subjected to stress infers the idea that juveniles in this study were investing energy in growth as opposed to spending energy on maintenance or recovering processes. This study showcases metabolomics as a valuable tool for investigating the altered metabolic processes related to growth in abalone, and hence, is a valuable tool for identifying biomarkers for growth and health monitoring to support a growing and more sustainably abalone fishery. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s11306-022-01907-6. Supervision: AA; Project Administration: LV, TN; Funding Acquisition: AA. All authors have read and agreed to the published version of the manuscript. Funding Open Access funding enabled and organized by CAUL and its Member Institutions. This project was funded by The Pāua Industry Council, New Zealand. This work was also supported by the New Zealand Ministry for Business, Innovation, and Employment, through the Aquaculture Health Strategies to Maximise Productivity and Security programme (contract no. CAWX1707). Data availability Data supporting the findings of this study can be obtained from the authors upon reasonable request. Code availability Not applicable. Declarations Conflict of interest No potential conflict of interest was reported by the author(s). Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The abalone (Haliotis iris) used in this research were obtained from a fishery quota under Special permit (720, client number 9791209) issued by Fisheries New Zealand. No ethical approval was required under New Zealand's Ethical Guidelines for research on invertebrate molluscs. Consent for publication Not applicable. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.	2022-07-14T06:16:15.729Z	2022-07-01T00:00:00.000	{ "year": 2022, "sha1": "18345c513b98534042354eea8d77aac1c53f65ef", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s11306-022-01907-6.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "fcfcb5e4c6e531fd6a01eb4b127e7495f96f7cc1", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
250275309	pes2o/s2orc	v3-fos-license	Fad Diets: Facts and Fiction The global prevalence of obesity is alarmingly high and is impacting both developed and underdeveloped countries, beyond the borders of ethnicity, sex, and age. On the other hand, the global interest in dieting has increased, and people are obsessed with certain fad diets, assuming them as a magic bullet for their long-term problems. A fad diet is a popular dietary pattern known to be a quick fix for obesity. These diets are quite appealing due to the proposed claims, but the lack of scientific evidence is a big question mark. Such diets are often marketed with specific claims that defy the basic principles of biochemistry and nutritional adequacy. These diets may have protective effects against obesity and certain chronic diseases like cardiovascular diseases, metabolic syndrome, and certain cancers. Limited evidence exists to support the proposed claims; rather certain studies suggest the negative health consequences of long-term adherence to such dietary patterns. Many fad diets have emerged in the previous few decades. This review article will explore the current evidence related to the health impacts of some most popular diets: Atkins diet, ketogenic diet, Paleolithic diet, Mediterranean diet, vegetarian diet, intermittent fasting and detox diet. INTRODUCTION Obesity is one of the major public health concerns in this modern era. It is now considered a global epidemic due to the gradual but continuous increase in its prevalence. The global prevalence of obesity is alarmingly high and is impacting both developed and underdeveloped countries, beyond the borders of ethnicity, sex, and age. Worldwide obesity has tripled from 1975 to 2016, while childhood obesity is increasing dramatically (1). Excessive calories from fats and sugars, large portions of food, routinely junk food intake, availability of fast foods at the doorstep and limited physical activity are some of the contributing factors to obesity (2). Obesity is an independent risk factor for morbidity and mortality. Being obese or overweight puts a person at greater risk of developing cardiovascular diseases, hypertension, insulin resistance, diabetes, reproductive issues, liver and kidney diseases (3). Despite the growing global prevalence of obesity, there is always a group that is highly obsessed with dieting. The global interest in dieting has increased in the last two decades. A study indicated that internet searches related to weight loss queries had immensely increased between the years 2004 to 2018 (4). In the meantime, people rush toward certain fad diets (FD), assuming them as a magic bullet for their long-term problems. FD is not a scientific terminology but rather a popular or trendy dietary pattern that is known to be a quick fix for obesity (5). FD can be easily differentiated from a healthy and balanced diet based on its characteristic features: (i) promises rapid weight loss (ii) absence of physical activity guidelines (iii) promotes shortterm changes rather than achieving lifelong sustainable goals (iv) focuses on one type of food or eliminates any food group (v) cannot be maintained for life long period (vi) nutritional adequacy is questionable (vii) fails to provide health warnings for those with chronic diseases (viii) lacks scientific evidence to support the claims (5,6) (Figure 1). AIM OF THE STUDY A wide range of FDs has been proposed to date, ranging from low carbohydrate diets to low-fat diets, high-fats to high-protein diets, those with detoxification claims, and others of the Mediterranean or Paleolithic origin. These diets are followed blindly but are associated with certain negative health outcomes as one size does not fit all. This review article will explore the current evidence related to the health impacts of some popular diets, including Atkins diet, ketogenic diet, Paleolithic diet, Mediterranean diet, vegetarian diet, intermittent fasting, and detox diet. ATKINS DIET (AD) In the 1970s, a low carbohydrate, high protein (LCHP) regimen was developed by cardiologist Dr. Robert Atkins, which was published in his book "Dr. Atkins' New Diet Revolution" (7). This diet was promoted as a quick weight loss plan based on a lifetime change in eating habits. Atkins believed that metabolic imbalance resulting from carbohydrate consumption is the major cause of obesity. He claimed that this is the easiest, high-energy diet that mobilizes fats more than any other diet for weight loss maintenance. The AD involves an extreme reduction of carbohydrates, i.e., less than 5% of total calorie intake, ad libitum intake of proteins and fats, adequate fluid intake with vitamin and mineral supplementation, and regular exercise (8). The diet has four phases: induction phase, ongoing weight loss phase, pre-maintenance phase, and lifetime maintenance phase ( Table 1). The modified version of the AD (MAD) is currently available with the same four phases but slightly modified net carbs consumption in each phase. The MAD is less restrictive, allowing the person to choose the number of net carbs in phase 1, i.e., 20, 40, or 100 g of carbs and fats are not just allowed but encouraged. The primary goal is not weight loss rather it has shown promising results in seizure reduction in intractable epilepsy (9)(10)(11)(12)(13). Effectiveness of Atkins Diet There is substantial evidence suggesting that AD promotes more weight loss than conventional diets. One of the first AD research was published in The New England Journal of Medicine in 2003. Brehm et al. (14) in a study allocated 53 healthy, obese women to two groups, i.e., low carbohydrate ketogenic diet (LCKD) or energy-restricted low-fat diet (LFD) (carbs: 55%, protein: 15%, fats: 30%). Over 6 months, the LCKD subjects lost 8.5 kg versus 4.2 kg in the LFD group. There were no comparable differences between the groups in serum glucose, lipids, leptin, and insulin excluding triglycerides that showed a significant reduction in the LCKD group. In another randomized trial, 132 severely obese individuals (43% had metabolic syndrome while 39% had type 2 diabetes) were assigned to two groups. One group followed AD and the other followed LFD for 6 months. The results showed that LCD individuals lost 3.8 kg more weight than those on LFD. No significant difference was observed in both groups after 12 months (15). In another controlled trial of 1 year, 63 obese participants were randomly assigned to either the AD or conventional LFD. After 6 months, results showed that the LFD group lost less weight, i.e., 3.2 ± 5.6% than the AD group, i.e., 7.0 ± 6.5%. The AD group lost 4% more weight, had higher levels of high-density lipoprotein cholesterol (HDL-c) and lower levels of triglycerides (TG) than the other group. No significant differences between groups were noted in low-density lipoprotein cholesterol LDL-c (16). Several meta-analyses and systemic reviews reported the promising effects of low carbohydrate diets on weight loss and cardiometabolic risk factors. Mansoor et al. (17) demonstrated that the LCD group had a significant increase in HDL-c and LDLc, and had a greater weight loss and TG reduction in contrast to those following LFD. Hashimoto et al. (18) reported that LCD resulted in a greater reduction of body weight and body fat mass than the control diet. LCD was linked with moderately more significant advancement in weight loss and reduction of atherosclerotic cardiovascular diseases (ASCVD) risk, compared to LFD (19). Naude et al. (20) concluded that both LCD and balanced diets had shown weight loss. After 2 years of follow-up, there was no significant difference between the diets in terms of cardiovascular and diabetes risk factors. Bueno et al. (21) found that after 12 months or more, the individuals that followed an energy-restricted very low carbohydrate diet (VLCD) (carbs: <50 g/day or 10%) compared to LFD (fats: <30%) had a more significant improvement in HDL-c, LDL-c, TG and diastolic blood pressure (DBP) as well as the reduction in body weight. Hu et al. (22) compared LCD and LFD and concluded that both diets were efficient at reducing waist circumference, body weight, total cholesterol (TC), total to HDL-c ratio, LDL-c, TG, blood glucose, serum insulin, and blood pressure. LCD showed a greater decrease in TG, and less reduction in LDL-c and TC but increased HDL-c in comparison with LFD. Health Consequences Atkins diet has not been extensively studied while those studies that have been mentioned earlier have high dropout rates and are sometimes non-conclusive. Despite the rapid weight reduction, there are some concerns for those with comorbidities. There are some considerable potential complications associated with LCHP diets. There is conflicting evidence on the urinary stone formation tendency of LCHP diets (23). A short-term study showed that healthy subjects followed the LCHP diet for 6 weeks, decreased urine pH, increased urinary-acid excretion, and decreased calcium balance was observed in them. Therefore, they had a greater risk of stone formation (24). A prospective cohort study was conducted in Iran, involving 1,797 participants that were followed up for almost 6 years. Results showed that a higher tertile of LCHP diet correlates with a greater risk of chronic kidney disease (CKD) (25). Metabolic acidosis is a common complication of LCHP diets. A case of 40 years old obese woman was reported, who was presented with nausea, vomiting, dehydration, and dyspnea. Investigations revealed that she was following AD, lost 9 kg in 1 month, and laboratory findings were consistent with ketoacidosis Chen et al. (26). Pregnant and lactating mothers should be cautious when following such a diet as there is a reported case of LCD-associated ketoacidosis in a non-diabetic lactating mother (27). AD provides several benefits including weight reduction and cardio-metabolic health improvement, but limited evidence exists as compliance is the major barrier to this dietary regimen. Strict supervision by health professionals is advised as adverse metabolic sequelae can result from this type of diet. KETOGENIC DIET (KD) In 1923, Dr. Russell Wilder designed the classic KD for the treatment of epilepsy. The classic keto is a strict regime comprised of a 4:1 ratio, which means one part of carbs and proteins combined for four parts of fats. The use of KD for treating different diseases has increased over the past few decades. All the currently available versions are modified forms of classic KD. There are five types of KD published in the medical literature: (i) classic keto (ii) modified keto (iii) Medium-chain triglycerides oil (iv) Low glycemic index treatment (v) Modified Atkins diet. The macronutrient ratio is the major difference between these diets. In a nutshell, KD is a VLCD that relies on a moderate amount of proteins, high fat, and low carbohydrates that provide approximately 5-10% of calories from carbohydrates, 20-25% of calories from proteins, and 65-80% of calories from fats (28). KD includes fasting, proper hydration, physical activity, and intake of electrolytes and nutritional supplements (29). The KD works by bringing certain metabolic changes to the body. Glucose is the body's primary energy source. Carbohydrate deprivation resulting from KD causes a metabolic shift toward gluconeogenesis and ketogenesis. The preliminary shortage is managed by endogenous production of glucose from glycerol, glutamine, alanine, and lactic acid (gluconeogenesis). To keep up with the needs of the body, ketone bodies come into play and serve as an alternate energy source for the body (ketogenesis). At this stage due to low blood glucose feedback, secretion of insulin is also low, which further reduces the stimulus for fat and glucose storage. This ketotic state remains active until the body's carbohydrates needs are fulfilled (30). Effectiveness of Ketogenic Diet Literature is consistent with these findings that KD is an effective intervention for improving quality of life, seizure severity, and seizure frequency in epileptic patients (13,31). KD is known for its neuroprotective action in various neurological illnesses like Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, ischemic brain injury, traumatic brain injury, depression, autism, and narcolepsy (32). In the modern era, KD is recognized as a weight loss intervention but studies suggest mixed findings. A study compared the weight loss, appetite, and hunger responses of obese men who were fed a medium carbohydrate (35%) nonketogenic diet (MCNKD) and low carbohydrates (4%) ketogenic diet (LCKD) in a crossover manner. After 4 weeks period, significantly greater weight loss and lower ad libitum energy intake were observed in the LCKD group because of reduced hunger (33). A meta-analysis concluded that KD contributes to greater long-term weight loss than LFD (21). Another study compared the impact of KD and hypocaloric diet (HCD) on metabolic parameters in obese subjects. Fifty-eight subjects followed either of the two diets for 6 months. Greater differences in fat mass, weight, waist circumference, and fasting insulin were observed in the KD group as compared to the HCD group and only KD group showed significantly increased high molecular weight (HMW) adiponectin (34). The mechanism behind successful weight loss by KD is still a scientific debate. However, certain mechanisms have been hypothesized including appetite reduction due to the action of appetite-regulating hormones, fulfilling the effect of proteins, or appetite suppressing effect of ketone bodies (33,(35)(36)(37). Weight loss can also be due to the increase in lipolysis, reduction in lipogenesis, and ease in utilizing fats due to the increased metabolic efficiency as indicated by a reduction in the respiratory quotient at rest (38)(39)(40)(41)(42). In a non-randomized controlled trial, type 2 diabetes mellitus patients received either an intervention diet (KD) or served as controls. The KD group lost 10-15% of body weight, had a reduction in inflammatory markers like hsCRP, decreased WBCs, and increased TGs, HDL-c, and LDL-c (43). A recent review summarized that despite the efficacy of KD for rapid weight reduction and improved HbA1c values, KD raise LDL-c and had no superiority over other diets in terms of safety, effectivity, and sustainability (44). In a prospective study, KD promoted negative changes in lipoprotein sub-fractions. After 6 months, KD contributed to increasing the small LDL-c and decreasing the small HDL-c, thus increasing atherogenic risk in patients (45). In another study, 12 months of KD treatment was found to be associated with decreased carotid distensibility and increased LDL-c, TC:LDL-c, and LDL-c:HDL-c ratios. No significant changes were observed in hsCRP and BMI (46). Khodabakhshi et al. (47) evaluated the effect of KD on physical activity (PA), quality of life (QOL), and biomarkers in 80 metastatic breast cancer patients. In the 12week trial, subjects were randomly allocated to either control or KD group. No significant differences in PA and QOL scores between the groups were reported. However, the KD group showed decreases in ALP and lactate levels. Health Consequences Short-term minor side effects of KD are quite common, that include vomiting, nausea, gastrointestinal discomfort, fatigue, dizziness, feeling faint, decreased energy, and heartbeat alterations (48). KD initiation mostly results in hypoglycemia and lethargy (49). KD should be initiated with caution in combination with other treatments. A case report showed that the use of Valproate along with KD resulted in the development of hepatic dysfunction in a patient. The hepatotoxic effect was completely reversible as discontinuation of Valproate normalized the liver enzymes (50). Ketogenic diet may negatively impact the lipid profile. A case report showed that following strict KD for 30-40 days, resulted in a rapid increase in LDL-c and TC. Fasting lipid profile showed HDL-c of 59 mg/dL, LDL-c of 199 mg/dL, TC of 283 mg/dL, and TG of 124 mg/dL. After discontinuation of KD and the use of statins for 4 weeks, there was a significant improvement in LDLc (106 mg/dL) and TC (190 mg/dL). Furthermore, the patient maintained the optimal LDL-c levels after the discontinuation of statin therapy (51). A recent case report demonstrated KD induced severe hyperlipidemia in an overweight 41 (52). A retrospective cohort study showed that those on KD therapy had low normal bone mineral density, 8.8% of study subjects got kidney stones and 8.8% got a fracture during treatment (53). A newly recognized complication of KD is hypercalcemia. A series of case studies described the development of acute hypercalcemia about 2.1 years after initiating KD. Out of 14 patients, 13 had low levels of 1, 25-dihydroxyvitamin D, while all had low parathyroid hormone levels. Moreover, low alkaline phosphate (ALP) levels were noted in all subjects except the two oldest, while seven had impaired renal function (54). PALEOLITHIC DIET (PD) The PD also referred to as the Stone Age, caveman, or huntergatherer diet was initially introduced in 1985 by Eaton and Konner, and published by Dr. Loren Cordain in 2010 (55). It is marketed with the claims to improve health and cure diseases like obesity, cardiovascular disease, diabetes, cancer, and osteoporosis. Proponents of this dietary pattern believe that the modern diet (mainly processed foods, dairy products, grains, and legumes) is the cause of modern diseases and the obesity epidemic. Moreover, humans have evolved before agricultural development while the human diet has revolutionized more rapidly than our genetics; thus Paleolithic foods are more suited to our genetic makeup than the current modern diet (55,56). Apart from this theory, anthropological research provides evidence that Paleolithic people used to eat a varied diet comprising of plants, grains, legumes, and game meats (57,58). Cordain's PD has a basic set of rules, i.e., there is no restriction on the consumption of lean meats, fruits, and non-starchy vegetables while dairy products, legumes, cereals, and processed foods are strictly restricted ( Table 2). There is little to no focus on portions, and calories. There are three adherence levels to the PD: entry-level, maintenance level, and maximal weight loss level ( Table 3). One has a choice not to advance to the next level if satisfied with the results of this level (55). Effectiveness of Paleolithic Diet Metabolic syndrome and insulin resistance are the prime focused areas in most of the literature related to PD. It does provide benefits but only to specific groups, i.e., eliminating dairy products can help people with digestive disorders. 'Liberal consumption of fruits and vegetables can have a preventive effect for inflammatory bowel diseases (IBD). At the same time, this diet being high in meat increases the risk of IBD (59). Paleolithic diet is powerful at advancing weight reduction for the time being, even at the point when the weight reduction is unintentional (60)(61)(62). Initially, weight loss is due to the loss of water weight as this diet is low in carbohydrates. Previous studies suggest that the study participants lost 4-6% of total body weight within 10-12 weeks (63,64). Most of the studies are based on short-term interventions and there is only one study that followed the subjects for over 2 years. In a randomized trial, 70 post-menopausal obese women were divided into the ad libitum PD group or Nordic Nutrition Recommendations (NNR) diet group. After 24 months, the reductions in waist circumference, fat mass, and weight were observed in both groups irrespective of the dietary regimen followed (65). Most of the studies reported the TC reduction properties of this diet while there are mixed results for HDL-c (61,62,64,66). A study was conducted to evaluate the physiological and metabolic impacts of PD in healthy adults. After 10 days of intervention, reduction in TC, LDL-c, TG, and mean arterial pressure were observed (66). In another trial, participants were randomized to PD and reference diet groups. After 2 weeks of intervention, there were greater reductions in TC, TG, and diastolic blood pressure in the PD group (61). In another study, healthy subjects followed this dietary intervention for 10 weeks, which resulted in increased LDLc, TC, TC:HDL-c, along with a decline in HDL-c values (64). No significant changes in fasting blood glucose were seen in most studies (65,66). While, some studies were short-term, where HbA1c was not measured as per protocol (67). Modest reduction, i.e., 3-4 mmHg in systolic or diastolic blood pressure was reported in most studies (60,61,66,67). No significant change in inflammatory markers (CRP) was reported (61,67). Health Consequences The PD not only requires a big budget but is also very challenging to follow as compared to other diets (68,69). Despite weight reduction and some favorable impact on cardiometabolic profile, this diet can have long-term consequences. Some studies suggest that this diet is not nutritionally balanced as it discourages certain food groups like whole grains, legumes, and dairy products. The micronutrient deficiencies can have long-term adverse outcomes. Those who follow PD have inadequate calcium intake. A study was conducted to check the nutritional adequacy of this diet. In addition to a low intake of carbs, fats, and total calories that could have promoted weight loss, this diet provided about 50% less calcium than the daily requirement (60). Decreased HDL-c has also been observed among healthy adults and those with comorbidities. In a study comprising 28 type 2 diabetic patients, 14 followed the PD and 10 followed the American Diabetes Association (ADA) guidelines. Results showed that there was a significant reduction in HDL-c in the PD group (62). In another study, healthy subjects followed this dietary intervention for 10 weeks, which resulted in increased LDL-c, TC, TC:HDL-c, along with a decline in HDL-c values (64). More randomized trials need to be done to highlight the consequences of such diets that eliminate one or more food groups. PD is powerful at advancing weight reduction for the time being but its efficacy in cardiovascular events is not well established as limited long-term data is available. MEDITERRANEAN DIET (MD) The concept of the MD emerged in the 1950s by Dr. Ancel Keys. In one of the first research that related diet and heart health, it was revealed that CVDs associated mortality rates are different in Westerns and Europeans. Lower mortality rates were observed in Europeans, even though they typically consume a moderately high-fat diet (70). Their dietary pattern can be linked to lower mortality and incidence of CVDs (71). In 1975, Ancel Keys described this diet in his book as a complex of dietary choices followed by those living in Mediterranean regions. Whole grains, legumes, fruits, vegetables, olive oil, fish, and nuts are key components of this diet with a moderate allowance of alcohol, dairy products, and meat [Keys,(72)]. Traditionally, this diet derives its most calories from fish and plant-based foods. Fats account for 30% of calories which are mostly polyunsaturated fatty acid (PUFA) and monounsaturated fatty acids (MUFA), while carbohydrates provide 50-55% of calories from low glycemic index carbohydrates and proteins provide 15-20% of calories (73). Effectiveness of Mediterranean Diet Mediterranean diet is the most extensively studied diet to date. In a previous review, it has been summarized that MD is nutritionally adequate for the general public and may have the potential of preventing micronutrient deficiencies (74). Research shows that it has preventive and therapeutic potential for many chronic diseases like non-alcoholic fatty live disease (NAFLD), CVDs, metabolic syndrome, and certain cancers like colorectal and breast cancer (75). In a 2-year trial, weight loss by LFD, AD, and MD was compared and results showed that the AD group had the highest mean weight loss, i.e., -4.7 ± 6.5 kg, while the MD group stood second with a mean weight loss of -4.4 ± 6 kg and LFD group lost -2.9 ± 4.2 kg. Following changes were recorded in the MD group: increased molecular adiponectin reduced serum leptin, and CRP levels (76). In another controlled trial, 259 subjects were randomly allocated to American Diabetic Association (ADA) diet, traditional MD, or low carbohydrate Mediterranean diet (LCM) group. After 12 months, the LCM group had the highest weight reduction, increased HDL-c, improved LDL-c, TG and HbA1c (77). Another study described the effectiveness of MD in the primary prevention of CVDs [Estruch et al. (78)]. A study investigated the protective effect of MD against cancer and found that greater compliance with MD patterns reduces the risk of non-tobacco linked cancers in both men and women (79). Most MD studies are short-duration studies, only a few studies focused on the long-term impacts of following MD. In a study, non-diabetic elderly subjects (n = 3,541) at higher risk of CVD were randomized to three intervention groups: control diet, MD with nuts or MD with extra virgin olive oil. New cases of diabetes were recorded after regular intervals (median followup duration = 4.1 years) and results showed that MD with extra virgin olive oil was associated with a reduced risk of diabetes (80). In another 5 years clinical trial, subjects (n = 7,447) who were type 2 diabetics or those with risk factors of CVDs, were randomized to three intervention groups: control diet, MD with nuts, or MD with extra virgin olive oil. After the specified period, there was no significant weight reduction in all the groups, while the MD group had a significant reduction in central obesity [Estruch et al. (81)]. Health Consequences No evidence of adverse effects associated with MD is available in the literature. Rather, MD has preventive and therapeutic potential for many chronic diseases. It is highly suitable for the general public for the prevention of micronutrient deficiencies and specifically for those patients who are more health-conscious than just weight loss oriented. VEGETARIAN DIET (VD) The VD is a dietary pattern characterized by no consumption of meat and meat products, seafood, poultry, and sometimes other animal products like eggs, animal milk, and honey. Some studies have linked meat intake with an increased risk of chronic diseases, while others indicate a positive association between low meat intake and life expectancy (82,83). VD are of four main types: (i) a lacto-ovo-vegetarian does not consume any meat product but consumes eggs and dairy products (ii) a lactovegetarian does take dairy products but does not consume eggs and meat products (iii) an ovo-vegetarian does not eat meat products and dairy products and are free to consume eggs (iv) a vegan does not consume any animal products, including meat, eggs, dairy products, and honey (84). Vegan diets include different subtypes: raw vegan, vegan (general), and whole-food vegan (Figure 2). Each subtype has its own set of foods allowed and restricted with one thing in common, i.e., meat products restriction. This dietary pattern is gaining much popularity in the general population, especially in the Western world (85). There are various reasons for adopting this dietary profile, including religious beliefs, ethical motivation, cultural aspects, and health considerations (85,86). Effectiveness of Vegetarian Diet Several epidemiological studies reported a lower cardiometabolic risk in the vegan population. A study concluded that nonvegetarians have a higher type 2 diabetes prevalence (7.6%) than vegetarians (2.9%). While, the prevalence rate also varies with the type of VD, i.e., 3.2% in lacto-ovo vegetarians, 4.8% in pescovegetarians and 6.1% in semi-vegetarians. This can be explained by the low-glycemic-response associated with these diets as vegetarian diets typically include foods that have a low glycemic index such as beans, legumes, nuts, some fruits and vegetables (87). Glycemic control via a VD is quite controversial, as these are high carbohydrate diets. Some studies have shown that vegetarians also have increased life expectancy (82). Generally, vegetarians are more health-conscious and have lower BMI than the general population (88). The Seventh Day Adventist study showed a lower mean BMI, i.e., 23.6 kg/m 2 in the vegan population (89). In a 5-year prospective study, 22,000 subjects having different dietary patterns were checked for their weight gain during this period. Vegans had the lowest weight gain as compared to meat-eaters and fish eaters (90). Red meat and poultry intake were most strongly linked to increased risk of esophageal adenocarcinoma and gastric cardia or non-cardia adenocarcinoma, respectively (91). On the other hand, lower rates of heart diseases and cancers have been observed in vegetarians in comparison with those following other dietary patterns (92,93). A better cardiometabolic risk profile is generally present in vegetarians, i.e., lower BMI, TC, and LDL-c [Chen et al. (94); De Biase et al. (95)]. A crosssectional study investigated the lipid profile of fish-eaters, meateaters, and vegetarians. Not only the vegans have a lower BMI but also favorable serum lipid levels: lower LDL-c, TC, and apolipoproteins (96). In a study, out of 26,346 participants, 1,079 cases of prostate cancer were identified and results showed the protective effect of vegan diets against prostate cancer in the white population (97). This protective effect against prostate cancer may be due to the higher fiber intake. Some other studies are either short-term or have a very small sample size, showing mixed findings related to colorectal cancer and breast cancer. In a study, 2,304 patients from 10 European countries were assessed for their dietary intake to find the impact of diet on the risk of cancers. Not poultry but red meat intake was found to be associated with an increased risk of esophageal cancer and upper aerodigestive tract (UADT) cancer. Furthermore, vegetable and fruit intake are significantly associated with a reduced risk of UADT cancer (98). Butler et al. (99) demonstrated that the higher the intake of vegetable-fruit -soy dietary pattern, the lower the breast cancer hazard ratio among postmenopausal women. Another study showed a significant association between consumption of vegetables and risk of esophageal adenocarcinoma. Elimination of potentially harmful dietary components like animal protein, saturated fats, and cholesterol can be the reason for these benefits. These benefits can also be due to the addition of dietary fiber, phytochemicals, and antioxidants rich in beneficial dietary components like whole grains, legumes, nuts, fruits, and vegetables (91). Health Consequences This diet is associated with fluctuations in micronutrients intake because of the day-to-day variation in the menu. Depending upon the type of VD, vegetarians are potentially at increased risk of micronutrient deficiencies such as calcium, zinc, iron, vitamin E, vitamin B12, essential fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) (100). A study reported that half of the vegan participants were micronutrient deficient as compared to omnivores (101). Vegetarians have lower serum vitamin B12 levels as plant sources are deficient in this vitamin (101,102). As VD is generally low in calcium due to the suboptimal intake of dairy, vegetarians are at greater risk of bone fractures due to the lower bone mineral density (103). Thus supplementation of certain vitamins like vitamin B12 and vitamin D is needed to avoid these deficiencies among vegetarians (84). Vitamin B12 supplements are especially important for vegan pregnant and lactating mothers as a preventive therapy for deficiency in their babies (104). VD can be nutritionally adequate, so it may be helpful in chronic disease prevention and treatment. Benefits and harms depend upon the dietary choices so the individualized plan fulfilling the micronutrient requirements must be carefully developed by a professional. INTERMITTENT FASTING (IF) The IF is gaining much popularity and is widely adopted as an effective weight loss intervention. Contrary to the conventional weight loss programs that are based on calorie restriction, IF is more about scheduled eating. Some of the key features of IF are abstinence from food for a certain period, followed by a period of normal eating. There are various versions of IF but the most popular of these are alternate day fasting (ADF), 5:2 diet or periodic fasting (PF), and time-restricted feeding (TRF). The frequency and duration of fast cycles may differ among all types ( Table 4). Effectiveness of Intermittent Fasting The alternate-day fasting (ADF) approach has been tested for its metabolic effects. In a study, healthy young men (n = 8) were subjected to ADF for 20 h/day for 15 days. After the specified study period, weight remained unchanged (86.4 ± 2.3 kg) while the increase in glucose uptake, i.e., 7.3 ± 0.3 mg/kg/min that was previously 6.3 ± 0.6 mg/kg/min, and prominent increase in lipolysis of adipose tissues were observed (106). Another study showed that when non-obese subjects (8 women and 8 men) fasted for 22 days on alternate days, they lost 4 ± 1% of their initial fat mass and 2.5 ± 0.5% of their initial body weight. However, a decrease in fasting insulin and non-significant change in glucose and ghrelin were also reported (107). A randomized crossover trial was conducted to evaluate the fasting-induced acute changes in biomarkers. Healthy volunteers (n = 30) were randomized into two groups: (i) normal eating for 28 ± 4 h then water-only fasting for 28 ± 4 h (ii) 28 ± 4 h of water-only fasting then 28 ± 4 h of normal eating. Blood samples were drawn and analyzed at baseline, day 1 and day 2. Laboratory findings suggested that the fasting intervention acutely increased hemoglobin, hematocrit, red blood cell count, human growth hormone, and HDL-c; on the other hand, decreased body weight, bicarbonates, and TGs, as compared to the normal eating day. Moreover, cholesterol and human growth hormone returned to baseline after 48 h (108). Night-time fasting (NTF) has been linked to lower energy intake, consequently resulting in weight loss. In a study, twentynine healthy young men were subjected to 9 h of NTF for 2 weeks, then 1 week washout period followed by 2 weeks of controlled conditions. Results showed that the participants had less total calorie intake in the NTF phase as compared to controlled conditions. Significant differences in weight change were also reported, i.e., -0.4 kg for NTF and + 0.6 kg for control (109). In a randomized trial of 3 months, young overweight premenopausal women (n = 107) were randomly assigned to two groups: two consecutive days of fasting (25% energy restriction)/week or fasting for all days of the week. Both interventions were found to be equally good at a weight and showed improvement in risk markers of CVDs, cancer, and diabetes for example reduction in leptin, leptin to adiponectin ratio, inflammatory markers, fasting insulin, insulin resistance, blood pressure, and lipids (110). Fasting also impacts the appetite by influencing the appetiteregulating hormones (110,111). A previous systematic review summarized that IF may have the potential to provide metabolic benefits in terms of improving insulin resistance, thus providing better glycemic control as IF showed a significant decline in fasting glucose levels as compared to controls. Moreover, IF was associated with a decline in BMI, fat mass, and leptin while an increase in adiponectin (112). Headland et al. (113) evaluated the effectiveness of intermittent energy restriction (IER) in improving weight and biological markers in long-term studies. Irrespective of duration, IER was associated with weight loss. Toxins removal from fat stores Improved memory and intelligence quotient Better blood pressure and cholesterol levels (124) However, IER was not found to be superior to continuous energy restriction (CER) in terms of weight loss, blood lipids, glucose, and insulin levels. Health Consequences Some short-term studies highlighted the potential harms posed by IF among normal-weight subjects. IF induces lipolysis, resulting in increased free fatty acids (FFA). So whether it be ADF, periodic fasting, or else, a prolonged course of fasting can lead to large fluctuations in FFA in normal-weight individuals. A study showed that these fluctuations were three times greater than those typically seen after an overnight fast. Furthermore, it induced reductions in insulin sensitivity and acute glucose-simulated insulin response (114). Despite the effectiveness of IF in weight loss as indicated by several studies, the current evidence is nonconclusive. The prime focus of available literature is weight loss but little is known about its sustainability and long-term health effects. More long-term trials should be conducted to draw a clear conclusion. DETOX DIETS (DD) The popularity of detoxification dates back to Greek, Roman, Indian, and Native American cultures. Many effective approaches that are still used for the removal of toxins include fasting, saunas, herbs, rebounding, dry brush, water, rest, exercise, and meditation (115). However, detoxification or DD are interventional diets specifically designed for toxins elimination, health promotion, and weight management. These short-term dietary interventions involve multiple approaches, including total calorie restriction, dietary modification, or juice fasts, and often involve the use of additional minerals, vitamins, diuretics, laxatives, or cleansing foods. Some commercial DDs have been listed in Table 5. These are most commonly prescribed by naturopathic doctors to prevent or treat a number of conditions like gastrointestinal disorders, inflammation, autoimmune disorders, chronic fatigue syndrome, fibromyalgia, and weight loss (116). The DDs have not been extensively investigated; however, the handful of available studies have methodological limitations like sampling bias, small sample sizes, relying on self-reporting, and absence of control groups. Despite the emerging popularity, these diets fail to identify the mechanisms of eliminating toxins or even the specific toxins removed by a particular diet. Detox approaches defy the general principles of human physiology as the liver and kidneys are quite efficient in removing both exogenous and endogenous toxins from our body, along with extra-renal excretion of toxins in sebum and sweat (125). Effectiveness of Detox Diets Currently, there is no clinical evidence confirming or negating the effectiveness of commercially available detox regimes for losing weight. Because of its emerging popularity, this area needs attention. So, in the absence of scientific evidence, results can be extrapolated from other closely related studies. It is known that the success rate of dieting, in general, is only 20% (126). This may be possible because humans and animals have natural mechanisms to counter the weight loss as starvation can have negative health consequences like reduced fertility and even death. Calorie restriction alters the neuropeptides' expression in the hypothalamus; which reduces metabolic rate and stimulates appetite, resulting in a weight loss plateau (127). Furthermore, studies in mice have shown binge eating followed by a period of energy restriction, though this phenomenon is not established in humans yet (128). A study conducted by Mazurak et al. (129) showed that fasting raised cortisol levels in young healthy women. Another study reported an increase in stress hormone levels in females due to the restricted intake of 1,200 kcal/day (130). There is considerable evidence that stress stimulates appetite, thus promoting weight gain via elevations of cortisol (131). Many of the DD are liquid-based, low-calorie, and nutrientpoor. For example, a part of BluePrint Cleanse, Excavation Cleanse, provides only 19 g protein and 860 kcal/day which is far below the actual requirement. Food and Agriculture Organization (FAO) recommends a minimum of 0.83 g/kg body weight of high-quality protein and 1,680 kcal/day for an adult (132, 133). Based on the previous work, DD may induce stress, raise cortisol levels and increase appetite, resulting in difficulty in losing weight, followed by binge eating and weight gain (128)(129)(130). It is quite alarming that the components of detox products may not be according to the labels as there is no regulatory authority that approves such products. A case was reported in Spain that a 50 year old man with no history of relevant medical illness, presented with diffuse abdominal pain, lethargy, profuse diarrhea, and vomiting after ingesting Epsom salt during a liver cleansing diet. That person died within 72 h from the onset of symptoms. Forensic and clinical investigations concluded that instead of magnesium sulfate heptahydrate, the supplier had mistakenly added hydrated manganese sulfate resulted in manganese intoxication (134). Energy-restricted DDs are capable of short-term weight loss. But still, there is a high likelihood of health risks from detox products because of their nutritional inadequacy. As no convincing evidence exists in this domain so such diets and products need to be discouraged by health professionals and must be subjected to regulatory review and monitoring. CONCLUSION Fad diets facilitate fast and easy weight loss, improve appearance, and do not require a longer time to achieve the results. These diets are effective in improving health to some extent. However, compliance is always a significant concern because of the unrealistic combinations and nutritional inadequacy due to the complete elimination of one or more essential food groups. Despite the rapid weight reduction, there are some concerns for those with comorbidities. All these diets have not been extensively studied while those studies that have been mentioned in the literature have high dropout rates and are sometimes non-conclusive. More randomized controlled trials of prolonged duration need to be done to establish the safety of FDs for the public and to make people aware of the possible consequences of long-term adherence to such dietary patterns. AUTHOR CONTRIBUTIONS AT and AR: conceptualization. AT: writing -original draft preparation. AT, AN, RR, AR, RA, CS, and CM: writing -review and editing. AR and RA: supervision. All authors have read and agreed to the published version of the manuscript.	2022-07-05T15:57:39.418Z	2022-07-05T00:00:00.000	{ "year": 2022, "sha1": "49350c75c7fa74e5369b6e107dae352e4af69898", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Frontier", "pdf_hash": "49350c75c7fa74e5369b6e107dae352e4af69898", "s2fieldsofstudy": [ "Medicine", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
5534363	pes2o/s2orc	v3-fos-license	Volunteer Participation in the Health eHeart Study: A Comparison with the US Population Direct volunteer “eCohort” recruitment can be an efficient way of recruiting large numbers of participants, but there is potential for volunteer bias. We compared self-selected participants in the Health eHeart Study to participants in the National Health And Nutrition Examination Survey (NHANES) 2013–14, a cross-sectional survey of the US population. Compared with the US population (represented by 5,769 NHANES participants), the 12,280 Health eHeart participants with complete survey data were more likely to be female (adjusted odds ratio (ORadj) = 3.1; 95% confidence interval (CI) 2.9–3.5); less likely to be Black, Hispanic, or Asian versus White/non-Hispanic (ORadj’s = 0.4–0.6, p < 0.01); more likely to be college-educated (ORadj = 15.8 (13–19) versus ≤high school); more likely to have cardiovascular diseases and risk factors (ORadj’s = 1.1–2.8, p < 0.05) except diabetes (ORadj = 0.8 (0.7–0.9); more likely to be in excellent general health (ORadj = 0.6 (0.5–0.8) for “Good” versus “Excellent”); and less likely to be current smokers (ORadj = 0.3 (0.3–0.4)). While most self-selection patterns held for Health eHeart users of Bluetooth blood pressure cuff technology, there were some striking differences; for example, the gender ratio was reversed (ORadj = 0.6 (0.4–0.7) for female gender). Volunteer participation in this cardiovascular health-focused eCohort was not uniform among US adults nor for different components of the study. Methods Health eHeart Study Sample. The Health eHeart Study is a cardiovascular focused eCohort, with enrollment, consent and participant occurring entirely using the internet. We analyzed cross-sectional baseline examination data and follow-up data from Bluetooth-enabled blood pressure measurement devices obtained between March 8, 2013 (enrollment initiation) and March 24, 2016 from consecutive participants enrolled in the Health eHeart Study. Participation in the Health eHeart Study is open to any person (world-wide) with a self-reported date of birth indicating age ≥18 years and an email address. Recruitment into the study occurred via several news media stories, social media and word-of-mouth in addition to being actively sought via email campaigns sent to persons associated with the American Heart Association (primarily via emails sent to participants in their Go Red for Women campaign 5 ), to adult patients at the University of California, San Francisco (UCSF) Medical Center (primarily via unsolicited email invitation), through various other specific referral sources (we track referral source by provided a special URL to referring partners), and from unspecified sources (through our general URL). After online registration (name, date of birth, email and password) and consent, participants were prompted to complete a series of online questionnaires pertaining to basic socio-demographics, family history, medical history, activity and well-being, habits and lifestyle, mental health, food and nutrition, and use of internet or social media. Participants were also invited to "connect" devices and apps (that they already own) from Fitbit, iHealth, Withings, Qardio, Alivecor, Azumio, Ginger.io and Google Fit and donate their data to the study. We limited our primary analysis to participants age ≥20 years (for comparability with NHANES) and with complete information and without "unknown" or "refused" responses on all baseline core survey instruments and survey items. For our secondary analysis, we additionally limited the sample to such participants who also contributed at least one blood pressure measurement via Bluetooth-enabled blood pressure measurement devices (iHealth, Withings and Qardio were all supported). NHANES Sample. We used NHANES 2013-2014 to represent the US population and compare against participants in the Health eHeart Study. NHANES is a program of the National Center for Health Statistics (NCHS) that aims to investigate the health and nutritional status of the US population. Since 1999, the survey has been released every 2 years in a continuous fashion. These cross-sectional data are representative of the non-institutionalized US population. Every year, approximately 5,000 individuals of all ages are interviewed in their homes and complete the health examination component of the survey. NHANES follows a complex, multistage sampling procedure where the primary sampling units are counties or small groups of contiguous counties, within which city blocks are selected. Within these blocks, households are then randomly selected, and then individuals are drawn at random 6 . All NHANES protocols were approved by the NCHS Research Ethics Review Board 7 . In 2013-2014, 14,332 persons were selected for NHANES from 30 different study locations. Of those selected, 10,175 completed the interview. NHANES provides study weights that account for both non-response and deliberate oversampling of particular segments of the population. Because various components of NHANES are only delivered to adults ≥20 years, we limited our analyses to these participants, leading to a sample size of 5,769. In order to maintain strict representativeness of the NHANES study sample ≥20 years and allow for direct comparisons with Health eHeart, we performed multiple imputation using chained equations to estimate missing and "unknown"/non-response values of all variables of interest (n = 13 variables) for all participants (n = 1,162 participants with at least one missing value) 8,9 . We used 10-fold multiple imputation to generate imputed datasets, each with complete data on all 5,769 NHANES participants included in our sample. This 10-fold imputed dataset was used for all subsequent analyses. Informed consent was obtained from all participants in both Health eHeart and NHANES. Our analysis of the Health eHeart Study data is covered by the UCSF Institutional Review Board (IRB); our analysis of the de-identified NHANES data is exempt from IRB Review. Methods were performed in accordance with the relevant guidelines and regulations. Statistical Method. We first used descriptive statistics to compare the demographic characteristics, medical conditions, and lifestyle factors of the Health eHeart sample by recruitment source, using ANOVA and chi-square tests for between-source differences. Then, to identify factors independently associated with participation in Health eHeart, we used a case-control approach, using pooled data for the combined NHANES and Health eHeart samples to estimate logistic regression models for the "outcome" of inclusion in the Health eHeart Study sample. We first fit single-predictor models for age, sex, race, income, marriage status, educational level, hypertension, hyperlipidemia, diabetes, stroke, coronary heart disease, heart failure, heart attack, general health, smoking and sleeping duration, and then fit a final multivariable model for Health eHeart participation that included this entire set of predictors. Results are summarized as odd ratios (ORs) and 95% confidence intervals (CIs). We accounted for the complex stratified survey design of NHANES using the sampling weights, pseudo-strata, and primary sampling unit (PSU) variables provided by NHANES, with weights normalized to sum to the NHANES sample size. In the pooled analyses, Health eHeart participants were each given unit weight, and randomly assigned to two PSUs with a distinct pseudo-stratum. Multiple imputation of the NHANES data was implemented using the mi package in Stata Version 14.0, and the case-control models were estimated using the Stata svy package for complex survey data, which accommodates multiply-imputed data. Two-sided P values less than 0.05 were considered to be statistically significant. Results At the time of our data lock, 42,828 participants had registered for the Health eHeart Study by providing their name, email and date of birth. Of those, 33,236 (78% of registered participants) signed the online consent, 28,420 completed at least one survey, (86% of consented participants), and 12,280 were participants age ≥20 years with complete core baseline survey data and without "unknown" or "refused" responses to any survey item (Fig. 1). These participants constitute our primary analysis sample. Of these, 251 contributed at least one blood pressure measurement via Bluetooth-enabled blood pressure measurement device; these participants constitute our secondary analysis sample (Fig. 1). As described in our Methods, all NHANES participants age ≥20 years were included after multiple imputation successfully imputed missing/unknown/refused items for the 1,162 participants missing at least one required data element. Baseline characteristics of Health eHeart Study participants differed by referral source (Table 1). For example, only 3% of participants referred by American Heart Association sources were male (consistent with the primary focus on the Go Red for Women program), compared with 37%-44% from other sources (p < 0.001). We also detected differences by recruitment source in age (more elderly participants from UCSF), race/ethnicity (more Black, non-Hispanic participants from AHA), income and education (higher in both from UCSF), general health (highest among participants from unspecified referral source), and sleep duration (lowest duration from AHA referrals, Table 1, all p-values < 0.001). Compared with all adults in the US, as represented by NHANES participants (applying sample weights), Health eHeart Study participants were more likely to be middle-aged: more likely to be female; less likely to be Black, Hispanic, or Asian versus White/non-Hispanic; more likely to be highly educated; more likely to have cardiovascular disease and risk factors but less likely to have diabetes; more likely to be in excellent general health; less likely to be current smokers; and more likely to report low sleep duration (Table 2). Associations with higher income and marital status did not persist in adjusted models. The higher prevalence of female participants Tables 1 and 2. # Health eHeart Study sample subset used in Table 3. in Health eHeart persisted even after excluding participants referred from the Go Red for Women program (OR adj = 1.6; 95% CI: 1.5-1.7). When we limited both the Health eHeart Study and NHANES population to participants with coronary heart disease (Health eHeart Study n = 1297; NHANES n = 293), characteristics of the sample were different (e.g., higher prevalence of cardiovascular risk factors), but predictors of participation in the Health eHeart Study were quite similar (Supplemental Table 1). Only a small subset of Health eHeart Study participants (n = 251, 2%) used a Bluetooth-enabled blood pressure measurement device, connected their device account to their Health eHeart Study account, and donated at least one blood pressure measurement to the study (median number of measurements per participant = 30; interquartile range 9-82). These highly self-selected participants showed mostly similar patterns of characteristics when compared with NHANES as the full Health eHeart sample, with some striking contrasts ( Table 3). Instead of a large female preponderance in the full Health eHeart sample (73%, Table 2), Health eHeart participants contributing device-measured blood pressure values were less likely to be female than the US population (35%, Table 3). Persons with hypertension and coronary heart disease were even more heavily over-represented in this subset. Also, in this subsample in which moderately expensive purchases were required (blood pressure cuff and smartphone), higher income persisted as a strong predictor even after adjustment for education and other factors. Discussion The Health eHeart Study used efficient electronic methods for recruitment and took advantage of partner organizations willing to refer patients to our study website. This resulted in extremely efficient recruitment into the study. The sample of recruited individuals, however, differs from the US population in a variety of ways. Not only does the study over-represent persons with cardiovascular diseases and risk factors (as expected based on the study focus), but it also appears to over-represent females and non-Hispanic Whites, higher educational level, persons with more prevalent medical conditions but better self-reported general health, and fewer current smokers than would be expected if participation were proportional from all segments of the US population. Patterns were different (e.g., reversal of the female predominance) in the highly selected subset of the Health eHeart Study who contributed blood pressure measurements from a Bluetooth-enabled device. Internet-and technology-enabled epidemiology can have major advantages in terms of efficiency. Consistent with the Health eHeart Study recruitment experience, one Danish internet-based study estimated more than 50% savings in their recruitment compared with a conventional approach ($160 vs. $322 per subject) 10 , and an internet-based clinical trial similarly reported that their web-based methods cost about half that of a hospital based approach 11 . Web-based questionnaires generally reduce cost substantially 12 , as do studies that invite participation by e-mail 13 . Aside from cost, web-based surveys can be more efficient in terms of response speed from respondents 14 , easier to adjust and modify by the research team 15 , quicker and less error-prone to process since data are entered electronically and coded automatically 16 , and easier to complete for disabled participants 17 . Our results, in terms of which characteristics predicted participation, were similar in some ways, but different in others when compared with prior studies. As with Health eHeart, women and those with higher socioeconomic status appear to be consistently more likely to participate in epidemiologic studies 18 , especially in eCohorts 14,19,20 . For example, the NutriNet-Santé study in France found a much higher percentage of women compared with the corresponding national figures (78.0% vs 52.4%); and both the NutriNet-Santé study and the Australian Longitudinal Study on Women's Health found higher participation rates in persons with higher educational levels. In contrast to the NutriNet-Santé study, however, which found higher proportions of married or partnered participants compared to their national data (70.8% vs. 62.0%), the unadjusted association we found in Health eHeart (69% married vs. 62% in NHANES) was not significant after adjusting for other selection factors. Also in contrast with Health eHeart, the Australian Longitudinal Study reported a higher percentage of study participants who rated their health in the online survey as fair or poor, and a higher percentage of study participants who were current smokers compared to their Census data. Their study, however, was limited to a very narrow demographic band (women age 18-23) so may not be comparable. We did not find another study describing self-selected participation in a study requiring use of sensor technology such as our analysis of participants in the Bluetooth-connected blood pressure cuff subsample. Several factors likely contribute to the differences we observed between the Health eHeart Study and NHANES. First of all, NHANES makes special efforts to recruit underrepresented minorities. In fact, such individuals are oversampled in NHANES (though sample weights correct this factor so results are generalizable to the US population). No such efforts are made in the Health eHeart Study. Second, the Health eHeart Study's focus naturally attracts participants at risk for heart disease, so the overrepresentation of people with cardiovascular diseases, such as coronary heart disease, stroke and heart failure, is to be expected. However, when we subset both samples to only participants with coronary heart disease, general selection patterns (e.g., for sex, race/ethnicity, education level and smoking) were consistent with those we found in the full Health eHeart sample. Clearly, the "digital divide" may explain differences in participation by education, and particularly also by income for the subset of Health eHeart using a Bluetooth-enabled blood pressure measurement device. As the digital divide diminishes 21 and technology diffuses through all segments of society, this participation selection factor may ameliorate to some degree. The Health eHeart Study is large and nationally-scoped and includes participants who complete extensive online surveys and device-associated data collection; and the NHANES study provides a near-ideal way to compare to the US population. However, our analysis has some limitations. Unlike NHANES, the Health eHeart Study does not limit participation to US residents. In contrast to Health eHeart, bias from self-selected non-participation in NHANES is minimized by post-stratification re-weighting based on the known demographic characteristics of the target sample; however, missing values arising from so-called item non-response in NHANES may not be missing at random (even conditional on other factors included in our imputation model), such that multiple imputation may be flawed. Finally, while both Health eHeart and NHANES collect many additional measurements, we were only able to evaluate measurements that were identically collected in both studies (or nearly so), preventing us from assessing the representativeness of Health eHeart on other potentially important dimensions. Our results have some clear implications. First, given that Health eHeart recruitment is ongoing, this analysis provides guidance for how the study team can refocus recruitment efforts to target thus-far under-represented subgroups of the US population. It also represents a roadmap for prospective targeting efforts that can be used by the Precision Medicine Initiative as it begins internet-based direct volunteer recruitment later this year. While some self-selection characteristics may be expected from prior work on participation in research (e.g., under-representation of racial/ethnic minorities 22 ), our findings regarding the technology product-dependent subsample (e.g., reversal of the sex ratio) are more surprising and potentially important to account for. The other clear implication relates to inference: it is clear that simple descriptive analyses of the self-selected Health eHeart Study (e.g., % technology use) will often not yield results that are representative of the US population, either on average or within strata defined by other covariates (e.g., gender). However, it is important to note that estimates of average adjusted associations are likely robust to over-or under-(mis-) sampling even on the variables included in the association, provided that the mis-sampling occurs independently for each variable, and that the association is not modified by factors associated with self-selection. For example, we might obtain valid adjusted estimates of the marginal association of technology use with gender, despite oversampling of technology users and of women in the Health eHeart Study, provided that the oversampling on each factor is independent, and that the effect of technology use on gender does not vary, for example, by education. Note, even in the presence of effect modification, estimates within strata of the effect modifier should remain valid (e.g., there is internal validity). Furthermore, the effects of these various aspects of selection bias may potentially be minimized by re-weighting the Health eHeart sample (similar to the post-stratification weighting performed by NHANES), based on an extension of the multivariable logistic model developed here, with the result that all included covariates have weighted distributions very close to those in NHANES. In conclusion, the Health eHeart Study demonstrates efficient internet-based recruitment, and allows remote data collection from online surveys and sensor/device technology. While it also clearly demonstrates that participants who volunteer for the study are different on average than the US population, this does not rule out its potential for providing valid estimates of adjusted associations. Whether this limitation can be overcome by future internet-based studies such as the planned Precision Medicine Initiative Cohort remains to be seen and will likely require more deliberate sampling, more costly targeted recruitment efforts, and application of post-recruitment standardization methods that correct for unrepresentative volunteer participation.	2018-04-03T02:33:55.362Z	2017-05-16T00:00:00.000	{ "year": 2017, "sha1": "472313095f5b0129fee15e3b77d041331d68a747", "oa_license": "CCBY", "oa_url": "https://www.nature.com/articles/s41598-017-02232-y.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "12402443f815783348daa57b77fed78646467294", "s2fieldsofstudy": [ "Medicine", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
14807000	pes2o/s2orc	v3-fos-license	Main Results Of Nuclear Danger Examination Of The "Shelter" Object The Shelter of the 4 th Unit of the Chernobyl Nuclear Power Plant object belongs to the category of nuclear dangerous objects. The main source of danger in the Shelter object (SO) is considerable concentration of fuel-containing materials (FCM) which appeared as a result of the nuclear accident of 26 April, 1986. Nuclear danger of this concentration is caused by the existence of a potential possibility (risk) of the beginning of the self-supporting chain reaction of fission (SCR). It is known that the reaction of the 235 U and 239 Pu fission has no lower limit and occurs during an interaction with any energy neutrons. There are always free neutrons in the fuel-containing medium. Their natural sources are cosmic radiation, spontaneous fission, as well as photo-neutron and (a, n) reactions. In the reaction of forced neutron-induced fission, instead of one neutron used for the fission, 2-3 new neutrons are released (the average output for 235 U-~2.43 neutron/fis., for 239 Pu-~2.89 neutron/fis.). It entails multiplication of the initial quantity of neutrons which can continue the chain of fission. Up to now the process of forced fission has been of a damping character, the multiplication property of the system can be characterized by the multiplication coefficient M: M=S/S source , where: S source (neutron/s) is a power of an independent neutron source; S (neutron/s) is a stabilized neutron power of the multiplication system, taking into consideration an additional inflow of neutrons due to the reaction of forced fission of nuclear fuel. In contrast to spontaneous nuclear transformations, the intensity of which is stable and determined by the only law of radioactive decay, intensity of the reaction of forced neutron-induced fission changes, depending on the multiplication properties of a fuel-containing composition. The index of the multiplication properties of the medium is the multiplication coefficient K ¥ characterizing the balance of neutrons in the reaction of forced fission at unlimited sizes of systems. If K ¥ ³1-the criticality becomes possible, i.e. reaching the level of neutrons reproducibility in the system by means of the reaction itself, sufficient for continuing forced fission of nuclear fuel without an inflow of neutrons from any other sources. In a critical state the multiplication coefficient M=¥ and neutron power may increase unlimitedly by means of the self-supporting chain reaction of fission. Pu -~2.89 neutron/fis.).It entails multiplication of the initial quantity of neutrons which can continue the chain of fission.Up to now the process of forced fission has been of a damping character, the multiplication property of the system can be characterized by the multiplication coefficient M: M=S/S source , where: S source (neutron/s) is a power of an independent neutron source; S (neutron/s) is a stabilized neutron power of the multiplication system, taking into consideration an additional inflow of neutrons due to the reaction of forced fission of nuclear fuel. In contrast to spontaneous nuclear transformations, the intensity of which is stable and determined by the only law of radioactive decay, intensity of the reaction of forced neutron-induced fission changes, depending on the multiplication properties of a fuel-containing composition. The index of the multiplication properties of the medium is the multiplication coefficient K ¥ characterizing the balance of neutrons in the reaction of forced fission at unlimited sizes of systems.If K ¥ ³1 -the criticality becomes possible, i.e. reaching the level of neutrons reproducibility in the system by means of the reaction itself, sufficient for continuing forced fission of nuclear fuel without an inflow of neutrons from any other sources. In a critical state the multiplication coefficient M=¥ and neutron power may increase unlimitedly by means of the self-supporting chain reaction of fission. V.I.Kupnyi, E.L.Belousov, A.S.Tovstogan Subcriticality reserve is generally evaluated by the value that is inverse to the multiplication coefficient -subcriticality level dK ef =1/M. Subcriticality dK ef =1-K ef (K ef <1), where K ef -effective coefficient of neutron multiplication in the system of limited sizes. K ef is used to characterize both subcritical (K ef <1), and supercritical systems (K ef >1).If K ef =1, then the system is in a critical state, i.e. the process of forced neutron-induced fission becomes undamping.The level of deviation of the multiplication system state from the critical state is characterized by the reactivity value r: At the positive reactivity r > 0,5% the period of the neutron power doubling is reduced to the fractions of a second which makes the chain reaction of fission As for the FCM within the range K ¥ >1 (picture 1), the calculation of the critical masses parameters were performed.After the end of the active phase of the accident, a set of diagnostic measurements has been pointing out at subcriticality of all the fuel-containing materials in the Shelter object. Quantitative indices of the multiplication coefficient obtained experimentally on the basis of special passive neutron methods, for all the FCM are less than 0.4, and on the basis of active methods -less than the sensitivity limit (0.7). The calculations confirmed that all the FCM are in a deep subcritical state. Various natural calamity events which cause the shifting of construction fragments of the destroyed unit (without water flooding), do not entail the appearance of critical systems. Water can be the main factor causing the reduction of subcriticality of the FCM in the Shelter in the course of time.2) Examination of the fuel-containing lava samples from the premises 305/2 (under the control room), carried out in 1992 -1993, showed that some samples contained AZ (active zone) fragments in a non-melted form. Besides, visual observations showed zone fragments which were directly adjoining the lava.Thus, for calculations and assessment of the nuclear safety it is necessary to take into consideration the new composition Lava + AZF + water which is more dangerous than the Lava + water composition. Preliminary calculations of critical parameters for such mixtures were performed in the Kurchatov Institute [2].Water content: chosen under condition K ¥ -max. The following data were obtained for the sphere geometry (table 3): At fuel enrichment 2% for Uranium-235 (fuel with low burning-out) the radius of critical spheres reduces two times. Assessment calculations for analogous compositions were also performed in SSC RF PEI [6].Previously verified complex REDUT was used as a software.When there is no water, the system remains deeply subcritical under any conditions.But, as it has been pointed out, in practice there are no working barriers for water inflow. Taking the following facts into consideration: • The considerable lowering of the barriers which used to present the selfsupporting chain reaction; • Detection of new, potentially dangerous compositions of FCM; • Classification (based on a conservative approach) of the Shelter premises according to the level of nuclear danger and a number of other reasonswe can arrive at the conclusion that: at present, in some premises of the Shelter object and with respect to those FCM concentrations about which there is no sufficient information, under certain initial conditions there exists a possibility for the appearance of FCM criticality. Such premises include the Central Room, where dozens of tons of nuclear fuel may be under the layer of thrown-off materials, the reactor pit, the premises under the control room where the most part of the fuel lava is located. It should be noted that until recently some abnormal phenomena were registered related to the increase in the density of a neutron flux which may have been caused by both the change in the multiplication properties of fuel massesdue to water inflow, and the increase in the instrumental error -due to the same reason. 4. Assessment of the self-supporting chain reaction consequences on the premises of the Shelter object [5,6] At present, the most dangerous scenario of the development of the local selfsupporting chain reaction (SCR) is connected with a fast increase of radioactivity when flooding the fuel-containing masses with water.Without protection barriers, the consequences of such an event will be irradiation of the Shelter object personnel with a powerful penetrating neutron g-radiation. Duration of the neutron burst during the SCR -0.3 s, energy release -10 As long as several hours will take to decrease the dose rate of g-radiation from fission products (formed during the SCR) from 300 R/s down to the level commensurable to background values at the FCM surface (800 ¸ 80 R/h). Assessment of the explosion energy that may happen in case of the SCR shows that with the existing geometry of FCM, the kinetic energy of scattering the critical mass fragments will not exceed 0.5 MJ.The trinitrotoluene equivalent of this value is no more than 0.5 kg of the explosive.Such an explosion is dangerous not because of the possibility of destroying building constructions, but because of the possibility of releasing radioactive dust and aerosol from inside the Shelter object into the environment.Water evaporation during the SCR may also cause formation of aerosols and result in the additional release of radioactivity into the atmosphere. First priority measures for the Shelter object water management Water is not only a factor of the potential nuclear danger of FCM, but it also determines the radio-and-ecological danger of the Shelter object in the longterm perspective.Different projects, elaborated at present for the object modernization, are called upon, first of all, to prevent the contact of the FCM with water. In parallel, in order to avoid radioactive leakage into the soil, it is necessary to solve the problem of collection, removal and treatment of the water accumulated in the northern and central parts of the Shelter object.The first stage of the work will be completed in 1998, after the final approval of the conceptual design and principal consent of the regulatory body. Nuclear safety during the treatment of the Shelter water In order to facilitate the designing of installations and selection of equipment, meeting the requirements of nuclear safety, scientific and research work was carried out in 1996 for the purpose of nuclear and radioactive safety during the management of water (LRW) containing fissionable materials [7].Below is given an assessment of minimum critical and safe parameters for the most dangerous case -a mixture of Uranium dioxide of 2% enrichment with water (table 5).Specified values of parameters will be regarded as outdated.The assessment of nuclear safety of individual equipment requires data and information on the processes parameters. Due to the possible formation of critical masses out of new formations the calculations were carried out for the assessment of critical parameters of water composition systems on the basis of salt Na 4 UO 2 (CO 3 ) 3 .These assessments, as well as the known experimental data on criticality of other Uranium compounds, show that minimum critical parameters of the systems with low-enrichment 3 . 1 ) Results of the 1991-1996 examinations It should be noted that conclusions in the Feasibility Study of Nuclear Safety (FSNS) were based on the examinations carried out on the surface of fuel lava concentrations, since there was no technology for the extraction of highly active cores to penetrate into the lava.During the last few years (after the publication of FSNS) numerous experimental and computing examinations were performed for the determination of nuclear-physical, mechanical physico-chemical characteristics of the FCM related directly to the nuclear safety of the Shelter object.Below we list the most essential findings which resulted in a need for a substantial addition to the FSNS conclusions.While earlier the penetration of water into lava-like FCM was impeded by its high temperature and water resistance of the matter, now the conditions have changed.Calculations and experiments reveal considerable cooling of the lava, its cracking and transformation into water-tight structure. . 6 R At this number of fissions, about 4 kKi of radioactive inert gases (RIG) and radionuclides of iodine are generated.The consequences of their release into the atmosphere will be irradiation of the personnel nearby the Shelter object with up to 1 rem doses[2].As to the personnel working inside the Shelter object, criticality of the FCM may result in irradiation with greater doses.Density of the prompt neutron flux in the zone of SCR development is assessed expected dose of neutron irradiation in the zone of SCR development is assessed to be at the level of ~10 7 rem, and at the distance of 20 m -10 4 rem.The exposed dose rate of a prompt g-radiation in the zone of SCR development is assessed as the value equal to ~10 /s, and at the distance of 20 m ~ 300 R/s, at the same time g-doses during the SCR (0.3 s) at these points (due to prompt g-quantum) may be equal to ~10 6 rem and 100 rem, correspondingly. will be conducted in compliance with the Procurement Policies and Rules for the Projects Financed by the EBRD and any special instructions issued by the EBRD for implementing the SIP.The total cost of first priority measures within the 13 th Task (WBS 1.3.02.03 -1.3.02.30) is estimated in the amount of $1 833 000.To reduce the impact of the aqueous medium of the Shelter object on its safety and environment it is envisaged to determine the sources, water migration routes, quantity of radioactive and fissionable materials contained in the water of the Shelter object.The plan of water management should be developed and implemented, which would include specification of the water collection points, determination of the strategy of the control and the monitoring of water, as well as technological schemes of pumping-out and the purification of water. Table 1 . The values of burning-out and the UO 2 share at which K ¥ virtually uncontrollable and entails a nuclear accident with grave radioactive consequences.In 1986 the chain nuclear reaction inside the 4 th reactor of the ChNPP was stopped by the total self-destruction of the active zone.But a large quantity of the nuclear fuel that remained inside the 4 th unit (almost 200 tons with the effective enrichment >1% for 235 U) still keeps the threat of a new formation of local critical masses and appearance of a self-supporting chain reaction (i.e. the secondary nuclear accident).It is not excluded that such critical compositions could have spontaneously appeared during the active phase of the accident and have been one of the causes of the intensification of accidental ejection of 2 in the mixture, which makes possible the existence of critical mass for optimum humidified LFCM (table1).The value of dry mixture density (SiO 2 +UO 2 ) was accepted to be equal to 2.5 gr/cm 3 . Table 2 . Critical parameters of homogeneous multiplication compositions Table 3 . Critical parameters for the mixture lava + active zone fragments + water with a concrete reflector. Table 4 shows results of calculations of critical parameters for an equally sized cylinder (H=D) with a 50 cm.thick concrete reflector. Table 5 . Values of minimum critical and safe parameters for the homogeneous mixture of UO 2 (2% enrichment) with water.Reflector -water.	2014-10-01T00:00:00.000Z	1997-01-01T00:00:00.000	{ "year": 1997, "sha1": "173cf65eadf6a65d78a3da09b0acd7df86d7abad", "oa_license": "CCBY", "oa_url": "http://www.icmp.lviv.ua/journal/zbirnyk.12/009/art09.pdf", "oa_status": "GOLD", "pdf_src": "CiteSeerX", "pdf_hash": "173cf65eadf6a65d78a3da09b0acd7df86d7abad", "s2fieldsofstudy": [ "Engineering", "Environmental Science", "Physics" ], "extfieldsofstudy": [ "History" ] }
9557099	pes2o/s2orc	v3-fos-license	Role of yeast glutaredoxins as glutathione S-transferases. The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, that are required for resistance to reactive oxygen species. We recently reported that Grx1 is active as a glutathione peroxidase and can directly reduce hydroperoxides (Collinson, E. J., Wheeler, G. L., Garrido, E. O., Avery, A. M., Avery, S. V., and Grant, C. M. (2002) J. Biol. Chem. 277, 16712-16717). We now show that Grx2 is also a general hydroperoxidase, and kinetic data indicate that both enzymes have a similar pattern of activity, which is highest with hydrogen peroxide, followed by cumene hydroperoxide and tert-butyl hydroperoxide. Furthermore, both Grx1 and Grx2 are shown be active as glutathione S-transferases (GSTs), and their activity with model substrates such as 1-chloro-2,4-dinitrobenzene is similar to their activity with hydroperoxides. Analysis of the Grx1 active site residues shows that Cys-27, but not Cys-30, is required for both the peroxidase and transferase activities, indicating that these reactions proceed via a monothiol mechanism. Deletion analysis shows that Grx1 and Grx2 have an overlapping function with yeast GSTs, encoded by GTT1 and GTT2, and are responsible for the majority of cellular GST activity. In addition, multiple mutants lacking GRX1, GRX2, GTT1, and GTT2 show increased sensitivity to stress conditions, including exposure to xenobiotics, heat, and oxidants. In summary, glutaredoxins are multifunctional enzymes with oxidoreductase, peroxidase, and GST activity, and are therefore ideally suited to detoxify the wide range of xenobiotics and oxidants that can be generated during diverse stress conditions. Glutaredoxins are small, heat-stable oxidoreductases, first discovered in Escherichia coli as GSH-dependent hydrogen donors for ribonucleotide reductase (1). They have proposed roles in many cellular processes, including protein folding and regulation, reduction of dehydroascorbate, protection against reactive oxygen species and sulfur metabolism (2)(3)(4). Glutaredoxins form part of the glutaredoxin system, comprising NADPH, GSH, and glutathione reductase, which transfers electrons from NADPH to glutaredoxins via GSH (5). Yeast contains two classic glutaredoxin genes, designated GRX1 and GRX2, that share 40 -52% identity and 61-76% similarity with those from bacterial and mammalian species (6). Strains deleted for both GRX1 and GRX2 are viable but lack heat-stable oxidoreductase activity using ␤-hydroxyethyl disulfide (HED) 1 as a model disulfide substrate. Previous studies have shown that the yeast glutaredoxins are active as antioxidants and are required for protection against reactive oxygen species. Mutants lacking GRX1 and GRX2 are sensitive to oxidative stress conditions (6,7), and overexpression of GRX1 or GRX2 increases resistance to various hydroperoxides (8). Furthermore, expression of GRX1 and GRX2 is up-regulated in response to stress conditions, including exposure to oxidants, with both genes regulated via stress-responsive elements (9). Differences in gene expression between GRX1 and GRX2 indicate that the two glutaredoxins may play distinct roles during different growth and stress conditions (9). Interestingly, a further difference between Grx1 and Grx2 is shown by recent findings indicating that two isoforms of Grx2 can be detected in cells that localize to the cytosol and mitochondria, respectively (10). Purified Grx1 was shown to have glutathione peroxidase activity and could reduce hydroperoxides directly, including hydrogen peroxide (H 2 O 2 ), tert-butyl hydroperoxide, and cumene hydroperoxide (CHP) (8). This reaction proceeds in a catalytic manner using reducing power provided by NADPH, GSH, and glutathione reductase. The glutathione peroxidase activity of yeast Grx1 seems to be conserved; a recent report has shown that a rice glutaredoxin has the same activity (11). Interestingly, yeast does not contain any classic glutathione peroxidases but expresses three phospholipid hydroperoxide glutathione peroxidases (PHGPx) encoded by GPX1-3 (12). These PHGPx enzymes have activity with phospholipid hydroperoxides as well as nonphopsholipid peroxides and are able to protect membrane lipids against peroxidation. Thus, Grx1 and Grx2 provide an overlapping defense system with Gpx1-3 protection against a range of hydroperoxides. The glutaredoxin-mediated resistance to hydroperoxides seems to be part of the glutathione conjugation/removal system of cells, because it is absent in strains lacking glutathione S-transferases (GTT1, GTT2) or the GS-X pump (YCF1) (8). Glutathione S-transferases (GSTs) are a major family of proteins involved in the detoxification of many xenobiotics compounds (13). They catalyze the conjugation of electrophilic substrates to glutathione before their removal from cells via glutathione conjugate pumps. Yeast Gtt1 and Gtt2 share only limited homology with GSTs from other organisms but are active in a GST assay using the model substrate 1-chloro-2,4dinitrobenzene (CDNB) (14). Strains lacking GTT1 and GTT2 are viable and are unaffected in growth during normal aerobic conditions. In addition, the gtt1 gtt2 mutant does not show any increased sensitivity to CDNB, which is surprising given that Gtt1 and Gtt2 would be expected to detoxify CDNB via conju-gation (14), and the Ycf1 GS-X pump is required for CDNB resistance (15). These findings led to the suggestion that both GST-dependent and -independent pathways for CDNB detoxification exist in yeast (14). To account for the involvement of Gtt1, Gtt2, and Ycf1 in glutaredoxin-mediated antioxidant activity, we proposed a model in which the glutathione peroxidase activity of yeast glutaredoxins converts hydroperoxides to their corresponding alcohols, which can then be conjugated to GSH by glutathione S-transferases and transported into the vacuole by Ycf1 (8). In the present study, we have extended these findings to show that Grx2, like Grx1, is a general hydroperoxidase. Furthermore, both Grx1 and Grx2 are shown to have glutathione S-transferase activity, and we provide the first evidence that glutaredoxins and glutathione S-transferases have overlapping functions in protection against stress conditions including heat shock, oxidative stress, and exposure to CDNB. EXPERIMENTAL PROCEDURES Yeast Strains and Growth Conditions-The Saccharomyces cerevisiae strains used in this study are described in Table I. Strains were grown in rich YEPD medium (2% (w/v) glucose, 2% (w/v) bactopeptone, and 1% (w/v) yeast extract) or minimal SD media (0.17% (w/v) yeast nitrogen base without amino acids, 5% (w/v) ammonium sulfate, and 2% (w/v) glucose) supplemented with appropriate amino acids and bases: 2 mM leucine, 0.3 mM histidine, 0.4 mM tryptophan, 1 mM lysine, 0.15 mM adenine, and 0.2 mM uracil. Media were solidified by the addition of 2% (w/v) agar. Plasmids-Multicopy plasmids containing GRX1 (mcGRX1) and GRX2 (mcGRX2) have been described previously (8). Plasmid pBAD-YGRX1 contains a His 6 -tagged version of GRX1 and was a kind gift from Barry Rosen (16). The GRX2 gene was amplified from mcGRX2 by PCR to introduce an NcoI site at the 5Ј-end and an EcoRI site at the 3Ј-end. The forward primer was 5Ј-CCAACCATGGTATCCCAGGAAA-CAGTTGCT-3Ј and the reverse primer was 5Ј-ATTTGAATTCTT-GAAATACCGGCTTCAATA-3Ј. The resulting 330-bp fragment was cloned into pGEM-T (Promega), before digestion with NcoI and EcoRI and insertion into the NcoI and EcoRI sites of pBAD/Myc-HisC (Invitrogen) in frame with the His 6 tag, creating plasmid pBAD-YGRX2. Sensitivity to Xenobiotics-Sensitivity to xenobiotics was determined by spotting strains onto YEPD plates containing various concentrations of H 2 O 2 , CHP, and CDNB. Cells were grown to stationary phase, and 5-l aliquots of diluted cultures were spotted onto appropriate plates. Sensitivity was determined by comparison of growth with the wild-type strain after 3 days. Dose-response curves to H 2 O 2 and CDNB were made by growing cells to exponential phase (A 600 ϭ 1) in SD medium at 30°C, and treating with oxidants for 1 h. Aliquots of cells were diluted in fresh YEPD medium and plated in triplicate on YEPD plates to obtain viable counts after 3 days' growth. Heat Shock Treatment-Cells were grown to stationary phase in YEPD medium at 30°C, before heat shock at 52°C for 10 min. Survival was determined by plating onto YEPD plates in triplicate to obtain viable counts after 3 days' growth. Protein Purification and Western Blot Analysis-His-tagged Grx1 and Grx2 were purified using Ni 2ϩ agarose columns as described previously (16). Protein extracts were electrophoresed under reducing conditions on 18% SDS-PAGE mini-gels, and Western blot analysis was performed as described previously (8). Enzyme Assays-Glutaredoxin activity was measured by the reduction of the mixed disulfide formed between HED and GSH (17). The components of the glutaredoxin system, NADPH (0.4 mM), GSH (1 mM), and glutathione reductase (6 g/ml), as well as HED (0.7 mM), were added to a reaction volume of 200 l in 0.1 M Tris HCl, pH 7.4. A mixed disulfide between HED and GSH is formed within 2 min, and the reaction was started by the addition of 5 pmol of Grx1 or Grx2. The reaction was followed by the decrease in A 340 because of the oxidation of NADPH. Peroxidase activity was measured with purified glutaredoxins as described previously (8). Glutathione S-transferase activity was measured with purified glutaredoxins and cell-free extracts using CDNB, 1,2-dichloro-4-nitrobenzene (DCNB), and ethacrynic acid as substrates. Reaction mixtures contained 0.1 M potassium phosphate buffer, pH 6.5, 1 mM GSH, and varying concentrations of substrate in a reaction volume of 200 l. Reactions were started by the addition of 5 pmol of purified glutaredoxin or 75 g of cell-free extracts and were followed by the increase in A 340 (CDNB and DCNB) or A 270 (ethacrynic acid) caused by the formation of GSH S-conjugates (18). Glutathione Oxidoreductase and Peroxidase Activities of Grx1 and Grx2-After our observation that Grx1 has glutathione peroxidase activity, we purified Grx2 to compare its catalytic ability. Glutaredoxins are routinely assayed for their ability to reduce the mixed disulfide formed between HED and GSH (17). The oxidoreductase activity of Grx2 (99.6 Ϯ 7.9 mol/min/mg) was somewhat lower than that of Grx1 (211.8 Ϯ 6.9 mol/min/mg), indicating that Grx1 is more efficient as a thiotransferase. We next examined whether Grx2 could act as a glutathione peroxidase, similar to Grx1 (8). Grx2 was able to reduce CHP in a catalytic manner in a reaction that depended on the presence of GSH, Glr1, and NADPH (data not shown). The ability of Grx2 to act as a general hydroperoxidase was investigated using various concentrations of hydroperoxides (tert-butyl hydroperoxide, CHP, and H 2 O 2 ). Grx2 could reduce all three hydroperoxides, and peroxidase activity was hyperbolic with respect to the concentration of peroxide used (data not shown). Comparing the kinetic constants of Grx1 and Grx2 (Table II) showed that Grx1 is catalytically more efficient than Grx2 as a general hydroperoxidase. The K m values for Grx2 (0.87-2.2 mM) were generally higher than for Grx1 (0.52-0.88 mM), indicating that Grx1 has a higher affinity for hydroperoxides than Grx2. However, both enzymes showed similar patterns of enzyme specificity (K cat /K m ); the greatest activity was directed toward H 2 O 2 , followed by CHP and tert-butyl hydroperoxide. Overexpression of GRX1 and GRX2 Increases Sensitivity to CDNB-Glutathione S-transferases from mammalian systems can act as general hydroperoxidases catalyzing the breakdown of alkyl hydroperoxides to their corresponding alcohols (19). Yeast Gtt1 and Gtt2 share little homology with mammalian glutathione S-transferases, and a gtt1 gtt2 mutant is unaffected in sensitivity to the model GST substrate CDNB (14). We therefore examined whether Grx1 and Grx2 might function in the detoxification of CDNB. Overexpression of GRX1 and GRX2 was achieved using multicopy plasmids and was confirmed by means of Western blot analysis (8). Wild-type and ycf1 mutant transformants were grown to stationary phase and spotted onto plates containing various concentrations of CDNB. No strains were able to grow on plates containing CDNB concentrations higher than 0.1 mM (data not shown). Surprisingly, elevating the level of Grx1 or Grx2 was found to increase the sensitivity of both strains to 0.075 mM CDNB compared with the vector-alone controls (Fig. 1). This is similar to findings that glutaredoxins increase the sensitivity of a ycf1 mutant to CHP (8), and rat liver GSTT1 causes increased bioactivation of dihaloalkanes (20). These results indicate that glutaredoxins have some activity toward CDNB and may convert it to a more toxic product. We therefore examined whether the yeast glutaredoxins could act as glutathione S-transferases. Glutathione S-transferase Activity of Grx1 and Grx2-The ability of glutaredoxins to catalyze the formation of GSH conjugates was tested in vitro using purified Grx1 and Grx2. Reaction mixtures contained GSH and CDNB and were followed by the increase in absorbance at 340 nm caused by the formation of the conjugate. Both Grx1 and Grx2 were able to catalyze conjugate formation, and reactions were dependent on the presence of both GSH and glutaredoxins because there was little or no activity when each was omitted from the assay ( Fig. 2A). The conjugation of GSH to CDNB was analyzed using differing amounts of Grx1 and Grx2 and was found to show strict linearity, confirming that glutaredoxins have efficient catalytic activities as glutathione S-transferases (Fig. 2B). We next determined the kinetics of glutathione S-transferase activity with GST substrates, including CDNB, DCNB, and ethacrynic acid. Grx1 and Grx2 showed no measurable activity with ethacrynic acid, whereas both enzymes were active against CDNB and DCNB (Table II). Comparing the kinetic constants (Table II) showed that Grx2 is catalytically more efficient than Grx1 as a glutathione S-transferase. K m values for Grx1 (1.3-4.2 mM) were generally higher than for Grx2 (0.99 -1.1 mM), and enzyme specificity values (K cat /K m ) for Grx2 (4.2-5.9 ϫ 10 3 M Ϫ1 s Ϫ1 ) were higher than for Grx1 (1.7 ϫ 10 2 to 3.1 ϫ 10 3 M Ϫ1 s Ϫ1 ). Cellular Glutathione S-transferase Activity-Given that purified recombinant Gtt1 and Gtt2 (14) and Grx1 and Grx2 (Fig. 2) have glutathione S-transferase activity, we determined their relative contributions to cellular GST activity. The quadruple gtt1 gtt2 grx1 grx2 mutant was viable and was unaffected in growth during normal aerobic conditions compared with the wild-type strain (data not shown). GST activity was therefore assayed in cell-free lysates from the wild-type, gtt1 gtt2, grx1 grx2, and gtt1 gtt2 grx1 grx2 mutants using CDNB and DCNB as substrates (Fig. 3). The wild-type strain showed similar GST activity with both CDNB and DCNB. Loss of GTT1 and GTT2 had little effect on GST activity assayed with CDNB, and caused a slight increase in activity toward DCNB. In contrast, loss of GRX1 and GRX2 reduced the cellular GST activity with CDNB and DCNB by ϳ50%. GST activity measured from the quadruple mutant was ϳ8-fold lower than the wild-type strain with both CDNB and DCNB. These data indicate that Grx1, Grx2, Gtt1, and Gtt2 account for the majority of glutathione S-transferase activity detected within yeast cells, and glutare- 1. Overexpression of GRX1 and GRX2 increases sensitivity to CDNB. Sensitivity was determined by spotting strains onto plates containing various concentrations of CDNB. Cultures of wildtype and ycf1 mutant strains containing YEp24, mcGRX1, and mcGRX2 were grown into stationary phase and diluted (A 600 ϭ 1, 0.5, and 0.1) before spotting onto appropriate plates. Plates were incubated at 30°C for 3 days before scoring growth. Results are shown for plates containing no CDNB (YEPD) and 0.075 mM CDNB. FIG. 2. Glutathione S-transferase activity of Grx1 and Grx2. A, glutathione S-transferase activity of Grx1 and Grx2 was measured in vitro using purified Grx1 and Grx2. The components of the complete reaction mixture were GSH (0.1 mM) and CDNB (0.3 mM) in 0.1 M potassium phosphate buffer, pH 6. Reactions were started by the addition of 5 pmol of Grx1 or Grx2 and followed by an increase in A 340 . GST activity is dependent on the presence of glutaredoxin and GSH; minimal activity was detected in reactions in which each component was omitted individually. B, GST activity with CDNB catalyzed by Grx1 and Grx2 was found to show strict linearity at concentrations ranging from 5 to 25 pmol. doxins seem to be more active than glutathione S-transferases under the conditions tested. Overlapping Functions of Yeast Glutaredoxins and Glutathione S-transferases-Strains deleted for GTT1 and GTT2 are unaffected in growth during normal aerobic conditions but exhibit increased sensitivity to heat shock in stationary phase (14). We therefore investigated the heat shock sensitivity of the quadruple gtt1 gtt2 grx1 grx2 mutant. Cells were grown to stationary phase at 30°C and subjected to heat shock at 52°C for 10 min. This treatment resulted in 35% loss of viability in the wild-type strain (Fig. 4A). Stains deleted for GRX1 and GRX2 were no more sensitive to heat shock than the wild-type strain. In agreement with previous findings (14), the gtt1 gtt2 mutant was sensitive compared with the wild-type strain, with ϳ54% survival after the heat treatment. Interestingly, the quadruple gtt1 gtt2 grx1 grx2 mutant was even more sensitive, and survival was reduced to ϳ36%. Mutants lacking GRX1 and GRX2 are sensitive to oxidative stress conditions (6), whereas mutants lacking GTT1 and GTT2 are unaffected in sensitivity to a range of oxidants and xenobiotics (14). We therefore compared the gtt1 gtt2, grx1 grx2, and gtt1 gtt2 grx1 grx2 mutants for sensitivity to stress conditions. Cells were grown to stationary phase and spotted onto plates containing various concentrations of H 2 O 2 , CHP, and CDNB (Fig. 4B). For all three treatments, the quadruple gtt1 gtt2 grx1 grx2 mutant was more sensitive than either the wild-type or gtt1 gtt2 and grx1 grx2 mutant strains. These data indicate that glutaredoxins (Grx1-2) and glutathione S-transferases (Gtt1-2) have an overlapping function in protection against stress conditions induced by heat, oxidants, and CDNB. Cys-27, but Not Cys-30, Is Required for Peroxidase and Transferase Activity of Grx1-The structure of glutaredoxins has been highly conserved throughout evolution, particularly in the region of the active site (3,17) including two redox-active cysteine residues (positions 27 and 30 in yeast numbering (6)). The two active site Cys residues in Grx1 were replaced with Ser residues to determine their role in peroxidase and transferase activities. These mutations were constructed in plasmid mcGRX1 to create mcgrx1-C27S and mcgrx1-C30S, respectively (see "Experimental Procedures"). Peroxidase activity was tested by examining resistance to H 2 O 2 and transferase activity by resistance to CDNB. Overexpression of GRX1 and grx1-C30S increased the resistance of a wild-type strain to H 2 O 2 , whereas grx1-C30S had no effect relative to the vector control (Fig. 5A). Similarly, overexpression of GRX1 and grx1-C30S increased the sensitivity of a wild-type strain to CDNB, whereas, grx1-C30S had no effect relative to the vector control. These data indicate that a single active site Cys residue (Cys27) is required for both peroxidase and transferase activities. FIG. 3. Cellular GST activity. GST activity was determined from cell-free lysates of the wild-type (wt), gtt1 gtt2, grx1 grx2, and gtt1 gtt2 grx1 grx2 mutant strains grown into stationary phase. Specific activities are shown for CDNB and DCNB using the assay described for Fig. 2. FIG. 4. Sensitivity to stress conditions. A, glutaredoxins and glutathione S-transferases have an overlapping function in protection against heat stress. Cultures from the wild-type (wt), gtt1 gtt2, grx1 grx2, and gtt1 gtt2 grx1 grx2 mutant strains were grown into stationary phase at 30°C and subjected to a heat shock at 52°C for 10 min. Cells were diluted and plated in triplicate on to YEPD medium to monitor cell viability, and percentage survival is expressed relative to the untreated controls. B, glutaredoxins and glutathione S-transferases have an overlapping function in protection against oxidative and CDNB stress. Sensitivity of wild-type (wt), gtt1 gtt2, grx1 grx2, and gtt1 gtt2 grx1 grx2 mutant strains was determined by spotting stationary phase cultures onto plates containing 7 mM H 2 O 2 , 0.9 mM CHP, and 0.05 mM CDNB. FIG . 5. Cys-27, but not Cys-30, is required for peroxidase and transferase activity of Grx1. A, peroxidase activity was tested by examining resistance to H 2 O 2 . B, glutathione S-transferase activity was tested by examining resistance to CDNB. The wild-type strain containing YEp24, mcGRX1, mcgrx1-C27S, and mcgrx1-C30S was grown to exponential phase (A 600 ϭ 1) and treated with 4 mM H 2 O 2 or 0.1 mM CDNB for 1 h. Cells were diluted and plated in triplicate on to YEPD medium to monitor cell viability, and percentage survival is expressed relative to the untreated controls. DISCUSSION Yeast, like all organisms, must be able to adapt to changes in their external environment, such as exposure to xenobiotics and oxidants. In most cases, these responses depend on gene activation, such as the response to H 2 O 2 , where a substantial number of proteins is induced (21). Furthermore, genome-wide analysis has shown that there are differences as well as similarities among the many sets of genes activated in response to different stress conditions (22). For example, the cellular responses to a diverse range of stress conditions, including oxidative, osmotic, heat, and starvation, are mediated by a number of genes containing stress-responsive elements (23,24). GRX1 and GRX2 form part of these responses, with both genes activated by multiple stress conditions via the Hog1 MAPK and the Ras-protein kinase A pathways (9). Our studies show that glutaredoxins are multifunctional enzymes with oxidoreductase, peroxidase, and GST activity and are therefore ideally suited to detoxify the wide range of xenobiotics and oxidants that might be generated during diverse stress conditions. The antioxidant activity of Grx1 and Grx2 provides protection against hydroperoxides (6,8). The localization of Grx2 in the mitochondria may account for the apparent redundancy of yeast glutaredoxins, indicating that Grx1 and Grx2 play complementary roles in the cytoplasm and mitochondria, respectively (10). Glutaredoxins have long been thought to protect against oxidative stress by catalyzing the reduction of protein mixed disulfides formed with GSH (25). Mixed disulfides can protect protein-SH groups against irreversible oxidation, and in vitro studies have shown that they can be reduced by glutaredoxins (26,27). However, in vivo data indicate that the levels of protein-GSH mixed disulfides are unaffected in yeast mutants lacking GRX1 and GRX2 during both normal growth conditions and exposure to H 2 O 2 (6,28). In this present study, we show that both Grx1 and Grx2 have oxidoreductase activity against the mixed disulfide formed between HED and GSH, although it remains unclear whether this activity serves a protective function against physiological substrates formed during stress conditions. We originally showed that Grx1 is a glutathione peroxidase that can reduce various hydroperoxides with activity comparable with that of yeast PHGPx2 and various eukaryotic glutathione peroxidases (8). Similarly, in the present study, we show that Grx2 can directly reduce hydroperoxides, albeit with a lower catalytic efficiency than Grx1. Because glutathione peroxidase activity can be associated with glutathione S-transferases in mammalian cells (19), we analyzed whether Grx1 and Grx2 have GST activity. Both Grx1 and Grx2 were active in a GST assay using CDNB and DCNB as substrates, with Grx2 showing a higher catalytic efficiency than Grx1. Furthermore, the activity of Grx1 and Grx2 with GST substrates was similar to their activity with hydroperoxides. Mutant analysis revealed that Grx1 and -2 have an overlapping function as GSTs with Gtt1 and Gtt2, with loss of all four genes substantially reducing cellular GST activity and leading to sensitivity to stress conditions, including exposure to xenobiotics, heat, and oxidants. Two reaction mechanisms have been described for glutaredoxins, which differ in their requirement for active site cysteine residues. Protein disulfide reductase activity with substrates such as ribonucleotide reductase requires both active site cysteine residues and proceeds via reaction of the dithiol form of glutaredoxin with the oxidized disulfide substrate (30,31). In contrast, the glutathione-disulfide oxidoreductase activity observed with low molecular weight disulfides, such as HED, proceeds via a monothiol mechanism that is initiated by the nucleophilic N-terminal cysteine residue (30). These reaction mechanisms seem to be conserved for the yeast glutaredoxins because alkylation studies have shown that the N-terminal cysteine residue is the nucleophile in disulfide reduction chemistry catalyzed by yeast glutaredoxin (32). In the present study, we show that Cys-27, but not Cys-30, is required for both the peroxidase and transferase activities, indicating that these reactions proceed via a monothiol mechanism. Based on structural and functional data, three glutaredoxin subfamilies have been proposed (33). Yeast Grx1 and Grx2 are part of the first class, which includes classic dithiol glutaredoxins, such as E. coli Grx1 and Grx2 and human Grx1. Members of the second subfamily of glutaredoxins, which differ from classical glutaredoxins in that they contain a single cysteine residue at their active sites, were originally identified in yeast (GRX3-5) but are conserved throughout evolution from bacterial to mammalian species (7). These enzymes are able to reduce protein-mixed disulfides in a reaction that proceeds via a monothiol mechanism and have been implicated in protection against oxidative stress (7). The third class of glutaredoxins contains an N-terminal Grx domain but shows low overall sequence identity with classic glutaredoxins. Interestingly, despite a lack of sequence homology, structural analysis of E. coli Grx2 has revealed that these glutaredoxins are homologous to the GST superfamily of proteins (33). This structural homology is particularly shown toward enzymes belonging to theand -classes of GSTs. Sequence analysis of yeast Grx1 and Grx2 reveals significant homology with the -class of GSTs (Fig. 6). Comparison with human GSTO 1-1 and Schistosoma mansoni GSTO identifies a number of conserved residues that are mainly clustered in the N-terminal portion of Grx1 and Grx2. Identical residues include the Cys-Pro active site residues of the yeast glutaredoxins. The corresponding Cys residue in -class GSTs (Cys-32 in GSTO 1-1 and Cys-25 in SmGSTO1) has been shown to form part of the glutathione binding site (the G-site), which makes a mixed disulfide with GSH (29, 34). Glutathione S-transferases FIG. 6. Alignment of the amino acid sequences of Grx1 and Grx2 with -class GSTs. The amino acid sequences of Grx1, Grx2, human GSTO 1-1, and Schistosoma mansoni GSTO1 were aligned using ClustalW (www.ebi.ac.uk/clustalw) and displayed using GeneDoc (www.psc.edu/ biomed/genedoc/). The sequences are aligned for maximal homology; dashes are used to denote gaps introduced for maximal alignment. Residues that are identical in all four sequences are denoted with an asterisk. Residues that are conserved in all four sequences are boxed in black shading, and residues that are conserved in at least three sequences are boxed in gray shading. contain a substrate binding site (H-site), adjacent to the G-site, that allows conjugation of the thiol group of GSH to an electrophile. This site is constructed from elements of both the Nand C-terminal portions of GSTs, and differences in substrate specificity are thought to be caused by differences in the confirmation of the H-site. The H-site in GSTO 1-1 (29) is a well defined cavity that includes Pro-33 (Pro-28 in Grx1 and Grx2) and Phe-31 (Tyr-26 in Grx1 and Grx2) flanking the catalytic Cys-32 residue. The -class of GSTs are multifunctional enzymes that have activity with a broad range of substrates (13,34). For example, human GSTO1-1 exhibits GST activity, glutathione-dependent oxidoreductase activity, and dehydroascorbate activity characteristic of glutaredoxins (29). The range of glutathione-dependent antioxidants that has been described in yeast differs from that in other eukaryotic organisms. Unusually, yeast expresses three PHGPxs in the absence of conventional glutathione peroxidases (12). In addition, only two GST isoenzymes have been found in yeast, which share little homology with GSTs from other organisms (14). Our observations suggest that the multiple antioxidant activities of Grx1 and Grx2 have evolved to complement the peroxidase activities of PHGPx1-3 and the transferase activities of Gtt1-2 in protection against stress conditions. In this view, glutaredoxins are able to protect against a wide range of toxic compounds (xenobiotic or endobiotic) to which yeast cells might be exposed. It remains to be determined whether glutaredoxins from other eukaryotes have a similar range of biochemical activities, but the peroxidase activity seems to be a conserved activity for at least one plant glutaredoxin (11).	2018-04-03T00:15:29.816Z	2003-06-20T00:00:00.000	{ "year": 2003, "sha1": "7df16ee1a6004c4c4551e7756ef588d1d152f11a", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/278/25/22492.full.pdf", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "27089c79c4d798a9800222fb2ed67c08aaaf8eb8", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
234743377	pes2o/s2orc	v3-fos-license	Risk of SARS-CoV-2 Infection in Previously Infected and Non-Infected Cohorts of Health Workers at High Risk of Exposure The objective of this study is to assess the risk of newly acquired RNA detection-proven SARS-CoV-2 infection after previous SARS-CoV-2 infection. This is a prospective study conducted from March to September 2020 in Barcelona, Spain. Healthcare workers caring for SARS-CoV-2 infected patients were divided in two cohorts: (a) previously RNA-proven SARS-CoV-2 infected cohort with mild symptoms (IC) and (b) healthy cohort (HC). Weekly SARS-CoV-2 RNA detection assays from nasopharyngeal swabs were performed. Serology status was assessed at the beginning and at the end of the study. Twenty participants were included in each group. The median age was 30 (IQR 27–34.75) years, and 55% were female. The median time of follow up was 49 (IQR 49–51) days. Fifteen out of 246 (6%) nasopharyngeal swab samples were positive for SARS-CoV-2, all in the IC. The percentage of participants in the IC with a probable newly acquired SARS-CoV-2 RNA-proven infection was 20% (95% IC 5.7–43.6%) at the end of the 7-week follow up period. The incidence reinfection rate was 28.6 (95% IC 7.8–73.2) cases per 1000 person-week. Despite detectable IgG antibodies against SARS-CoV-2 participants highly exposed to SARS-CoV-2 may develop a newly acquired SARS-CoV-2 RNA detection episode during the first months after the initial infection. Introduction The new coronavirus, SARS-CoV-2, is having devastating consequences in health systems, global economy, and social dynamics and behavior. Despite the huge efforts invested to fight the pandemic, SARS-CoV-2 still runs wild with the current death toll as high as the first months of the pandemic [1]. Non-pharmacological strategies are effective to curb the pandemic, although cannot be held too long without causing a negative impact in the economy and social health [2,3]. It is well described that herd effect can control highly infective diseases. However, the protection generated by the immune system needs to fulfil several criteria in order to adequately protect the society. Firstly, it should be a long-standing immunity. Secondly, it should have the capacity to reduce transmissibility (reducing the basic reproduction number) by limiting the contagious power or hindering the acquisition of the infection. Thirdly, antigens stimulating the immune system should be highly conserved hampering the appearance of new strains able to escape the immune response [4]. Immunity against SARS-CoV-2 can be naturally acquired or vaccine-induced. There is still scarce evidence on whether the immunity against SARS-CoV-2 reduces disease severity, infection and/or transmissibility. Equally important is the duration of the immunity, as short duration of protection will jeopardize proper control of the disease. To date, time from infection to decay of anti-SARS-CoV-2 antibodies and the role of these anti-bodies to prevent a new infection in naturally infected subjects are a matter of intense debate. Based on the current evidence it seems probable that naturally acquired immunity against SARS-CoV-2 confers early strong protection that may wane over time [5][6][7]. No data is available on whether vaccines can develop a longer immune response and protection than natural infection [8]. Long standing immunity after exposure to SARS-CoV-2 antigens, naturally or artificially via vaccine, with capacity to reduce disease severity and transmissibility remains the cornerstone of the strategy to fight the pandemic. For that reason, performing a thoroughly investigation of newly acquired infections after previous SARS-CoV-2 infection is of utmost importance. Reinfection cases have been anecdotally reported all over the world ranging from asymptomatic to severe cases requiring hospitalization and oxygen supplementation [9]. Unfortunately, it has been scarcely evaluated in a cohort of participants highly exposed to the virus [10]. Healthcare workers in Spain were badly hit by the pandemic during the first wave with infection proportions ranging from 25.8% to 33.8% in a cross-sectional study performed in May 2020 [11]. Healthcare workers highly exposed to the SARS-CoV-2 represent an outstanding opportunity to study risk of reinfection. In our study we aimed to assess assessed the risk of newly acquired RNA detectionproven SARS-CoV-2 infection in two highly exposed cohorts of healthcare workers, one with a previous history of RNA detection-proven SARS-CoV-2 infection and another with no previous history of RNA detection-proven SARS-CoV-2 infection during a period of 7 weeks. Secondary objective was to assess the severity of a newly acquired RNA detectionproven SARS-CoV-2 infection and the correlation with IgG antibodies levels. Study Setting and Population This is a prospective cohort study conducted from March 2020 to September 2020 at Vall d'Hebron University Hospital, Barcelona, Spain. Participants were health care workers accompanying SARS-CoV-2 infected patients. According to the previous history of SARS-CoV-2 infection, participants were divided in two cohorts: (a) previously infected cohort (IC), which included subjects with viral symptoms 7 days prior to detection of SARS-CoV-2 RNA from a nasopharyngeal swab and (b) healthy cohort (HC), which included subjects without history of viral symptoms and no record of previous RNA detection-proven SARS-CoV-2 infection or antibodies against the SARS-CoV-2. Subjects from IC cohort were invited to participate during the first two weeks after returning to work from the isolation period. Study Procedures After signing the informed consent, all participants fulfilled a structured questionnaire to collect demographic data, previous medical history and medication, viral symptoms in the previous weeks and work conditions and locations. Participants were instructed how to report symptoms during the study period in an individual's symptoms diary. All participants were followed for 7 weeks. Additional consent was requested to gather self-reported data after the end of the study. According to the study protocol, a blood sample for serology assessment was collected at baseline and at the end of the 7-week follow up. A nasopharyngeal swab was collected at baseline and every week until the end of the study. Nasopharyngeal swab collection could be either performed by a member of the research team or self-collected by the participant after training. Samples were received at the laboratory within the first 4 h after sample collection for their immediate storage in a −80 • C freezer. Blood samples were kept at −20 • C before the serological testing. An extra nasopharyngeal swab collection was performed if the patient reported viral symptoms during the study period. In the IC cohort, a probable newly acquired SARS-CoV-2 infection was suspected if a participant had a positive SARS-CoV-2 RNA detection assay at least 30 days after symptoms onset and had at least one negative SARS-CoV-2 RNA detection assay between the initial and the new positive assay. Statistical Analysis and Sample Size Calculation Continuous variables were expressed as median and interquartile range and categorical variables as absolute numbers and percentages. Longitudinal results are depicted in a timeline graphic for clarity. PCR-proven SARS-CoV-2 infection incidence comparison among groups was performed using proportion difference test. Tests were considered significant when the two-tailed p-value was <0.05. For the sample size calculations, we assumed a proportion of new PCR-proven SARS-CoV-2 infection after the 7-week period of 25% and 0% in the HC and the IC group, respectively. Given an alpha error of 0.1, a beta error of 0.2, and an exposure ratio of 1 the sample size was 21 participants per arm. Analysis was performed with SPSS software (IBM ® , Armonk, New York, USA). Study Oversight and Ethical Statement The institutional review board provided ethical clearance (PR (AG) (195/2020)). All patients signed a written informed consent. The detection of SARS-CoV-2 in nasopharyngeal samples was performed using the commercial Aptima ® SARS-CoV-2 Assay (Hologic, Marlborough, Ma, USA), which utilizes transcription-mediated amplification (TMA) for nucleic acid amplification on the Panther Fusion ® System (Hologic, USA). This amplification technique is known to have a higher sensitivity than conventional PCR-based assays in other RNA-viral infections [12]. TMA was the first-line assay due to the high throughput of the system. As the results obtained by TMA-based techniques do not have correspondence with viral quantification load, and additional real time PCR-based assay was performed for the determination of the cycle-threshold (Ct). Moreover, this technique was also used to assess the sample collection, integrity of extracted nucleic acids and presence of PCR inhibitors by detecting of a human housekeeping gene. Thus, the positive samples by TMA-based assay were additionally tested using an in-house RT-PCR assay based on the CDC 2019-nCoV Real-Time-PCR Diagnostic Panel, with two sets of primers and probe targeting the specific SARS-CoV-2 nucleocapsid protein (N1) gene and the human RNase P gene. Population Description Twenty participants were included in each group. The median age of the participants was 30 (IQR 27-34.75) years, and 55% were female. One participant in the IC was under anti-TNF treatment due to a joint inflammatory disease and one participant in the IC became pregnant at the end of the study but adhered to the protocol schedule. In the IC cohort, the median time from the first PCR-proven SARS-CoV-2 episode to inclusion in the study was 22.5 (IQ 18-25) days. All participants in the IC cohort reported mild symptoms, and none of them required hospitalization. During the follow up, all participants continued working at COVID19 high-risk hospital areas. The median time of follow up was 49 days (IQR 49-51) with five participants lost to follow up. No participants in either group reported new symptoms during the 7-week study period. Seven participants spontaneously reported extra data beyond the study time. More information can be found in Table 1. SARS-CoV-2 RNA Detection Overall, 15 out of 246 (6%) nasopharyngeal swab samples were positive by TMA. All the 123 follow up samples from the HC were negative. In the IC, 15 out of 123 (12%) follow up samples were positive (including the post study follow up self-reporting period). Seven out of 20 (35%) participants in the IC had at least one positive result during the follow up. Seven participants (35%) had a positive SARS-CoV-2 RNA detection beyond 30 days after inclusion in the study. At the time of the inclusion in the study, 4 participants had a positive SARS-CoV-2 RNA detection, which was considered as remnant from the first PCR-proven infection. According to our case definition, the percentage of participants in the IC with a probable newly acquired SARS-CoV-2 RNA-proven infection was 20% (95%IC 5.7-43.6%) at the end of the 7-week follow up period, with 2 additional cases in the self-reporting period, which depicted 30% (95% IC 11.9-54.3%) of probable newly acquired SARS-CoV-2 RNA-proven infection in 8 months. The incidence reinfection rate, taking into consideration the 7-week study period, was 28.6 (95% IC 7.8-73.2) cases per 1000 person-week in health care workers at high risk of SARS-CoV-2 exposure. None of them had symptoms at the time of RNA detection of the second probable infection, and no subsequent infections in close contact were declared. Figures 1 and 2 depict the timeline for symptoms and testing results of participants in the IC. Additional information regarding these cases is available in Appendix A. When comparing the percentage of newly acquired SARS-CoV-2 RNA detection proven infection between HC and IC we did not find any statistically different (0% vs. 20%; p = 0.11). Serology Results At the time of inclusion, 18 out of 20 participants in the IC had an available initial serology, and 55.6% (10/18) had specific IgG antibodies against SARS-CoV-2. At the end of the 7-week study 11 out of 17 (64.7%) had positive IgG antibodies against SARS-CoV-2. Four participants had negative IgG antibodies against SARS-CoV-2 throughout the duration of the study, 4 participants seroconverted, and 1 participant had undetectable IgG antibodies against SARS-CoV-2 at the end of the study period. All participants in the HC had negative IgG antibodies against SARS-CoV-2 at the beginning and at the end of the study, except for one participant who had a positive anti-SARS-CoV-2 IgG at the end of the study, although all 8 RT-PCR from nasopharyngeal swabs were negative during the study period. This participant had positive total antibodies against SARS-CoV-2 at the time of inclusion with negative IgG antibodies, emphasizing the possibility of a recent asymptomatic SARS-CoV-2 infection before participating in the study. Discussion In this report, we describe a prospective study involving 40 health care workers taking care of COVID19 patients. Twenty participants had been previously diagnosed with symptomatic SARS-CoV-2 infection confirmed by molecular tests and 20 participants without evidence of previous SARS-CoV-2 infection were included as controls. In the HC group, all participants had RNA-detection assays and IgG antibodies against SARS-CoV-2 negative at the beginning of the study. Based on our definition of probable newly acquired SARS-CoV-2 infection, 4 participants could be classified probable newly acquired SARS-CoV-2 infection during the 7-week study period; therefore, the incidence rate of reinfection in the 7-week study was 28.6 cases 1000 person-week in our study population. When including the self-reporting period, 6 participants fulfilled the probable newly acquired SARS-CoV-2 infection. Persistence of detectable of SARS-CoV-2 RNA in nasopharyngeal samples has been reported for long periods, especially in patients with impaired immune systems. However, follow up periods are usually short, covering only the first weeks of the infection [13,14]. In a recent report by Kim et al., hospitalized participants with COVID19 were repeatedly sampled to assess viral shedding and viability in viral culture. From symptoms onset, the median time to SARS-CoV-2 RT-PCR clearance occurred on day 34 (lower limit of 95% CI being the 24 th day, upper limit was not computable) in 50% of the patients [15]. In our study the 4 participants with probable newly SARS-CoV-2 infections during the 7week study had at least 55 days between the onset of symptoms and the SARS-CoV-2 RNA detection assay that led to reinfection suspicion, and 3 out of these 4 probable cases Serology Results At the time of inclusion, 18 out of 20 participants in the IC had an available initial serology, and 55.6% (10/18) had specific IgG antibodies against SARS-CoV-2. At the end of the 7-week study 11 out of 17 (64.7%) had positive IgG antibodies against SARS-CoV-2. Four participants had negative IgG antibodies against SARS-CoV-2 throughout the duration of the study, 4 participants seroconverted, and 1 participant had undetectable IgG antibodies against SARS-CoV-2 at the end of the study period. All participants in the HC had negative IgG antibodies against SARS-CoV-2 at the beginning and at the end of the study, except for one participant who had a positive anti-SARS-CoV-2 IgG at the end of the study, although all 8 RT-PCR from nasopharyngeal swabs were negative during the study period. This participant had positive total antibodies against SARS-CoV-2 at the time of inclusion with negative IgG antibodies, emphasizing the possibility of a recent asymptomatic SARS-CoV-2 infection before participating in the study. Discussion In this report, we describe a prospective study involving 40 health care workers taking care of COVID19 patients. Twenty participants had been previously diagnosed with symptomatic SARS-CoV-2 infection confirmed by molecular tests and 20 participants without evidence of previous SARS-CoV-2 infection were included as controls. In the HC group, all participants had RNA-detection assays and IgG antibodies against SARS-CoV-2 negative at the beginning of the study. Based on our definition of probable newly acquired SARS-CoV-2 infection, 4 participants could be classified probable newly acquired SARS-CoV-2 infection during the 7-week study period; therefore, the incidence rate of reinfection in the 7-week study was 28.6 cases 1000 person-week in our study population. When including the self-reporting period, 6 participants fulfilled the probable newly acquired SARS-CoV-2 infection. Persistence of detectable of SARS-CoV-2 RNA in nasopharyngeal samples has been reported for long periods, especially in patients with impaired immune systems. However, follow up periods are usually short, covering only the first weeks of the infection [13,14]. In a recent report by Kim et al., hospitalized participants with COVID19 were repeatedly sampled to assess viral shedding and viability in viral culture. From symptoms onset, the median time to SARS-CoV-2 RT-PCR clearance occurred on day 34 (lower limit of 95% CI being the 24th day, upper limit was not computable) in 50% of the patients [15]. In our study the 4 participants with probable newly SARS-CoV-2 infections during the 7-week study had at least 55 days between the onset of symptoms and the SARS-CoV-2 RNA detection assay that led to reinfection suspicion, and 3 out of these 4 probable cases of reinfection had several negative RNA tests in between, reinforcing the likelihood of reinfections rather than remnants from the first infection. The cases 8, 17 and 27 deserve special attention since they had TMA-positive swab nasopharyngeal samples several months after the first episode. In patient 17, RT-PCR was also positive with Ct values consistent with an acute infection. Patient 8 recalled a close contact with a positive COVID19 case before having a new positive SARS-CoV-2 RNA detection assay. Interestingly, viral shedding of the newly acquired SARS-CoV-2 infection in patient 17 and 27 was very short compared with data from the first episodes, suggesting that an early reinfection may have low infectivity potential. Besides, participants with a probable reinfection did not have any symptoms, unlike what happened in the first episode when all of them have mild symptoms. Our data suggest that reinfection in highly exposed subjects with a mild first episode can appear within the 12 weeks after the first episode, supporting observation from other coronaviruses. Reinfections with seasonal coronaviruses have been reported even with the presence of high antibody titres [16]. In the HC cohort we did not find any newly acquired SARS-CoV-2 infection, suggesting that uninfected healthcare workers may act with higher precaution at hospital and in the community, limiting the chances of acquiring a SARS-CoV-2 infection. In a population-based study conducted in Qatar, newly acquired SARS-CoV-2 infection in patients with previous history of infection in the last 6 months was 0.02% and the incidence reinfection rate was 0.36 per 10,000 person-week [17]. These results should be interpreted with caution, since asymptomatic patients were not systematically tested. A recent publication in health care workers found an incidence reinfection rate of 1.09 per 10,000 days at risk in anti-Spike seronegative participants and 0.13 per 10,000 person-day in anti-Spike seropositive participants [10]. Our study depicts a highly exposed population of health care workers dealing with the peak of the first wave of the pandemic in Spain [18]. Although, our results may not be informative of the risk of reinfection in the community, they highlight the possibility of early newly acquired infections even with the presence of antibodies. Many healthcare workers during the first pandemic wave acquired the infection in the community, although hospital acquired infections were more frequent in workers caring from COVID19 patients [19]. Outstanding efforts to develop a vaccine with long-term protection against SARS-CoV-2 infection have been made during the last months. Currently several vaccines against SARS-CoV-2 are being administered worldwide, however the long-term protective effects are still unknown [20]. The success of the vaccine relies on the long-term persistence of neutralizing antibodies and the development of immune memory cells [21]. It has been proposed that high titres of neutralizing antibodies could prevent infections or rapidly sterilize the virus. Furthermore, neutralisation activity has been associated with high anti-receptor-domain antibodies titres 3 weeks after the infection [22,23]. Unfortunately, first reports and experiences from previous coronaviruses suggest waning over time [21,24]. To date, between 6 to 8 months after symptoms onset 90% of the infected patients had detectable spike-binding IgG antibodies. On the other hand, disease severity is expected to depend, to a large degree, on the persistency of immune memory cells [25]. From a public health point of view, knowing the potential infectivity of reinfected patients is of utmost importance to design intervention strategies. There is little information regarding SARS-CoV-2 transmission blocking effect in previously immunized subjects, however no cases of secondary infection have been described from sequence-proven reinfection cases. Our data suggest that SARS-CoV-2 RNA detection long after symptoms onset is possible, regardless the presence of IgG antibodies, although none of this reinfection led to symptoms or secondary cases, opening a window of hope that vaccination could wane the impact of the pandemic. Several limitations of this study should be mentioned. Firstly, the time of the sample collection between the first episode and the first follow-up nasopharyngeal swab were slightly heterogeneous among participants. However, all of them except one were recruited within the first month after the first PCR-proven episode. Secondly, the sample size of the study was calculated assuming a higher infection rate among uninfected professionals. Surprisingly, our results did not show any infection in the HC group. Accordingly, our study was not aimed to study the incidence rate of reinfection. Another limitation was the insufficient sample volume or the low viral load to perform sequencing studies of the patients suspected to be re-infected, so absolute certainty of reinfection cannot be obtained. However, the time lapse between the first infection and the posterior positive molecular test eliciting a probable newly acquired infection, the evidence of negative results in between, and CT results from PCR tests support our interpretation of newly acquired infections. Persistent infection in our cases is unlikely due to the resolution of symptoms and negative results between episodes, limiting the possibility of viral remnants. We did not assess infectivity by means of viral viability in cell culture isolation, which prevent us from knowing whether reinfections have the potential to infect other subjects. It is noteworthy that our participants were young healthcare workers, and the subjects from IC group had mild symptoms during the acute initial episode, other populations may behave differently. In support of our study is the used of TMA tests, which have shown greater sensitivity than the conventional RT-PCR [26]. Subsequent cohort studies with healthcare workers should consider sequencing and infectivity studies, as well as prolonged follow up schemes with regular respiratory and blood samples. Conclusions Our study shows SARS-CoV-2 serology and RNA detection dynamic in previously infected and uninfected cohorts of healthcare workers caring for COVID19 patients. Despite, detectable IgG antibodies against SARS-CoV-2, participants highly exposed to the virus may develop newly acquired SARS-CoV-2 RNA detection episode during the first months after the initial infection. These results may help to design vaccination strategy and highlight the need of a follow up of the pandemic transmission dynamic. Case Description of the Probable Reinfection Cases Participant 4 had a positive SARS-CoV-2 PCR from a nasopharyngeal swab on March 20th. His symptoms started 2 days before the swab collection and consisted in high fever, asthenia, headache, myalgia, diarrhoea and anosmia. All symptoms resolved in 10 days. The participant did not recall contact with COVID19 patients outside the Hospital. The participant had 3 consecutive positive SARS-CoV-2 RNA detection assays after symptoms resolved. Last positive SARS-CoV-2 RNA detection assay was on April 29th. Afterward, the samples on May 5th and May 13th were negative. The last SARS-CoV-2 RNA detection assay on May 25th was again positive. The participant did not report any symptom or secondary cases. Participant 5 started had viral symptoms on March 21st including asthenia, headache, arthromyalgia, low-grade fever and anosmia. Symptoms resolved in 4 days, except for anosmia that lasted for up to 4 months. This participant showed a possible episode of reinfection on May 23th due to a SARS-CoV-2 RNA detection assay by TMA. This sample could not be retested by RT-PCR due to insufficient volume. The participant did not report any symptom or secondary cases derived from the last positive result. The participant 8 showed anosmia on March 26th that was resolved after 7 days but reported having thoracic ache for one month. During the next 2 months the participant had 8 weekly negative SARS-CoV-2 RNA detection assays. Five months later, this participant reported having had close contact with a person infected with SARS-CoV-2 and was retested with TMA test that was positive. The participant had detectable IgG antibodies at least from June 6th, and were retested during the reinfection episode and persisted positive. The participant was not aware of secondary cases, and reported no symptoms. The patient 17 started viral symptoms on March 22nd and lasted for 10 days. Two months after the diagnosis and after five negative samples during the follow-up (confirmed by RT-PCR), he had two consecutive SARS-CoV-2 RNA detection assay (one by both TMA and RT-PCR and one by TMA). Again, no symptoms or secondary cases were reported. IgG antibodies at the beginning of the inclusion were positive, the second determination two months later showed indeterminate results. The participant 27 had low-fever and anosmia on March 31st. Anosmia lasted for two weeks. During the next 2 months the participant had 8 weekly negative SARS-CoV-2 RNA detection assays. IgG antibodies against SARS-CoV-2 were detected neither at the initial nor at the end of the follow up. Six months later, during a hospital massive screening the participant had a positive for SARS-CoV-2 infection by TMA. The participant had no other sign of infection. There were no secondary cases from the second episode. One month later, the IgG antibodies remained negative. Participant 33 had symptoms on March 11th consisting in high fever, myalgias and cough. Symptoms resolved in one week. On March 24th participant was included in the study and had a negative RNA SARS-CoV-2 detection assay and positive IgG antibodies against SARS-CoV-2. On May 8th the participant had a positive RNA SARS-CoV-2 detection assay by TMA. Again no symptoms or secondary cases were reported. At the end the 7-week period the participant persisted with positive IgG antibodies against SARS-CoV-2.	2021-05-14T13:14:37.998Z	2021-05-01T00:00:00.000	{ "year": 2021, "sha1": "b2f037e78eaa18c0d9ecb75d04c6944fb53d277d", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2077-0383/10/9/1968/pdf", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "cbaeaa4fc5eaf27f3bdccd6e150f467939d18ec4", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
14040610	pes2o/s2orc	v3-fos-license	Impacting tumor cell-fate by targeting the inhibitor of apoptosis protein survivin Survivin (BIRC5), a member of the inhibitor of apoptosis protein (IAP) family that inhibits caspases and blocks cell death is highly expressed in cancer and is associated with a poorer clinical outcome. Functioning simultaneously during cell division and apoptosis inhibition, survivin plays a pivotal role in determining cell survival. Survivin has consistently been identified by molecular profiling analysis to be associated with higher tumor grade, more advanced disease, abbreviated survival, accelerated rates of recurrence, and chemotherapy and radiation resistance. Survivin's differential expression in cancer compared to normal tissue and its role as a nodal protein in a number of cellular pathways make it a highly flexible therapeutic target, suitable for small-molecule inhibitiors, molecular antagonists, and vaccination-based therapies. By targeting survivin it is hoped that multiple tumor signaling circuitries may be simultaneously disabled. This effect may be applicable to many tumor histologies irrespective of specific genetic makeup. To date, survivin inhibitors have shown modest activity as single agents, but it is anticipated that when given in combination with cytotoxic chemotherapy or monoclonal antibodies they may exhibit enhanced efficacy. This review discusses the complex circuitry of survivin in human cancers and highlights clinical trials involving novel agents that target this important protein. Introduction Survivin (BIRC5), is a member of the family of inhibitors of apoptosis proteins (IAPs) [1,2] of which eight members are known, including X-linked inhibitor of apoptosis (XIAP), cIAP 1 , cIAP 2 , NAIP (NLR family, apoptosis inhibitory protein), livin, ILP2 (IAP-like protein 2), BRUCE and survivin [3,4]. Survivin, the smallest family member, is a 142-amino acid, 16.5 kDa protein encoded by a single gene located on the human 17q25 chromosome, consisting of three introns, and four exons [2,5,6] and exists physiologically as a functional homodimer [7,8]. Alternative splicing of survivin pre-mRNA produces five different mRNAs with the potential to encode up to five distinct proteins, survivin, survivin 2B, survivin ΔEx3, survivin 3B and survivin 2α [9][10][11]. Survivin has been implicated in both control of cell survival and regulation of mitosis in cancer [5,[12][13][14]. Survivin is preferentially and highly expressed in cancer cells, with little expression in most normal non-dividing adult tissues (Table 1) [5]. The integral role of survivin in cancer cell division and survival makes it an attractive therapeutic target to inhibit cancer cell growth [1,2]. It was originally suggested that survivin inhibits cell death induced via the extrinsic and intrinsic apoptotic pathways and confers resistance to apoptosis by directly suppressing caspase activity [14]. Although the exact mechanism of action is unknown, current evidence is that most IAPs, including survivin, block apoptosis by mechanisms other than by direct initiator or effector caspase inhibition [15][16][17]. Survivin is now thought to function upstream of the effector caspases by inhibiting caspase 9 [18], by forming a survivin-hepatitis B X-interacting protein (HBXIP) complex bound to pro-caspase-9 thereby preventing the recruitment of apoptotic protease activating factor 1 (Apaf-1) to the apoptosome [19]. Additionally survivin associates with XIAP enhancing its inhibition of caspase-9 activation [20]. Survivin is inhibited by SMAC/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI) which results in the displacement of bound IAPs, which, may then bind to and inhibit caspase function [21,22]. Some investigators have suggested that the primary function of survivin is in controlling cell division, rather than apoptosis inhibition [23,24]. Survivin is up-regulated during cell division and is closely associated with centrosomes and mitotic spindle microtubules. It controls chromosome spindle-checkpoint assembly, thereby ensuring normal cell division. Survivin is maximally expressed during the G 2 M phase of the cell cycle and exists predominantly as a multi-protein complex, known as the chromosomal passenger complex (CPC) [25][26][27]. By functioning in this complex survivin can facilitate accurate sister chromatid segregation and stabilization of the microtubules in late mitosis [23]. In addition to its direct role in carcinogenesis, survivin may also play a key role in tumor angiogenesis as it is strongly expressed in endothelial cells during the proliferative phase of angiogenesis [12,28,29]. Manipulating the survivin pathway may facilitate endothelial cell apoptosis and promote vascular regression during tumor angiogenesis [29]. Increased expression of survivin also appears to be associated with an increased risk of tumor progression and chemoresistance in many tumor types [30][31][32][33][34][35][36][37][38][39][40][41]. Results of in vitro and in vivo studies have shown that survivin down-modulation reduces tumor-growth and sensitizes tumor cells to chemotherapeutic agents such as taxanes, platinum agents, etoposide, gamma-irradiation, and immunotherapy [42]. As an example, resistance to docetaxel is associated with increased levels of survivin [43], and response is often associated with the degree of expression of the various survivin splice variants [44]. Mechanism of Action of Survivin Cellular apoptosis is controlled by two pathways. The extrinsic pathway is critical for immune selection and inflammation. It is initiated by the activation of cell death receptors, such as tumor necrosis factor alpha (TNF-α) receptor, located at the cell membrane. The intrinsic pathway is initiated by toxic insult such as radiation or chemotherapy (DNA damaging and antimicrotubule agents) [45]. The two pathways converge at caspase-3 and follow a common process of activating caspases that cause cell death by cleaving essential substrates for cell survival, such as cytoskeletal proteins, DNA repair proteins, and inhibitory subunits of endonucleases [46]. Upon activation of the intrinsic pathway, mitochondrial permeability is increased resulting in the release of both cytochrome C and SMAC/DIABLO. Cytochrome C activates the apoptosome, which in turn activates caspase-mediated proteolysis involved in cell death [47]. SMAC/DIABLO acts as an inhibitor of the IAPs. Upon activation of pro-apoptotic cell signaling, survivin is released from the mitochondria and inhibits caspases-3 and -9. This function requires association with hepatitis B X-interacting protein (HBXIP) and/or with Xlinked IAP (XIAP) and is inhibited by SMAC-DIABLO [47]. The regulation of survivin expression and function is complex and occurs at various levels, including transcription, differential splicing, protein degradation, and intracellular sequestration via different ligands [47]. Survivin expression is up-regulated at a transcriptional level by both nuclear factor-appa β (NF-B) which in turn, can be activated indirectly by growth factors via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and by TCF-4/β-catenin pathway [48][49][50]. The insulin like growth factor-1/mTOR/RAS and Wnt-2 signaling pathways have also been reported to up-regulate survivin via rapid changes in mRNA translation [51][52][53]. Survivin degradation occurs via the ubiquitin-proteasome pathway in the G 1 phase of the cell cycle and is stabilized when bound to heat shock protein 90 (Hsp90) [54]. The survivin protein is closely associated with Cdc2/Cdk1 and it is phosphorylated at the threonine-34 (T 34 ) residue. This phosphorylation stabilizes the protein and allows it to interact with the mitotic spindle and inhibit caspase-9 [55]. There is accumulating evidence that molecular chaperones play a key role in the regulation of survivin. Binding of survivin to the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) [56] or to heat shock protein-90 (Hsp90) [54] maintains its stability against proteasome dependent destruction. Heat shock protein-60 (Hsp60) has also been identified as a molecular chaperone for survivin [57]. Acute ablation of Hsp60 by small interfering RNA (siRNA) has been shown to destabilize the mitochondrial pool of survivin leading to enhanced mitochondrial dysfunction and caspase-dependent apoptosis. This response involves disruption of the Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and subsequent Bax-dependent apoptosis [57]. Table 1 Over-expression of survivin in common human malignancies Survivin as a Regulator of Cell Division Survivin plays a central role in cell division, where its expression is coordinated within the cell cycle [2,47]. Survivin levels increase in G 1 and peak in the G 2 M phase. During mitosis, survivin functions as a regulator of microtubule dynamics and as part of the chromosomal passenger complex (CPC). Survivin functions both at the centrosomes and the microtubules of the metaphase and anaphase spindle providing stabilization and ensuring accurate separation of sister chromatids [2,58,59]. Survivin also localizes to the kinetochores, the mid-region or centromeric portion of the metaphase chromosomes. Here survivin is associated with regulators of cytokinesis, such as Aurora B kinase, the inner centromere protein antigens (INCENP), and Borealin/Dasra [60,61]. This supports the hypothesis that survivin acts as a subunit of the CPC which is required for proper chromosome segregation and cytokinesis [62]. It follows that if survivin is removed from the system, the kinetochore-microtubule system is not properly formed, cell division is either halted or improperly completed, and ultimately cell death occurs. Survivin is also bound to microtubules located in the mitotic apparatus during cell division. Through its association with cyclin-dependent kinase 1 (CDK1), microtubule-bound survivin becomes phosphorylated on Thr34 [1]. This leads to stabilization of the protein and efficient counter-activation of apoptosis in dividing cells. Elimination of survivin leads to apoptosis of dividing cells. Survivin Expression in Cancer Cells Survivin is undetectable in most non-proliferating adult tissues. Exceptions are CD34+ hematopoietic stem cells, placenta, basal cells of the colonic epithelium, and thymus [5]. On the other hand, survivin is over-expressed in a wide variety of cancers (see Table 1) [5]. Overexpression is correlated with advanced disease, accelerated time to recurrence, reduced survival, and resistance to therapy [38]. Survivin has been identified as one of 16 genes within an mRNA expression signature that, in patients with breast cancer, correlates with a poor response to tamoxifen, but a good response to chemotherapy [63]. Furthermore, inhibition of in vitro or in vivo survivin expression by antisense oligonucleotides, or inhibition of survivin function by in vitro inhibition of CDK1-mediated phosphorylation leads to cell death [58]. This is particularly apparent when CDK1 inhibition is combined with taxanes [58]. Recently the mTOR pathway, which can act as a sensor network in stressed conditions, has been implicated in elevating survivin levels [51]. In prostate cancer cells, insulin-like growth factor-1 (IGF-1)-mediated mTOR pathway activation positively modulated survivin levels by increasing the translation of a survivin mRNA pool [51]. The role of survivin in malignant melanoma Genes that are highly expressed in aggressive melanomas includes many with roles in cell cycle regulation/proliferation, DNA replication/repair, and apoptosis pathwayrelated genes. Survivin is variably expressed in the cytoplasm across the spectrum of melanocytic lesions, with nuclear expression detectable in a subset of malignant melanomas, but not in benign or dysplastic nevi [64]. Nuclear expression has been reported to be an independent predictor for disease recurrence and decreased overall survival in patients with early stage cutaneous melanoma [65]. Patients with nuclear immunoreactivity for survivin showed an increased risk of melanoma recurrence during the first three post-operative years and an increased risk of death. Cytoplasmic survivin staining has not demonstrated a correlation with patient survival [65]. This suggests that the nuclear localization of survivin may play a role in the transformation process. Chen and co-workers demonstrated the up-regulation of survivin in primary melanomas as compared to benign nevi [66]. Similarly, Nasr et al found immune-histochemistry (IHC) nuclear staining for survivin was present in 12 of 18 cases (67%) of malignant melanoma with an average index of 7% (range 0%-15%), and no nuclear staining was present in any benign lesions examined [67]. Expression of survivin, bcl-2, bax, and bcl-X in sentinel lymph node (SLN) biopsies was assessed in 36 patients with stage I and II melanoma using RT-PCR and Southern blotting and correlated to overall survival. Survivin expression correlated with the outcome of patients in a statistically significant way (P < 0.005), whereas the expression of bcl-2, bax, and bcl-X, did not seem to correlate to progression of disease [68]. In this study, 61.5% of patients expressing survivin in the SLN progressed or died of melanoma. In patients negative for survivin expression, 100% were disease-free at a median follow-up time of 52.9 months suggesting that survivin gene expression in SLNs may be a useful prognostic indicator. There was no correlation between bcl-2, bax, and bcl-X gene expression and outcome [68]. Survivin expression has been studied in a limited number of non-cutaneous melanomas. In uveal melanomas, expression of survivin was reported to be low and did not correlate with outcome, resistance of the tumor to brachytherapy, or the presence of liver metastases [69]. Survivin expression has also been reported in a case of esophageal melanoma [70], but its impact is unclear. A phase II study investigated YM155 (section 5.1), a small molecule survivin suppressor as a single agent first line treatment in 34 patients with metastatic melanoma. One patient had a complete response, one had a partial response, and 11 had stable disease 97 . The role of survivin in solid tumors Survivin expression has been detected in a wide variety of benign and pre-neoplastic lesions including polyps of the colon, breast adenomas, Bowen's disease and hypertrophic actinic keratosis [71]. Survivin has been shown to play an important role in colorectal tumorigenesis by stimulating the transition of adenomas with mild dysplasia into highly dysplastic lesions [72]. This transition was associated with a significant decrease in the apoptotic index (AI) and significant increases in the Ki-67 labeling index (LI) and microvessel density (MVD) (P < 0.001). The expression of survivin inversely correlated with AI and was positively correlated with Ki-67 LI and MVD (P < 0.001). In esophageal cancer, survivin expression has been correlated with a poor prognosis whereby the median survival for patients with high levels of survivin expression was reduced compared to patients with low expression levels [73]. In this study the median survival for patients with advanced esophageal cancer and with high levels of survivin expression was 9.0 months compared to 30.0 months for those patients with low survivin expression (p = 0.0023) suggesting that survivin expression may provide prognostic information. Similarly patients with advanced gastric cancer with survivin-positive tumors have a significantly lower 5-year survival rate compared to patients with survivin-negative tumors (P < 0.05) [74]. In non-small cell lung cancer (NSCLC), survivin is over-expressed in approximately 80% of tumors and its presence is associated with a reduced survival in patients with resected neoplasms [75,76]. Expression of survivin also correlates with resistance to therapy. In prostate cancer cells treated with docetaxel, apoptosis is induced by the binding of survivin to SMAC/DIABLO and to the mitotic spindle [77]. Nakahara et al have reported that YM155 (Section 5.1) induced regression of established hormone refractory prostate cancer (HRPC) xenografts [78]. In vitro, docetaxel resistance was reversed in gastric cancer cells when messenger ribonucleic acid (mRNA) expression of survivin was down-regulated by gambogic acid, a survivin inhibitor [43]. Preliminary results are available for a phase II trial investigating 168-hour continuous iv infusion of YM155 (Section 5.1) in patients with metastatic HRPC who have received prior taxane therapy. The primary endpoint of this trial is PSA response rate. Data on 32 patients has been reported and 2 PSA responders were seen indicating YM155 may have some activity in HRPC [79]. An ongoing study is investigating the combination of YM155 and docetaxel in patients with HRPC and preliminary data appear promising. Survivin overexpression has been associated with platinum and taxane-based chemotherapy resistance in a number of solid tumors [80,81]. It is postulated that vascular endothelial growth factor (VEGF) induced angiogenesis may lead to a transient increase in survivin levels [82] suggesting a role for combining survivin inhibitors with anti-VEGF strategies. The role of survivin in hematological malignancies Survivin expression has been observed in the majority of malignant lymphoproliferative disorders with the exception of chronic lymphocytic leukemia in which it is expressed at levels significantly lower than that found in normal lymphocytes [83]. Different techniques for detecting survivin expression have been used including immunohistochemistry (IHC), immunoblotting, and messenger RNA (mRNA) detection; however, no studies comparing these methods have been performed. Three trials have utilized IHC to determine expression of survivin in diffuse large B cell lymphoma (DLBCL) and one study used mRNA expression. The largest study included 222 patients from four separate randomized trials [31]; however, the interpretation of the effect of survivin expression on outcome is complicated by the fact that different chemotherapy regimens were used in the different trials. Although most regimens contained an anthracycline, usually doxorubicin, or mitoxantrone, some regimens did not. In this report, tumors were characterized as being positive for survivin expression if cytoplasmic staining was demonstrated in 70-90% of cells, and negative if less than 5% of the tumor showed staining. Survivin expression was observed in 60% of the patients' tumors and was found to confer an inferior outcome. Patients whose tumors did not show expression of survivin had a 5-year survival of 54%, whereas only 40% of survivin-positive cases were alive at five years (P = 0.02). The complete remission rate was lower in the survivin-expressing group, but not significantly different from the survivin-negative cases at 61% versus 68%, respectively (P = 0.29). This resulted in a lower event-free survival in the survivin-positive cases, but the difference did not achieve statistical significance (p = 0.09). The survival difference was examined in a multivariate analysis incorporating the International Prognostic Index (IPI) and was found to be independent of the IPI score. In two other reports that included 60 and 39 patients, respectively, the nuclear localization of survivin was associated with an inferior outcome in one, but not the other [84,85]. The relatively small numbers of patients in these studies may have accounted for this disparity. The statistical significance was lost when the patients in the larger study were grouped according to whether they represented a germinal center or nongerminal center DLBCL. There was a trend towards a worse outcome in both subgroups, but the difference did not achieve significance. Survivin expression has also been evaluated using an RNAse protection assay [86]. Here the authors reported that survivin mRNA expression was increased in 80% of the tumors examined, but it did not impact on overall survival. Survivin protein expression was not simultaneously examined and it is possible that translational regulation could have resulted in a disparity in the mRNA and protein expression levels. A recent report examining the relationship between survivin mRNA and protein levels in patients with HTLV-1associated adult T cell leukemia/lymphoma (ATLL) found a strong correlation between mRNA levels and protein expression [87]. There has been increasing interest in survivin as a potential therapeutic target in lymphoma. Antisense oligonucleotides (ASO) have been shown to inhibit survivin expression in a number of DLBCL cell lines. These ASO inhibited cell growth in vitro and slowed tumor growth in animals [88]. Clinical studies of investigational survivin inhibitors are ongoing in a number of hematological malignancies. There was great enthusiasm for the small molecule imidazolium-based survivin transcriptional inhibitor YM155 (Section 5.1) following the phase I trial as responses were observed in three of five patients with relapsed B cell lymphoma, including two patients with DLBCL [89]. These results were not reproduced in the phase II trial that included 41 patients with relapsed and refractory DLBCL [90]. At the time of the abstract submission, only one of the first 25 patients enrolled and evaluated for tumor response showed an objective response. YM155 was well tolerated with grade 3 or 4 anemia (16%), neutropenia (8%), fatigue (8%), and deep venous thrombosis (8%) as the only events that occurred in more than 4% of the patients. Due to the potential for synergism of YM155 with other agents, additional trials are underway or planned combining YM155 with chemotherapy or monoclonal antibodies including rituximab and alemtuzumab. Survivin expression may have even greater prognostic significance in patients with T cell lymphoma [50,87,91,92]. High levels of expression have been reported in patients with HTLV-1-associated ATLL. Survivin levels were greater in the aggressive acute subtype of the disease compared with the more indolent chronic form of ATLL. ATLL patients whose tumors expressed high levels of survivin had an inferior outcome, with a median survival time of 6.4 months compared to 18 months in those patients with low survivin levels [93]. These results have been reproduced in a number of other studies and are similar to the results seen in DLBCL. Inhibition of survivin expression in primary ATL cells using shRNA leads to decreases in tumor cell viability. Anaplastic large cell lymphoma (ALCL), another relatively uncommon T cell neoplasm, is separated into two prognostic groups based on the expression of the anaplastic large cell kinase gene (ALK). The expression of ALK is most frequently the result of a translocation between chromosomes 2 and 5, resulting in a unique fusion of the ALK and nucleophosmin genes, respectively. ALK-positive ALCL has a superior clinical outcome with about 80% of patients cured with current chemotherapy. In contrast, patients with ALK-negative ALCL demonstrate a worse prognosis with a 50% 5-year survival. Survivin expression was examined in ALCL patients by IHC and was found to be frequently positive with primarily cytoplasmic localization [92]. Approximately 63% of ALK-positive ALCL tumors expressed survivin compared with 47% of the ALK-negative ALCL. Absence of survivin expression in both subtypes conferred an improved prognosis with 100% of the ALKpositive, survivin-negative patients alive at 5 years, and 89% of the ALK-negative, survivin-negative patients alive at 5-years. In contrast only 34% of ALK-positive, survivin-positive patients were alive at 5 years. For the ALK-negative group, the 5-year overall survival was 60% for patients with survivin-positive tumors versus 92% for patients with survivin-negative tumors (P = .04). Survivin expression remained an independent adverse prognostic marker in a multivariate analysis that included IPI score. Current therapeutic approaches Transcriptional repressors YM155 (1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-ihydro-H-naphtho[2,3-d]imidazolium bromide) (Astellas Pharma Inc) is a small-molecule suppressor of survivin. YM155 interacts specifically with the 269 base pair survivin core promoter region and functions in a cell cycle independent manner as a transcriptional inhibitor. YM155 has demonstrated potent antiproliferative activity against various cancer cell lines and preferentially induces cell death in tumor cells. Preclinical data has also shown that it has the ability to serve as a radio-sensitizing agent and to potentiate the antitumor activity of various cytotoxic agents including carboplatin and paclitaxel [94,95]. In preclinical experiments, YM155 has shown promising activity in a wide variety of human tumor xenograft models including non-small cell lung cancer (NSCLC) [96]. YM155 has been investigated in six phase I or II clinical trials in solid tumors and non-Hodgkins lymphoma. It has been shown to be well tolerated with the most common toxicities being reported as grades 1-2 in severity and consisting primarily of stomatitis, pyrexia and nausea [97][98][99][100]. In a phase I study conducted in the United States, 41 patients received YM155 at doses ranging from 1.8 to 6.0 mg/m 2 /d by a 168-hour (7 days) continuous intravenous infusion every 3 weeks. The maximum tolerated dose (MTD) was determined as 4.8 mg/m 2 . In a second phase I trial conducted in Japan [101], patients with advanced refractory solid tumors were treated with escalating doses of YM155 administered by continuous i.v. infusion for 168-hours in 21-day cycles. Of the 34 patients enrolled, 33 (median age, 59 years) received at least 1 dose of YM155 and the MTD was determined to be 8.0 mg/ m 2 /d. The most common adverse reactions judged to be related to YM155 were microalbuminuria, fever, injectionsite phlebitis, fatigue, and decreased hemoglobin/anemia, serum albumin, and lymphocyte count. As monotherapy, YM155 has shown modest antitumor activity in a phase II trial in patients with advanced NSCLC who had failed one or two prior chemotherapies [98,102]. Two partial responses were observed in 37 patients (5.4%) enrolled in this study, where YM155 was administered as a 7 day continuous infusion at 4.8 mg/m 2 / day, repeated every 3 weeks. Fourteen patients (37.8%) achieved stable disease resulting in a disease control rate of 43.2% (95% CI, 27.1% to 60.5%). Median PFS was 1.7 months (95% CI, 1.3 to 2.8 months). Median overall survival was 6.6 months (95% CI, 4 to 12.2 months), with a 1-year survival rate of 35.1%. Treatment was well tolerated with the majority of treatment discontinuations not YM155 related. A phase I/II study evaluating the combination of YM155 in combination with carboplatin and paclitaxel has now been initiated. YM155 is also being investigated in malignant melanoma, HER-2/neu-negative breast cancer, and in combination with docetaxel in hormone refractory prostate cancer (see Table 2). Terameprocol (EM-1421, Erimos Pharmaceuticals) is a semi-synthetic small molecule with antitumor activity occurring via selective targeting of Sp1-regulated proteins, including survivin and cyclin dependent kinases (cdc2) that control cell cycle and apoptosis [103,104]. Terameprocol is in clinical development as a site-specific transcription inhibitor in solid refractory tumors and in leukemias. mRNA Inhibitors LY2181308 (ISIS/Eli Lilly Pharmaceuticals), a novel modified ASO, is a specific inhibitor of survivin. In a phase I trial in which 24 patients were enrolled, LY2181308 showed a safety and pharmacokinetic (PK) profile consistent with previously described ASOs. Side effects were mild to moderate and no grade 3 or 4 toxicities were described at the MTD of 750 mg [105]. Additionally, a pharmacodynamic study has been conducted in 34 patients, including 22 patients with available pre-and post-treatment biopsies. IHC indicated that survivin expression was reduced in the nucleus and cytoplasm in 11 of 17, and 5 of 14 evaluable pairs, respectively. Gene expression analysis indicated a reduction in survivin expression of 20-50% in 11 of 15 evaluable pairs. Analysis of fresh tumor from endobronchial sampling revealed that 2 of 3 NSCLC patients had a near-complete elimination of survivin-positive cells accompanied by an increase in the fraction of cells with a sub-G1 DNA content, consistent with cell death [106]. Furthermore, a human micro-dosing imaging PK study of an ASO with LY2181308 using carbon-11 radiolabeled LY2181308 ([ 11 C]LY2181308) was conducted. In this study pharmacokinetic analysis confirmed that biologically active human tumor drug concentrations of [ 11 C]LY2181308 can be achieved. LY2181308 therapy saturated normal tissue kinetics and increased tumor uptake of [ 11 C]LY2181308 [107]. LY2181308 is currently being evaluated in phase II trials for relapsed and refractory acute myeloid leukemia, prostate cancer and NSCLC (see Table 2). Other approaches for inactivating specific mRNAs that remain predominantly in the preclinical setting involve the use of hammerhead ribozymes [108,109] and small interfering RNAs [110,111]. Small molecule survivin inhibitors Shepherdin is a small molecule inhibitor in early stage clinical development that acts as an antagonist of the survivin-Hsp 90 complex [112]. This molecule is a 5 amino acid peptide that antagonizes the binding between survivin and Hsp-90 as well as acting as a global inhibitor of Hsp90 function via competition with ATP. Immunotherapy Due to its differential expression by tumors, it has been hypothesized that cancer patients may recognize survivin as a "non-self" protein and mount an immune response against it [113]. Phase I trials using survivindirected autologous cytotoxic T lymphocytes acitvated with survivin-primed dendritic cells or survivin peptides have been performed. Treatment was well tolerated with limited toxicity [114,115]. Wobser et al, described an early example of survivin-based vaccination therapy in a patient with metastatic pancreatic cancer that achieved a complete regression of liver metastasis after receiving a HLA-A2 restricted survivin peptide plus adjuvant [116]. Recently a phase I-II trial of vaccination with HLA-A*0201-restricted peptides from prostate specific membrane antigen (PSMA) and survivin was carried out in 20 prostate cancer patients with biochemical failure after surgery or radiotherapy. The vaccine consisted of two peptides from PSMA (PSMA4-12 and PSMA711-719) and one from survivin (SVV96-104/97M) given by 4 biweekly (priming) and then 4 monthly administrations (boosting). To selectively eliminate regulatory T cells (Tregs) and possibly enhance immunization, the vaccinations were preceded by cyclophosphamide 300 mg/kq, i. These results indicate that the vaccine-induced Ag-specific CD8+ T cells had a reduced ability to cross-recognize prostate cancer cells which could explain why PSA control was achieved only transiently [117]. The tolerability, immunogenicity, and clinical efficacy of three survivin peptides restricted to HLA A1, A2 and B35 were addressed in a phase I/II trial which included 79 patients with metastatic melanoma (n = 61), pancreatic (n = 8), cervical (n = 5), colorectal (n = 2), adrenal gland (n = 2), and Merkel cell carcinoma (n = 1) who failed to respond to systemic standard therapy. Peptide vaccination was safe and vaccine-specific immune responses were induced in 50% of the patients. Objective responses and control of disease were essentially restricted to immunological responders. Three complete responses and three partial responses were observed (OR = 7.6%) with the duration of response ranging from 3 to 36+ months [118]. Due to these encouraging results, ongoing Phase II studies are investigating survivin based immunotherapy in patients with advanced pancreatic, colon and cervical carcinomas as well as in melanoma. Survivin as a radiosensitizer Survivin is known to play an important role in the sensitivity of cells to radiation with high survivin levels corresponding to reduced radiation sensitivity [119,120]. In a number of preclinical studies inhibition of survivin expression has been shown to sensitize tumor cells to ionizing radiation [121][122][123][124]. Although apoptosis does not play a major role in the response of solid tumors to radiation as a single agent, inhibition of survivin prior to irradiation leads to an increase in apoptosis and reduced tumor cell survival [121]. In addition, survivin inhibition interferes with normal cell cycle progression and DNA repair [125,126]. Consistent with these observations, a number of small molecule inhibitors of survivin have been shown to enhance the cytotoxic effects of radiation [127]. Collectively, these findings provide a strong rationale for the clinical evaluation of survivin inhibitors administered either concurrently or sequentially with therapeutic radiation. Conclusion Over the last decade it has become increasingly clear that inhibitor of apoptosis proteins play an integral role in maintaining cellular homeostasis. In particular, one of these proteins, survivin serves many functions involved in cell survival including complex intracellular signaling, stabilizing mitosis and facilitating cellular adaptation. Much remains to be learned regarding the biology of survivin and other IAPs in terms of how these molecules intersect with other pathways. Survivin is a highly expressed in many different tumors and its expression correlates with advanced disease, poorer survival, and chemotherapy and radiation resistance. Because of the role it plays, survivin is of increasing interest as a potential therapeutic target in cancer. Survivin antagonists may function not as single protein inhibitors, but rather as global pathway inhibitors that may disable multiple signaling circuits in tumors. Clinical trials have highlighted the problems with attempts to correlate survivin expression with clinical outcome. Small sample numbers, nonuniform treatments, the presence of multiple alternatively spliced survivin mRNAs with differing effects on apoptosis and the different methods of detection of survivin, all lead to difficulty in trial interpretation. Further efforts are required to achieve a greater understanding of the biology of survivin and the other IAPs and more effectively exploit strategies that target this protein in cancer. Authors' contributions RK: Conception and design, manuscript writing, final approval of manuscript AL: Manuscript writing, final approval of manuscript DC: Manuscript writing, final approval of manuscript JJ: Manuscript writing, final approval of manuscript JM: Conception and design, manuscript writing, final approval of manuscript Abbreviations ALCL: anaplastic large cell lymphoma; ALK: anaplastic lymphoma kinase; ATLL: adult T cell leukemia/lymphoma; HTLV-1: human T cell lymphotrophic virus-1; IAP: inhibitor of apoptosis protein; NSCLC: non-small cell lung cancer; VEGF: vascular endothelial growth factor; DLBCL: diffuse large B cell lymphoma; SMAC: second mitochondria-derived activator of caspases; DIABLO: direct inhibitor of apoptosis binding protein with low pI; mRNA: messenger ribonucleic acid; PK: phramacokinetics; TNF-α: tumor necrosis factor alpha; HBXIP: hepatitis B X-interacting protein; XIAP: X-linked IAP; Hsp90: heat shock protein 90; INCENP: inner centromere protein antigens; CDK1: cyclin-dependent kinase 1; CPC: chromosomal passenger complex.	2017-06-25T11:56:07.924Z	2011-04-06T00:00:00.000	{ "year": 2011, "sha1": "7e8ce3056c114e9940a8516d9d3b6cf07651f9dd", "oa_license": "CCBY", "oa_url": "https://molecular-cancer.biomedcentral.com/track/pdf/10.1186/1476-4598-10-35", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1a5c6d96661608a9d98b3a2256c31a4f39a65b6e", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
181426198	pes2o/s2orc	v3-fos-license	Block chain Enabled E-Voting System - The term “vote” is referred as to opt among the eligible candidates. The prime objective of registering a vote is to pick the best among the capable candidates who can serve the people in better way. Many nations like India have several obstacles during the elections. Several obstacles includes turn-over of votes at the time of election, uncertain or isolated polling terminals, poor polling stuff and lack of workforce skills. Developing a secure smart electing approach to offers simplicity & confidentiality over existing voting systems. It is challenging to provide flexibility through present voting systems. Using block chain as technique to enact this approach. Blockchain have few drawbacks in current approach and assess major block chain architecture for implementing a blockchain-enabled electing approach. This approach enhances safety and minimizes price of conducting elections. INTRODUCTION 2.  "ONLINE VOTING SYSTEM" is an online voting technique.The persons who hold citizenship of India and their age is greater than 18 year is eligible to register their choice.A specific database is preserved in which complete information about the users is kept. Voting and elections are necessary elements of democratic societies.  E-voting briefs about registering choice of users voting through electronic means to look after the routine tasks such as choice of users and sum up the choices.  It is significant widely known zone which is implemented using block chain approach.  Block chain is shared database which preserves the information which is keep on increasing supervised by several units.  In "Online Voting System" users who are eligible are given freedom to register their choice. Primarily, the users needs to register to elect their choice.Registration is majorly carried out by system admin for safety perspective.Users are asked to fill registration form to register their choice.Following Registration the users information will be validated to grant eligibility to elect and will be provided a unique voter identification which is helpful to log on to the portal and allowed to register their choice.  If the information provided by the users is found to be wrong then the user is not allowed to participate in the electing process. LITREATURE SURVEY According to NirKshetri et.al [1], Electronic-Voting is one of the essential public sector that can be implemented byblock chain approach.To use digital currency analogy, block chain enabled voting which provide every user a pocket consists of aspirant information.Every user receives a coin indicates single occasion to register choice. According to Fridrik P Hjalmarsson et.al [2], this paper directs to examine the block chain approach to build decentralized e-voting method.It analyses few of the major block chain configuration.More likely it analyse capability of decentralized log by detailed study. According to Ahmed Ben Ayed [3], Blockchain is offering new platforms to implement new kinds of digital solutions. The block chain approach is secured and provides reliability..It helps in maximize the users and also belief of peoples towards the voting system. According to Freya Sheer Hardwick et.al [4], this proposal hopefully offer a distributed design to aid voting system.It is configured in such a way that adhere to principles of electronic-voting characteristics as well as provides decentralization. EXISTING SYSTEM Currently many countries including india are using electronic voting machines which is generally called as EVM, which consists of mainly two components control unit which is supervised by polling officer in the voting booth and another one is balloting unit which is the point where people register their choice.The balloting unit accepts input only when polling officer enables it in control unit. Electronic voting machines has limitation that an individual ballot unit can consists of maximum of 16 candidates or party and for control unit 4 such ballot units are connected in series. While voting the peoples who cast their votes are marked with electoral ink which is made up of phosphor which is semi-permanent, this process is done to avoid double voting by same set of individual peoples.In some cases ballot paper is used for voting which contains a set of eligible candidates and seal is pressed in front of candidates name and dropped into the box which is opened during the counting where every papers should be opened and evaluate the vote. METHODOLOGY Using block chain technology as a method to implement the electronic voting which consists of blocks of particular block size and block header which is encrypted by the hash i.e unique for individual block and each block contain hash of the previous block.In this way the chain of blocks is created which is immutable i.e once entered data cannot be altered.The system also consists of transaction counter which counts the number of transactions that occurs in the system in our case each vote is considered as a single transaction. The block chain technology also protects the privacy of individual votes as the voted information cannot be revealed to third parties or any higher authorities and it www.helps to verify that the casted vote is counted or not during announcing of results.As the block chain is decentralized no central authority will be controlling the process.It is secured, distributed and cannot be corrupted. SYSTEM DESIGN The two major units of voting system using block chain technology are users the one who cast their vote and another is admin the one who supervise transaction and hashing algorithm.Primarily, the user needs to register by providing necessary documents.Only the registered users are allowed to cast their vote.Once user is registered, the user can carry out operations like login and logout, creating account etc.After registration the user will be provided with unique identification ID from admin side. To cast vote, user needs to logon to the portal with unique identification ID provided and register their choice among the eligible candidates by confirming their vote through unique identification ID.The vote casted by user is recorded at respective district.After successful casting of vote the user will receive a transaction ID and will be given an option to print their the same.The votes casted by users are authenticated and one vote will be appended to the candidate to whom the vote has been casted for.All casted votes which were received and authenticated are deployed onto the block chain with each new vote appended to the block chain and respective district zone will updates the copy of log. It consists of four layers: Client layer: It is designed using html, css, java script.Web layer: It has servlets and java server pages Business logic layer: It has configuration of classes, interface, implementation and logic instances Database layer: It is the last layer which contains the transactions in tables by using relationships among them. CONCLUSION Blockchain approach is helpful to overcome the drawbacks which are in the current voting approach.By using this technology election can be conducted with good percentage of voting and we can involve the number of citizens in voting which provides good results for elections.No one can miss the elections in our country. Cost for conducting the elections are also reduced to maximum.. Thus helping the voters to vote from anywhere using this system. The thought of having block chain voting approach to create the electorol process is speedy and simple in the modern society.The voting procedure is cheaper and quicker, simplifies in front of one who vote and eleminates power barrier that may exists between the user and the official who are elected.It opens up the door to the straight form of democracy, permiting one who votes to convey their will to individuals.	2019-06-07T20:44:05.621Z	2019-04-30T00:00:00.000	{ "year": 2019, "sha1": "b505c9fb2959d555a1cdca3fdabf50e1b1f0dc75", "oa_license": null, "oa_url": "https://doi.org/10.17148/ijarcce.2019.8412", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "5ac17cb3b4902b74f9c01fa69d64682435bc2a3f", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Computer Science" ] }
119216769	pes2o/s2orc	v3-fos-license	An O(N) and parallel approach to integral problems by a kernel-independent fast multipole method: Application to polarization and magnetization of interacting particles Large classes of materials systems in physics and engineering are governed by magnetic and electrostatic interactions. Continuum or mesoscale descriptions of such systems can be cast in terms of integral equations, whose direct computational evaluation requires O(N^2) operations, where N is the number of unknowns. Such a scaling, which arises from the many-body nature of the relevant Green's function, has precluded wide-spread adoption of integral methods for solution of large-scale scientific and engineering problems. In this work, a parallel computational approach is presented that relies on using scalable open source libraries and utilizes a kernel-independent Fast Multipole Method to evaluate the integrals in O(N) operations, with O(N) memory cost, thereby substantially improving the scalability and efficiency of computational integral methods. We demonstrate the accuracy, efficiency, and scalability of our approach in the contest of two examples. In the first, we solve a boundary value problem for a ferroelectric/ferromagnetic volume in free space. In the second, we solve an electrostatic problem involving polarizable dielectric bodies in an unbounded dielectric medium. The results from these test cases show that our parallel approach, which is built on a kernel-independent FMM, can enable highly efficient and accurate simulations and allow for considerable flexibility in a broad range of applications. I. INTRODUCTION Massively parallel computer hardware, and the corresponding software, have enabled solution of increasingly complex problems in science and engineering. Such problems are often cast in terms of partial differential equations (PDEs). In many cases, it is convenient to formulate the PDEs in terms of integral equations, which can then be solved by relying on a variety of numerical methods. For the particular case of boundary integral equations, the Boundary Element Method (BEM) 1 has found numerous applications in physical problems ranging from solids and fluids 2 , to multiphase materials 3 , heat transfer 4 , electrostatics 5 , and magnetostatics. For example, BEMs were used in fluid problems to study the motion and deformation of bubbles 6 and to determine the phase diagram of complex fluids 7 ; BEMs have also been used to address three-dimensional (3D) linear elasticity problems involving particles embedded in a binder 8 , and in crack analysis in 3D time harmonic elastodynamics 9 . BEMs are particularly well-suited for problems that involve bodies embedded in an infinite medium, with the boundary condition that the relevant potential decays to zero at infinity. If the embedding space is homogeneous and the governing equations for the underlying physics are linear, BEMs are frequently used in order to avoid explicitly including the embedding space exterior to the bodies. In this work, we consider two versions of this kind of problem as explicit test cases: interacting ferromagnetic bodies embedded in space, and dielectric bodies embedded in an infinite dielectric medium. However, our proposed method is widely applicable to problems in which the mathematical formulation can be cast in terms of a boundary integral equation. For the problem of interacting ferromagnetic bodies embedded in space, the interactions between the bodies are calculated using an open boundary condition on their boundaries. Specifically, the non-zero, finite magnetization of the bodies leads to a discontinuity in the magnetostatic potential at their boundaries. One approach to this problem is to use a hybrid finite element (FEM)-BEM to calculate the non-local magnetostatic interactions between the magnetic bodies 10 . The method has the advantage that it allows for arbitrary-shaped bodies and requires no mesh between them. A disadvantage is that the integral of the Green's function over the boundary of all magnetic bodies leads to a computational complexity of O(N 2 ) in direct implementations, where N is the number of degrees of freedom (DoFs) on the boundaries of all magnetic bodies. Several techniques have been proposed to increase the efficiency of the boundary integral calculation. Examples include the tree-code algorithm and the hierarchical matrices method 11,12 , which exhibit computational complexities of O(N log N) and O(N), respectively. In electrostatics, a similar class of problems is concerned with the electrostatic polarization of finite-size dielectric bodies embedded in an infinite dielectric medium. Free charges carried by these bodies, or free point charges in the medium, are sources of electrostatic fields. Because the relative permittivity of the medium is usually different from those of the bodies, the electrostatic field is discontinuous across the interface between a body and the medium. As a consequence, bound surface charges are induced on the interface to compensate for the discountinuity in the electrostatic field. In order to calculate the induced bound charges on the interface, a BEM was originally proposed using a variational approach 5 to solve the underlying Poisson equation. In numerical simulations of the polarizable bodies, BEMs have been used for stationary or mobile dielectrics [13][14][15][16][17] . A central aspect of this class of problems is the calculation of the distribution of bound surface charge density through solutions of a linear system of equations Ax = b, where A is a dense matrix whose entries depend on the geometry of the dielectric bodies, x represents the induced bound surface charge density, and b represents contributions from free charges on the dielectric bodies, or free point charges in the exterior medium. The iterative Generalized Minimal Residual Method (GMRES) 18 provides an efficient numerical method to solve the resulting dense linear system of equations 19 . However, the matrix-vector multiplication in each GMRES iteration has a computational complexity of O(N 2 ) in any direct implementation. In Ref. 17 and 19, the matrix-vector multiplication is accelerated using a fast Ewald solver, i.e., the particle-particle particle-mesh method (PPPM or P 3 M) 20 , which offers a computational complexity of O(NlogN). In this work, we have developed an efficient and scalable parallel computational approach for BEMs that is built on the scalable parallel libraries libMesh (http://libmesh.github.io) 21 and ScalFMM (http://scalfmm-public.gforge.inria.fr) 22 . The boundary integrals are accelerated using a kernel-independent fast multipole method (FMM) 23 with O(N) computational complexity. This kernel-independent FMM is an interpolation-based FMM that utilizes Chebyshev interpolation and low-rank approximation, and can also be used for kernels that are known only numerically. It was first developed for non-oscillatory kernels and later extended for oscillatory kernels 24 . Our approach to boundary integrals also adopts a matrixfree method that requires no explicit storage of a global matrix, thus enabling simulations with a memory cost of only O(N). The corresponding software is is made freely available to potential users. In the remainder of this paper, we first introduce the general boundary integral problem. We then discuss methods for the magnetostatic and electrostatic problems, and our implementations of BEM; We apply our computational approach to several model problems and test its accuracy, efficiency, and scalability. We conclude with a summary and suggestions for future directions for improvements of the proposed approaches, and elaborate on several research areas and physical phenomena to which our approach could be applied. p i , where P is the total number of bodies and p i is the region occupied by ith body. The boundary the interior region is denoted as ∂Γ. A. General integral problems Confined geometries can also be considered as shown in Fig. 6 Many integral problems share similar configurations to those shown schematically in Fig. 1. Space is divided into an exterior region Ω and an interior region Γ consisting of the union of different bodies with arbitrary shapes, for example dielectric or ferroelectric/ferromagnetic bodies. It is assumed here that there are no intersections or contacts between any two bodies. In most problems, the solution is obtained from functions f (r) obtained from the integral over the boundary of all bodies (∂Γ) of a kernel K(r, r ′ ) and a source function g(r). The kernel is derived from a Green's function that represents the underlying physics of a delta-function source; thus In numerical integration of the above integral using a Gaussian quadrature rule, f (r i ) can then in general be expressed as Equation (2) is exactly the type of summation for which the FMM is used, with O(N) computational complexity rather than the O(N 2 ) complexity that results from using the direct method over all points r i and r j . The kernel-independent FMM enables fast computation of the integral with arbitrary kernels for a variety of problems; two specific test cases are considered in the following sections. B. Magnetostatic problem The magnetostatic problem is ubiquitous for any system that involves patterned micronor sub-micron size magnetic bodies, such as read or write heads in magnetic hard disk drives, magnetic nanoparticles 25 , coupled magnetic disks 26,27 , or artificial spin ices 28 . In patterned ferromagnetic systems, there typically arises a competition between the short-range exchange interactions and long-range magnetostatic interactions. These latter originate in the volume pole density ∇·M(r) and the surface pole densityn·M(r). The magnetostatic fields from the surface pole density then involves solving a Poisson equation with sources on the boundary of a finite body and with the boundary condition that the magnetic scalar potential goes to zero far away from the body. In the framework of a hybrid FEM-BEM, the magnetic scalar potential φ is split into φ = φ 1 + φ 2 , where φ 1 solves the Poisson equation in the magnetic region (see Fig. 1) Γ: with the boundary condition Here, M(r) is the magnetization density of the magnetized bodies p i , and n is the outwardpointing (from the interior of the magnetic region) unit normal vector on the boundary of the magnetic region (∂Γ) . After φ 1 is obtained by solving the Poisson equation Eq. (3), φ 2 is calculated first on ∂Γ by the standard double layer integral where r is the coordinate vector on ∂Γ, and Ω(r) is the solid angle subtended by ∂Γ at r. C. Electrostatic problem A large class of systems in materials science involve polarizable metal oxide colloidal or nanoparticles in a dielectric continuum. The presence of a particle exerts an electrostatic field on nearby particles, leading to their polarization, which then propagates throughout the entirety of the system in a cooperative manner. Under some circumstances, polarization interactions can in fact lead to attractive forces between charged particles having the same charge, a feature that has been revealed in striking detail for metalic oxide micro-particles 29 . Here we consider polarizable dielectric bodies p i embedded in an infinite dielectric medium Ω. Dielectric polarization arises on the surface of the bodies when the relative permittivities of the bodies are different from that of the medium. Bound surface charges are then induced because of the discontinuity of the electrostatic field across the interfaces between bodies and medium. In the following, we first review key equations that facilitate implementation of the BEM. For details on the derivation of the method, we refer to Refs. 5 and 17. The relative permittivity of the medium is ǫ m , the relative permittivity of ith body is ǫ i , and the point charge carried by the i-th particle is Q i . In this method, Q i is represented by an equivalent surface free charge density σ f on the surface of each body (see section IV.I in Ref. 17). In the particular case of spherical bodies, σ f = Q i /(4πr 2 i ) on the ith body with radius r i . The induced bound surface charge density σ b can then be calculated by solving where with In the above equations, with where E f is the electrostatic field that arises only from the free charges, σ f is the surface free charge density on the surface of the dielectric bodies, and ρ f is the volume free charge density in the medium Ω. In our examples, only free charges carried by the dielectric bodies are considered, and there are no free volume charges in the medium, so the second term in Eq. (13) is absent. Once we obtain the total surface charge density σ = σ f + σ b and total electrostatic field E = E f + E b on the surface of the dielectric bodies (∂Γ), the force vector acting on the ith body, F i , can be calculated by a boundary integral over the surface of i-th body, where f(r) is the surface force density on the surface of the body, is solved using GMRES in a matrix-free form provided by libMesh and PETSc, i.e., we do not explicitly form the matrix A, rather, we define the action of matrix-vector multiplication Aσ b that is used in each GMRES iteration. The most computationally expensive part in the matrix-vector multiplication is calculating the electrostatic fields, which is a boundary integral with the kernel multiplied by the surface charge densities [see Eqs. (11) and (13)]. In a direct method, the computational complexity of the matrix-vector multiplication is O(N 2 ). In Ref. 17 and 19, a fast Ewald solver is used to reduce the complexity to O(NlogN); in our implementation, FMM is used to further reduce the complexity to O(N). In the following subsections, boundary integration and parallelization are presented. A. Integrals accelerated by FMM Magnetostatic problem Here, the relevant integral is given in Eq. (5). The boundary mesh is constructed for and applied only to the boundary integration, and it also enables us to couple the three solvers for Eqs. (3)-(6) in a single simulation. We transfer nodal values of φ 1 from the boundary of the volume mesh to the corresponding DoFs on the boundary mesh before executing the boundary integration. After φ 2 is evaluated on the boundary mesh, we transfer its nodal values from the boundary mesh to the boundary of the volume mesh before imposing the Dirichelet boundary condition. Note that the boundary mesh itself is 3D, but it uses elements with 2D linear shape functions. We evaluate φ 2 on nodal points of the boundary mesh using a five-point Gauss quadrature rule and the resulting discretized form of Eq. (5) is then where i is the Dof index on the boundary mesh, j is an index for the quadrature points, and N is the total number of quadrature points in all boundary elements. Note that N depends on the number of boundary elements and the order of the Gauss quadrature rule. Finally, w(r j ) is the Gauss quadrature weight at each quadrature point. We can see that the integral at the i-th Dof on the boundary mesh is discretized as a summation over all quadrature points, which is the first term in Eq. (16), with values of φ 1 and Gauss quadrature weights w(r i ) at quadrature points multiplied by the kernel We also denote the artificial physical value at j-th quadrature point as In the implementation of the summation in Eq. (16) using a kernel-independent FMM in ScalFMM, the normal unit vector at the quadrature points cannot be directly applied in the interpolation of the kernel. There are two methods to include the normal unit vector. In the first, the kernel in Eq. (17) is split into three components, and we rewrite the summation as In this method, the summation is then carried out using three kernels, and the components of the normal unit vector are incorporated in three artificial physical values associated with each kernel, as shown in Eq. (20). The second method incorporates the normal unit vector during the particle-to-multipole (P2M) stage in ScalFMM. According to the method of kernel-independent FMM and Eq. (6) in Ref. 23, the summation in Eq. (2) can be expressed where n is the order of Chebyshev interpolation, S n (x l , x i ) is the interpolating function for nodex l , and K r is the 1/r kernel such that the kernel in Eq. (17) can be written as The idea of the second method is to use the usual 1/r kernel, and to apply the normal unit vector as a normal derivative to the interpolating function (the last term in Eq. 21), which can be implemented in the P2M stage of FMM. One advantage with this approach is that ScalFMM is highly optimized for the symmetric 1/r kernel, which further reduces the computational cost of the boundary integration. However, this second method requires modifications to the current ScalFMM source code, which are beyond the scope of our work. Therefore, we currently incorporate the normal unit vector using only the first method. In our implementation of the boundary integral using ScalFMM, we used the target source model (TSM): the nodal points are treated as target points, and quadrature points in every element are treated as source points, so that target points are different from source points. These target and source points are then inserted into the octree 33 with information about their coordinates, particle type (target/source), and the artificial physical value [σ 1,j ,σ 2,j ,σ 3,j in Eq. (20)] associated with source points. We avoid the singularity of the integral by setting a tolerance radius around each target point: if the distance between target and source point is less than a small number, e.g., 10 −5 , then its contribution to the integral is set to zero. During the FMM computations, the height of the octree, which represents the balance between near-field and far-field calculations, is tuned to achieve optimal performance of the FMM calculation. In general, the more particles in the octree, the larger its height is. Electrostatic problem For the numerical implementation of the boundary integrals that arise when calculating electrostatic fields, we use Eq. (11) as an example for the discretization. The electrostatic field vector at the jth element that arises from the bound surface charge density is where N is the total number of boundary elements (here also the Dof because of the constant monomial shape function used here), a j and a k are the surface areas of the j-th and k-th element, H j is the mean curvature at j-th element, and n j is the unit outward-pointing normal vector at the centroid of the j-th element. The first term in Eq. (23) for flat surfaces, H j = 0, which means the correction term is zero. The kernel used in the summation in Eq. (23) is Note that this kernel is different from that in Eq. (17) for the magnetostatic problem, because it contains no component of the normal unit vector. It is also evident from Eqs. (10) and (12) that the normal unit vector is associated with target points rather than source points, and is outside of the boundary integral. We also note that the above kernel in Eq. (24) is the spatial derivative of 1/r kernel. Because the 1/r kernel and its spatial derivatives, i.e. the forces from the 1/r potential, are already implemented in ScalFMM, we can take advantage of its highly optimized algorithm specifically designed for the symmetric 1/r kernel to perform boundary integrals for the electrostatic problem, which is more efficient than our current implementation for the boundary integrals in the magnetostatic problem. B. Parallelization In our parallel implementation, we use a serial mesh, i.e. every processor has a copy of the boundary mesh (and volume mesh in the magnetostatic problem), but the data structures, such as matrices and vectors, used to solve the linear system of equations are partitioned and distributed among all processors automatically in libMesh during the parallel solution using PETSc. A parallel mesh is needed only if storing mesh data on a each processor consumes too much memory, which may be the case for problems with billions of DoFs. We use METIS 34 as the partitioner for data structures associated with the mesh. The magnetostatic problem has a volume mesh for solving the Poisson and Laplace equations [Eqs. (3) and (6)] and a boundary mesh for the boundary integral [Eq. (5)]. If we then were to use the same partitioner for the volume mesh and boundary mesh, poor load balancing would result. That is because even if the volume mesh is almost uniformly partitioned, the boundary mesh is generally not. Therefore, we assign one partitioner to the volume mesh and a separate partitioner to the boundary mesh, which significantly improves the parallel performance of the boundary integration, especially when the number of processors is large. For the electrostatic problem, only one partitioner is necessary and the main parallelization is implemented in building the right-hand side b, performing matrix-vector multiplication in each GMRES iteration, and calculating the force vectors on the dielectric bodies, i.e., every processor calculates its own contribution to the right-hand side, to the matrix-vector product, and to the force vectors. For the parallelization of FMM in the magnetostatic problem, one strategy is to partition only the source points, i.e., every processor has a copy of the coordinates of all target points on the boundary mesh, as well as coordinates, physical values of φ 1 , and normal unit vectors of only local source points. We then perform serial FMM calculations on every processor, which means that we first calculate the contributions from local source points to all target points. We then let all processors communicate and sum up contributions from every processor and assign integrated values to all target points. An alternative strategy would share a similar concept, but partition only target points and keeps on every processor a local copy of information from all source points. The specific choice between the above two strategies that is better suited for the problem at hand depends on whether the number of target points or number of source points is largest. If the number of source (target) points is larger than that of target (source) points, we choose to partition the source (target) points, i.e., the larger set of points, because this can reduce the computational time and the memory cost of storing particles on every processor. Most of the times we found the number of source points is larger than the number of target points, because the number of quadrature points is mostly larger than that of nodal points, so we choose to partition the source points in our parallel implementation. The strategy for parallelization of FMM in the electrostatic problem is similar to that in the magnetostatic problem. The only difference comes from the fact that we use different shape functions in this problem than those employed for the magnetostatic problem. Since We also note that ScalFMM has its own strategy for parallelization of FMM, in which both target and source points are partitioned based on their Morton ordering 35 . The idea is to map 3D points in the octree to those in a 1D array based on the z-value of each point, and then to partition all points based on the index of their 1D representation. After partitioning, local points are inserted into the octree on each processor, and each processor then performs FMM and communicates with other processors 36 . This strategy consumes less memory than the previous two methods, but its implementation is more involved. Therefore, we have not considered it here, and will examine it in future developments of our computational approach, where we will also compare its parallel efficiency with methods that partition only target/source points. In the following section, we test our computational approaches for the magnetostatic and electrostatic problems by applying them to several model problems, and we compare the results with analytical solutions. These comparisons serve to demonstrate the accuracy, efficiency, and scalability of our parallel approach. IV. RESULTS AND DISCUSSION A. Magnetostatic problem Two model magnetostatic problems are considered here to verify our proposed methods and their implementation: a uniformly magnetized unit radius spherical body, and a uniformly magnetized unit cubical body, with unit length side (see Fig. 2). For demonstration purposes, the magnetization density within each body is set to be M = (1, 0, 0). After solving the magnetostatic problem using our computational approach, the distribution of the magnetic potential within each body is obtained, as shown in Fig. 2. For the unit sphere, the magnetic potential varies linearly along the direction of the magnetization density, and the stray field (demagnetizing field) is calculated by which is the (negative) gradient of the magnetic potential, and it is uniform everywhere inside the sphere in this particular case. The analytical solution of the stray field along the direction of magnetization density is 4π/3 in Gaussian units, see Ref. 10. For the unit cube, the magnetic potential and stray field are nonuniform within the body. Instead, we compute the stray-field energy and compare our result with analytical solutions. The stray field energy is calculated as The analytical value of the stray field energy in a unit cube with unit homogeneous magne- Refs. 37 and 38), with M s the saturation magnetization density. We calculate the relative error for the stray field energy, for example, as Figure 3(a) shows the relative errors as a function of surface DoFs; they decay linearly in the log-log plot. For a certain number of surface DoFs, the relative error for the unit cube is smaller than that for the unit sphere; also the convergence rate for the unit cube is larger than that for the unit sphere from a posteriori estimates. The larger errors associated with the sphere calculations are due to the larger discretization error that arises when using flat elements and linear shape functions to describe a curved geometry. They do not arise because of selected settings in ScalFMM; indeed, Fig. 4 shows that the difference between the relative errors by FMM and and the direct method is small, and that using quadratic shape functions reduces the discretization errors. One way to decrease the relative errors in modeling curved surfaces is to adaptively refine the surface mesh so that surface DoFs and computational cost increase simultaneously; another way is to use curved elements or higher-order methods 39 to approximate variables on curved surfaces. the number of quadrature points (source points). We use a fifth-order quadrature rule for the boundary integration, so the number of source points in the octree is roughly 8 times that of target points. In using FMM to accelerate computation of boundary integrals, it is critical to choose the appropriate octree height to achieve optimal FMM performance, because it controls the balance between the near-field and far-field computation in FMM. The optimal octree height depends on the total number of points in the octree and the size of the cubic box, and they are determined by trial runs. As DoFs increases, the optimal octree height generally increases. In Fig. 3(b), two small humps are observed when the surface DoFs is 53K and 192K, and they indicate changes in the octree height. The trade-off between FMM settings (the octree height and the order of Chebyshev polynomial) and performance is shown in Fig. 5. The relative errors of the stray-field energy change little when octree height changes, and the accuracy in FMM is mainly determined by the Chebyshev order. Increasing the Chebyshev order improves accuracy, but the computational time increases. We also refer to Ref. 23 for more details on the trade-offs between FMM settings and performance. When the number of processor is larger than 64, we observe that the decay rate of time on the Poisson solver is slower than that on the Laplace solver and the boundary integral, while the decay rates of time on the Laplace solver and boundary integral are very similar. Algebraic multigrid preconditioners 32 , such as gamg and hypre, for the Poisson solver were also tested with the result that total computational time and strong scaling are similar to those using block-Jacobian pre-conditioner. Constructing pre-conditioners using gamg or hypre takes longer time than using a block-Jacobian method, but the number of GMRES iteration is much less using gamg and hypre than using block-Jacobian. For static problems, the total times are similar using different pre-conditioners but, for study of time-dependent problems, if the global matrix remains the same between different time steps, then pre-conditioning using gamg or hypre is recommended. B. Electrostatic problem We verified our method and implementation with a few model calculations for which there are analytical as well as numerical literature solutions: specifically, two dielectric spheres with surface-bound free charges, and a sphere inserted in a cylindrical cavity in a dielectric medium. In addition, we tested our method on a model problem for which there exists no analytical solution in closed form, namely two dielectric cubes with uniform surface free charges. The results are shown in Fig. 6, and Fig. 6(a) shows the solution for the problem of two spherical dielectric bodies. We also varied the center-to-center distance between two spheres and calculated the electrostatic forces at each distance using our numerical approach and an analytical expression 40,41 (see Fig. 7(a)). DoFs/Sphere, they are even larger when 2.7 < d/R < 3.0 than those when d/R ≥ 3.0. This is because the body forces are nearly zero at these locations as shown by analytical solutions, and they are orders of magnitude smaller than those at other locations and thus more sensitive to discretization errors, which makes the relative errors large while the absolute error is very small. Including polarization effects results in very different electrostatic interactions compared to those when only bare Coulombic interactions are included, especially when particle separations are small. These issues have been reported in past theoretical and numerical studies. In the particular case considered in Fig. 7, even though the surface free-charge densities on both bodies are all positive, the electrostatic interactions may exhibit attractive forces between the bodies when their center-to-center distance (d/R) is less than 2.74. Note that the bare Coulomb interaction consists of only repulsive forces, so it is crucial that the effects of dielectric polarization be included in calculations of electrostatic interactions for simulations of colloidal bodies, lest the underlying physics not be captured correctly. the body forces on one particle. As before, the order of Chebyshev polynomial is 5 in all cases. We can see that as surface DoFs increase, the computational time increases linearly, which is similar to that of the boundary integral using FMM in the magnetostatic simulation, and it confirms the O(N) complexity of our method. In each case, because the distance between two particles is different from those in other cases, the box size for the octree is also different. The octree height in each case is tuned to achieve optical FMM performance, and the height generally increases as the surface DoFs increase. Compared to PPPM and the particle-mesh Ewald method 43 large. One strategy to recover better scaling for larger numbers of processors would be to create a separate partitioner for FMM, and to distribute both target and source points to every processor according to their Morton index. There will then be two partitioners in the simulation: one for FMM, and one for boundary elements. The challenge is then to create a mapping between the two partitioners to couple FMM with the boundary element calculation. Because our current parallel implementation shows very good scaling up to 128 processors, and quite good scaling for 1.95 M DoFs up to 512 processors, we believe our implementation is more than adequate for most applications. Further improvements to our parallel implementation will be performed in future work. V. CONCLUSIONS We have developed accurate, efficient, and scalable parallel computational approaches for We note that FMMs have been used previously for boundary integrals and BEM [45][46][47] , as well as combining with GMRES 8, 48 . However, there are few publicly available parallel codes using these techniques, in particular highly efficient and scalable ones. Our work is aimed at providing such a code framework, which we developed with the following features in mind: first, the code must run in parallel for large-scale problems with good parallel performances; second, it must be available for general distribution, at http://ime-code.uchicago.edu as part for the boundary integral in the magnetostatic problem, the normal derivative in the Green's function could be implemented in the P2M stage in ScalFMM, which will take advantage of the highly-optimized symmetric 1/r kernel in ScalFMM to further accelerate the boundary integrations. sion, high-performance computing clusters operated by the Laboratory Computing Resource Center at Argonne National Laboratory.	2016-07-25T16:05:34.000Z	2016-05-05T00:00:00.000	{ "year": 2016, "sha1": "ee92aa53a755d181cde28ac0587c5af73cf9b249", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1605.01715", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "ee92aa53a755d181cde28ac0587c5af73cf9b249", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Physics", "Computer Science" ] }
208304311	pes2o/s2orc	v3-fos-license	On the Distribution of the Ratio of Products of Fisher-Snedecor $\mathcal{F}$ Random Variables and Its Applications The Fisher-Snedecor $\mathcal{F}$ distribution has been recently proposed as a more accurate and mathematically tractable composite fading model than traditional established models in some practical cases. In this paper, we firstly derive exact closed-form expressions for the main statistical characterizations of the ratio of products of $\mathcal{F}$-distributed random variables, including the probability density function, the cumulative distribution function and the moment generating function. Secondly, simple and tight approximations to the distribution of products and ratio of products of $\mathcal{F}$-distributed random variables are presented. These analytical results can be readily employed to evaluate the performance of several emerging system configurations, including full-duplex relaying systems operating in the presence of co-channel interference and wireless communication systems enhanced with physical-layer security. The proposed mathematical analysis is substantiated by numerically evaluated results, accompanied by equivalent ones obtained using Monte Carlo simulations. I. INTRODUCTION The performance of wireless communication systems is hampered by several factors, including multipath fading and shadowing. As such, accurate channel modeling of such phenomena is crucial for purposes of accurate performance analysis and design of wireless communications [1], [2]. In order to describe the statistics of the joint effect of multipath fading and simultaneous shadowing, various composite multipath/shadowing fading distributions have been proposed in the past decades, including the K [3] and the generalized-K distributions [4]. Recently, several generalized composite distributions have been presented in the open technical literatures which can better model the statistics of the mobile radio signal, such as the gamma shadowed Rician, κ-µ, η-µ, α-µ and α-κ-µ distributions [5]- [13]. Although these composite models can adequately characterize the incurred mobile signal fading phenomena, their mathematical representation is rather cumbersome or even intractable. The Fisher-Snedecor F distribution has been recently introduced as a mathematically tractable fading model that well describes the combined effects of multipath fading and shadowing, especially in the deep fading case [14]. This distribution can be reduced to some common fading models in some special parameter settings, such as Nakagami-m and Rayleigh fading channels. Furthermore, the F distribution can provide a better fit to experimental data obtained at device-to-device (D2D) communications with respect to the well established generalized-K distribution with lower computational complexity [14]. Because of its interesting properties, the performance analysis of digital wireless communication systems over F distributed channels has been analyzed in several recent research works, e.g. see [15]- [22] and references therein. On the other hand, for the performance analysis in many practical wireless applications, the statistics of the ratios and products of fading RVs is of significant importance. For example, the performance analysis of wireless communications systems operating in the presence of co-channel interference commonly involves the evaluation of the statistics of the ratio of signals' powers, i.e., the signal-to-interference ratio (SIR). Moreover, the distribution of products of random variables is useful for the performance evaluation of multi-antenna systems operating in the presence of keyholes or relaying systems with non-regenerative relays [23]. The statistics of the ratio and products of fading random variables (RVs) have been extensively studied in several past research works. For example, a very generic analytical framework for the evaluation of the statistics of products and ratios of arbitrarily distributed RVs using the so-called H-function technique has first been proposed in [24]. The H-function distribution is a very generic statistical model that includes as special cases several well-known distributions, such as the gamma, the exponential, the Weibull and the generalized gamma (α-µ) distribution. Several results on the distribution of the ratio of gamma, exponential, Weibull, and normal RVs have been derived in [25]- [28]. In a recent work [29], the so-called N * Fisher-Snedecor F distribution, obtained as the products of N statistically independent but not necessarily identically distributed (i.n.i.d.) F -distributed RVs, has been introduced. The statistical properties of the ratio of products of α-µ RVs have been investigated in [30] and [31]. In [32], the distribution of the product of two i.n.i.d. distributed α-µ, κ-µ, and η-µ RVs has been investigated. A major drawback of the above cited works, however, is that all corresponding analytical results are expressed in terms of Fox's Hor Meijer's G-functions, which are, in general, difficult to be evaluated numerically. In order to address this problem, approximate yet accurate closed-form expressions to the distribution of products and ratios have been proposed in several past research works. For example, [33], [34], highly accurate approximations to the distribution of products of generalized gamma and generalized normal RVs have been presented by employing a Mellin transform-based technique. On the other hand, the central limit theorem (CLT) can be efficiently used to approximate the products of RVs with a lognormal distribution. Using the CLT-based technique, in [35], a log-normal approximation to the distribution of the products of Nakagami-m RVs has been proposed. Additional results on the use of the log-normal distribution to the approximation of the products of Nakagami-m RVs (i.e. the so-called N * Nakagami fading distribution) have been obtained in [36]. The results presented in that work also revealed the connection between the log-normal distribution and the distribution of the products of RVs. Motivated by the facts outlined above, in this paper we first derive closed-form expressions for the main statistics of the ratio of products of i.n.i.d. squared Fisher-Snedecor F RVs, including the probability density function (PDF), the cumulative distribution function (CDF) and the moment generating function (MGF). Then, accurate log-normal approximations to the distribution of products, ratios and ratios of products of squared F RVs are presented. The parameters of the lognormal distribution have been obtained utilizing the classical moment matching theory. In order to further highlight the usefulness of the proposed analysis, two important system configurations are assessed, namely a secure wireless communication link and a full-duplex relaying system employing the decode-and-forward (DF) protocol with co-channel interference. Extensive numerically evaluated results accompanied with Monte-Carlo simulations are further presented to validate the proposed analysis. The rest of the paper is organized as follows. In Section II, an overview of the statistical properties of the Fisher-Snedecor F distribution is presented. In Section III, closedform expressions for the PDF, the CDF and the MGF of the ratio of products of squared F -distributed RVs are derived in closed-form. Section IV presents the proposed log-normal approximations to the ratios of products of squared F -distributed RVs. In Section V, the application of the proposed analysis to physical layer security and full-duplex relaying with cochannel interference is presented. Numerical and computer simulation results are presented in Section VI, followed by Section VII concluding the paper. II. PRELIMINARIES The Fisher-Snedecor F distribution is a composite fading model where the received signal's small-scale variations follow a Nakagami-m distribution whereas its root mean square power follows an inverse Nakagami-m distribution. A squared F -distributed RV, γ ∼ F (γ; m; m s ), can be mathematically obtained as the ratio of two gamma distributed RVs, X 1 and X 2 , having PDFs given by where a 1 = m is the fading parameter, a 2 = m s is the shadowing parameter, b 1 =γ is the average SNR, and b 2 = m s /(m s − 1). The PDF of γ is given as [21, eq. (6)] where m > 1/2 and m s > 1. Note that for m s → 0, heavy shadowing is attained whereas shadowing vanishes as m s → ∞ (i.e., only Nakagami-m small-scale fading). Finally, the n th moment of the Fisher-Snedecor F distribution can be derived in closed-form as [21, eq. (9)] The CDF of Z can be obtained as Finally, the MGF of Z can be obtained as and employing [38, eq. (2.24.1.1)] yields (4c), thus completing the proof. ) and γ 2 ∼ F (γ 2 , m 2 , m s2 ). The PDF, the CDF and the MGF of X can be deduced in closed-form as Because of the fact that the Fisher-Snedecor F RV can be obtained as the product of a gamma and an inverse gamma RV, ratios and products of F RVs can be treated as products of RVs. Motivated by the CLT-based technique mentioned in the introduction section, in this work we propose to use the lognormal distribution as an accurate closed-form approximation to the distribution of the products and ratios of F RVs. Furthermore, extensive experiments carried out by using the Matlab distribution fit tool have revealed that the log-normal distribution can indeed serve as an efficient approximation to a single F distribution as well as to the ratio of products of F RVs. The parameters of the log-normal distribution are obtained using the moment matching method. A. Log-Normal Approximation to a Single Squared Fdistributed RV Hereafter, Y ∼ L (µ, σ) denotes that the RV Y follows the log-normal distribution with parameters σ and µ. The PDF of Y is given as The n th moment of Y can be expressed as The application of the moment matching method for the first two moments of the Fisher-Snedecor F distribution and the approximated log-normal distribution yields By combining (8) and (9), the parameters σ and µ can be deduced as where . It is noted that the expressions of distribution parameters given by (10) may result in a relatively large approximation error, especially in the lower and upper tail regions. This is because a good fit happens only around the mean by matching the first and second moments. In order to address this issue, we adopt a modified form by considering an adjustment factor (ε), i.e., where ε is bounded as −Y f ≤ ε ≤ Y f . To obtain ε, the numerical measure of the different (or absolute difference of two continuous distributions, i.e., Kolmogorov distance) between the exact and approximate PDFs (or CDFs) are commonly recommended. B. Log-Normal Approximation to the Products of Two Squared F -distributed RVs In what follows, we propose to use the log-normal distribution as an accurate approximation to the statistics of the product of two F -distributed RVs. Let Z = X Y , where X ∼ F (γ x ; m x ; m s,x ) and Y ∼ F (γ y ; m y ; m s,y ). The first and second moments of Z is given by and respectively. Matching the first and second moments of Z yields the following system of equations The parameters of the approximating log-normal distribution can therefore be expressed as where The adjusted forms of (16) can be written as Note that the expressions in (18) can be extended to the products of N independent F -distributed RVs as (16), where The parameter ε i can be computed in a similar manner as the one proposed in Section IV-A. Finally, assuming independent and identically (i.i.d.) factors with m x = m y = m, m s,x = m s,y = m s and γ x = γ y = γ, the above expressions simplify to C. Log-Normal approximation to the Ratio of Two Squared F -distributed RVs Hereafter, we use the moment matching method to approximate the statistics of the ratio Z = X/Y , where X ∼ F (γ x ; m x ; m s,x ) and Y ∼ F (γ y ; m y ; m s,y ), with a log-normal distribution. The first and second moments of Z, can be expressed as and The moment matching method yields estimators for σ and µ as the solution of the following system of equations The solution of this system can be obtained in closed-form as where After considering the adjustment factor ε, eq. (25) can be rewritten as The adjustment factor ε can be obtained as before. D. Log-Normal Approximation to the Ratio of Products of Squared F -distributed RVs In what follows, the ratio of products of squared Fdistributed RVs is approximated with the log-normal distribution using the moment matching method. ℓ1 , m 1,ℓ1 , m 1,s ℓ 1 (ℓ 1 = 1, · · · , L 1 ) and γ 2,ℓ2 ∼ F γ 2,ℓ2 , m 2,ℓ2 , m 2,s ℓ 2 (ℓ 2 = 1, · · · , L 2 ). Again, we can match the first two positive moments by using an adjustable form for the parameters obtained in (IV-B) and (IV-A). In particular, one obtains and The application of the moment matching method yields The above equations can be written as where The adjusted forms of (32) can be deduced as For the i.i.d. case, (32) can be expressed as (35b) E. KS Goodness-of-fit tests The accuracy of the proposed approximations can be measured by using the Kolmogorov-Smirnov (KS) goodness-offit statistical test [39]. The KS test is defined as the largest absolute difference between the two cumulative distribution functions. Mathematically speaking, the KS test is expressed as where F Sγ (z) is the empirical CDF of RV S γ and FŜ γ (z) is the analytical CDF of the approximate RVŜ γ . In what follows, we consider the ratio of products of L i.i. i.e., the distribution of the ratio of products of F -distributed RVs, belong to the CDF of the approximate distribution FŜ γ (·), i.e., the log-normal distribution. The null hypothesis is accepted if T < T max , where T max = −(1/2v) ln(α/2), is the critical value which corresponds to a significance level of α [39]. For α = 5% one obtains T max = 0.0136. Figs. 1 and 2 depict the test statistic, T , with 5% significance level for various values of m, m s and L = 1 or L = 2, respectively. The adjustment factor achieving the minimum gap between the exact and the approximated distributions is depicted in Figs. 3 and Fig. 4 for L = 1 and L = 2, respectively. It is clearly observed from Figs. 1 and 2 that the hypothesis H 0 is always accepted with 95% significance for different combinations of parameters. Hence, the log-normal distribution can be regarded as a highly accurate approximation to the exact one. INTERFERENCE In this section, applications of the proposed analytical framework to physical layer security and full -duplex relaying with co-channel interference are presented. A. Exact Analysis 1) Physical Layer Security: Physical layer security (PLS) has received considerable attention due to its ability to fortify secrecy in wireless links. A point-to-point channel is assumed where a transmitter (S) sends confidential messages to a legitimate receiver (D). This system operates in the presence of an eavesdropper (E). According to [36], it is assumed that nodes S and D are separated and surrounded by many stationary and moving objects such that the signal transmitted by the source terminal can propagate to the receiver only through electromagnetically small apertures, i.e., keyholes among obstacles. Each keyhole behaves like a source terminal to the next keyholes. Thus the resulting wireless channel can be modeled as a cascaded fading channel. Moreover, it is also assumed due to high shadowing and path loss between S and D, the direct link is highly attenuated, and thus it can be neglected. Finally, all links are subject to block i.n.i.d. F fading. Next, the performance of the considered system is assessed in terms of the secrecy outage probability and the probability of non-zero secrecy capacity. The secrecy outage probability (SOP), is defined as the probability that the instantaneous secrecy capacity falls below a target secrecy rate threshold, R th [40], [41]. The secrecy capacity is given by [40] where are the Shannon capacities of the main and wiretap channels. Hence, SOP can be expressed as. A exact closed-form expression for the average SOP is, in general, difficult to be deduced in closed form. A lower bound for average SOP can be deduced as where τ 2 Rth , Y = γ D /γ E and F Y (·) is given by (4b). As it will become evident, this bound is tight in the entire SNR region. Another fundamental performance metric of PLS is the probability of non-zero secrecy capacity (PNSC) which can be readily deduced as 2) Full-Duplex Relaying: Let us consider a two-hop fullduplex (FD) relaying network consisting of three nodes, namely one single-antenna source (A), one single-antenna destination (B) and one DF relay (R) equipped with one transmit antenna and one receive antenna to operate in fullduplex mode. Throughout this analysis, it is assumed that A, R and B are far away from each other and surrounded by many obstacles so that the resulting communication channel can be considered as a cascaded fading channel. In addition, it is assumed that all links are subject to i.n.i.d. F fading. By further assuming that the considered system employs the DF relaying protocol as well as an interference-limited scenario, the outage probability (OP) of the considered system can be formulated as [42] P out = Pr min where is the CDF of γ AR /γ RR given by (4b) and F γ (·) is the CDF of γ RB given by [29, eq. (20)]. B. Asymptotic Analysis Although the previously derived analytical results have been obtained in closed-form, they provide little insight as to the factors affecting system performance. As such, an asymptotic performance analysis that becomes tight at high SNR values will be presented next. Since our newly derived formulas for the SOP, PNSC and the OP of FD relaying system require the CDF of ratio of products of F RVs, it suffices to derive an asymptotic expression for F Y (σ), where F Y (·) is given by (4b) and σ is a fixed value. VI. NUMERICAL RESULTS In this section, some numerical results are presented to illustrate the proposed analytical framework. All numerical results are substantiated by semi-analytical Monte Carlo simulations. Figures 5 and 6 depict the PDF and the CDF of the ratio of products of F -distributed RVs, respectively, and their proposed log-normal approximation for different values of fading parameters with m 1,s ℓ 1 = m 1,s (ℓ 1 = 1, · · · , L 1 ), m 2,s ℓ 2 = m 2,s (ℓ 2 = 1, · · · , L 2 ), m 1,ℓ1 = m 1 and m 2,ℓ2 = m 2 . As it can be observed, analytical results perfectly match Monte Carlo simulations for all considered test cases, which validate the derived analytic expressions. Furthermore, it can be observed that the approximate PDFs and CDFs, obtained using the log-normal distribution, are practically indistinguishable to the exact ones. Figures 7 and 8 depict the PDF and the CDF of the ratio of products of squared F -distributed RVs, respectively, as well as their log-normal approximation for different values of L with m 1,s ℓ 1 = m 1,s (ℓ 1 = 1, · · · , L 1 ), m 2,s ℓ 2 = m 2,s (ℓ 2 = 1, · · · , L 2 ), m 1,ℓ1 = m 1 and m 2,ℓ2 = m 2 . Again, a perfect agreement between analytical and simulation results is observed, which confirms the validity of the proposed mathematical analysis. Furthermore, the approximate PDFs and CDFs obtained using the log-normal distribution closely match the exact analytical ones. has been computed by using the proposed moment matching method. It is noted that in order to obtain a the best match to the exact solution, an appropriate adjustment factor for each SNR value has been taken into account, as described in Section IV. In addition, the asymptotic expressions match well the exact ones in the high-SNR regime thus proving their validity and versatility. Finally, it can be observed that secrecy performance deteriorates as m D,s decreases and γ E increases. This fact implies that fading conditions can be exploited to prevent the information from being overheard by an eavesdropper. secure transmission with a higher probability. This because the channel state randomness (fading) can be exploited to enhance the security. Figures 13-15 depict the outage performance of a FD relaying network as a function of the first hop average SNR, γ AR , for m AR,ℓ1 = m AR (ℓ 1 = 1, · · · , L 1 ), m AR,s ℓ 1 = m AR,s , m RB,ℓ2 = m RB (ℓ 2 = 1, · · · , L 2 ), m RB,s ℓ 2 = m RB,s and σ = 1. For each test case, approximate results using the lognormal distribution are also depicted. Again, the adjustment factor with the best matching performance to the exact outage performance has been introduced, which is also dependent on the operating SNR. As it can be observed, analytical and approximate results agree well with Monte Carlo simulations thus confirming the correctness of the proposed analysis. In Fig. 13, different values of m AR,s have been considered. From the observation of Fig. 13, it becomes evident that the OP increases as m AR,s decreases, i.e., when shadowing becomes more severe. The impact of m AR,s on OP performance becomes more significant for large values of γ AR . In Fig. 14, OP is depicted for different values of γ RB . It is evident that OP increases as this γ RB decreases. Finally, in Fig. 15, OP is depicted for different values of the residual self-interference, γ RR , and as it can be observed, γ RR has a detrimental impact on OP. Thus, to exploit the potential benefits of FD relaying, self-interference mitigation matters, especially for medium-tohigh SNRs. Further guidelines for self-interference mitigation on FD relay systems are available in [44], [45]. Finally, from the observation of Figs. 13, 14 and 15 it is obvious that for large SNR values OP asymptotically becomes a constant. This constant value depends on the value of the second hop average SNR, γ RB . The reason behind this behavior in the high-SNR regime is that large values of γ AR do not significantly affect F Y (σ), as is also evident by observing eq. (41). Therefore, for large SNR values, (41) and henceforth OP is mainly determined by γ RB . VII. CONCLUSIONS In this paper, exact as well as approximate closed-from expressions for the PDF, the CDF and the MGF of the ratio of products of Fisher-Snedecor F RVs have been derived. The log-normal distribution has been selected to accurately approximate the exact PDFs and CDFs by using the momentmatching method and a properly selected adjustment factor. It has been shown that the proposed log-normal approximation is very accurate, as it yields results that are very close to the ones obtained using the exact distributions. The presented mathematical analysis is useful for assessing the performance of practical wireless communication systems operating in the presence of composite multipath fading and shadowing, including secure wireless communication systems and fullduplex relaying systems. The proposed framework may also be used in the study of several areas of wireless communications such as spectrum sharing, interference-limited scenarios, high resolution synthetic aperture radar clutter and multihop systems. Finally, numerical results and Monte Carlo simulations have been presented to validate the proposed analysis and an excellent match has been observed.	2019-11-26T09:36:22.000Z	2019-11-26T00:00:00.000	{ "year": 2019, "sha1": "966b8a46f7ed74e99f0be330213bd206beff4184", "oa_license": null, "oa_url": "https://arxiv.org/pdf/1911.11418", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "966b8a46f7ed74e99f0be330213bd206beff4184", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
55971841	pes2o/s2orc	v3-fos-license	Robust Optimal Attitude Controller for MIMO Uncertain Hexarotor MAVs: Disturbance Observer-Based 1Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, 54100 Kuala Lumpur, Malaysia 2Mechatronic Systems Laboratory, Robotic Systems Enterprise, 75250 Melaka, Malaysia 3Department of Mechanical Engineering, Graduate School of Engineering, Chiba University, Chiba Prefecture, Chiba 263-0022, Japan 4Autonomous Control Systems Laboratory Ltd., Chiba-ken, Chiba 263-8522, Japan Introduction In recent years, a great deal of research has been conducted on the control of multirotor micro aerial vehicles (MAVs) around the world as their ability to perform certain task that are unable or difficult to complete by humans.The interest for these aerial vehicles is growing in civilian and military applications such as search and rescue mission, inspection (for wind turbines, power lines, pipelines, and etc.), surveillance, security, and agriculture.For most recent applications, multirotor MAVs have been used to create damage assessment maps for disaster efforts.In contrast to traditional helicopters and fixed wing aerial vehicles, multirotor MAVs are much safer to operate, are highly manoeuvrable, can easily hover above target, are able to do vertical takeoff and landing, are able to fly at low altitude, and are a low cost solution.However, the multirotor MAV is an underactuated system because it has six degrees of freedom but only four control inputs are available.Thus, the autonomous flight control design of multirotor MAV is a challenging task. Quadrotor is the most popular multirotor MAVs among researchers in the previous studies.Based on reported results, the quadrotor MAVs have successfully achieved their stability in hovering condition by the proportional-integral-derivative (PID) control method [1,2].In general, the PID control design is straightforward and can easily be implemented in a real-time embedded system.However, the performances of the PID controller will be degraded under the effects of nonlinear dynamics and coupling.Therefore, many modelbased control methods have been extensively studied to restrain the influence of nonlinearities and coupling in dynamic model of multirotor MAVs such as a flatness-based nonlinear control method [3], a dynamic inversion control method [4], a hierarchical control method [5], a nonlinear control method using nested saturations [6], a backsteppingbased control method [7], a model predictive controller (MPC) [8], a quaternion-based nonlinear feedback controller [9], an optimal LQR controller [10], and a backstepping-based inverse optimal controller [11].Actually, those controllers require an accurate model of hexarotor in order to restrain the effects of nonlinear dynamics and coupling.Based on the accurate model, the steady-state and dynamical tracking performances can be achieved.However, the effects of parametric uncertainties and external disturbances were not fully covered in those model-based controllers.In reality, an exact mathematical model of a complex dynamic system like the multirotor MAVs cannot be obtained because the model is only an approximation of the real system.The multirotor MAVs are small and lightweight vehicles and, thus, it is easier to be affected by the payload mass variation.Furthermore, the exact vehicle parameters are difficult to be measured or obtained and normally regarded as constants thus subject to parametric uncertainties.In practice, the closed-loop system can be affected by the external disturbances (e.g., wind disturbances) and instability may happen. Therefore, many flight control methods have been proposed in literature to encounter the effect of parametric uncertainties and external disturbances.In previous studies, many researches focused on parametric uncertainties problems only such as an adaptive command-filtered backstepping controller [12], a model reference adaptive controller [13], an adaptive dynamic feedback-linearization controller [14], and a nonlinear adaptive controller [15].However, those controllers are not suitable for practical applications in outdoor environments since the closed-loop system will be affected by the external wind disturbances.The new approaches of adaptive scheme called robust adaptive control have been proposed in the presence of modelling errors and external disturbances [16][17][18][19][20].The proposed method in [16,18] does not require a priori knowledge of the modelling errors and external disturbances.However, the implementation issues of adaptive law need to be addressed.In [17], the proposed method introduced a new approach using robust integral of the signum of the error (RISE) and immersion and invariance-(I&I-) based adaptive control method to compensate the modelling errors and external disturbances.The proposed method in [20] has considered the presence of constant wind disturbances only.In general, those adaptive controllers could achieve the robust asymptotically tracking performances for the closed-loop system, but the desired robust properties of the controller cannot be specified.Based on sliding mode control (SMC) method, the new developed and higher order SMC schemes were proposed in [21][22][23][24] to deal with parametric uncertainties and external disturbances.In [21], the SMC is driven by sliding mode disturbance observer to provide robustness against external disturbances and uncertainties while [24] has proposed a second-order sliding mode observer to reconstruct disturbances.In [22,23], the use of higher order sliding mode control and adequate controller tuning was developed to decrease the chattering phenomenon encountered with SMC approach.However, their algorithms are very complicated and difficult to implement.The attitude control problem of quadrotor subject to time-varying disturbances was considered in [25] by combining an extended observer to estimate disturbances and a feedback controller with sliding mode term for stabilization.But the effects of parametric uncertainties were neglected. In this paper, a new robust optimal attitude control design for multiple-input multiple-output (MIMO) uncertain hexarotor micro aerial vehicles (MAVs) in the presence of parametric uncertainties, external time-varying disturbances, nonlinear dynamics, and coupling is proposed.The parametric uncertainties, external time-varying disturbances, nonlinear dynamics, and coupling are treated as the total disturbance in the proposed design.The proposed controller is achieved in two simple steps.First, an optimal linear-quadratic regulator (LQR) controller is designed to guarantee that the nominal closed-loop system is asymptotically stable without considering the total disturbance.After that, a disturbance observer based on [26,27] is integrated into the closed-loop system to estimate the total disturbance in the system.Then, the total disturbance is compensated from the system by the compensation input based on the estimated total disturbance.Robust properties analysis is given to prove that the state is ultimately bounded in specified boundaries.The simulation results are presented to demonstrate the robustness of the proposed control design for hovering and aggressive flight missions in the presence of the total disturbance, to prove that the state is ultimately bounded in specified boundaries and to demonstrate excellent steadystate and dynamic tracking performances of the closed-loop system. Compared to previous studies in [1-6, 8-23, 25], this paper focuses on another potential multirotor MAV platform called hexarotor which has better stability and fault tolerance as shown in Figure 1.Two additional rotors can increase payload, reliability, and manoeuvrability compared to quadrotor because at least four rotors will contribute to each angular rotation.Therefore, the forces and torques generated by two additional rotors will result in different torques about each axis and thus affect the dynamical response.Compared to several researches on hexarotor [28][29][30], the present paper treats hexarotor MAV as the MIMO system rather than the combination of multiple single-input single-output (SISO) systems.This paper also demonstrates the effectiveness of the proposed method for aggressive manoeuvres under the effects of multiple uncertainties.Compared to [3][4][5][6][7][8][9][10][11], the proposed controller does not require an accurate model and is able to compensate the total disturbance.In addition, the proposed controller can improve the steady-state and dynamic attitude tracking performances by introducing the disturbance observer to estimate the total disturbance acting on the system.Compared to [16-20, 29, 30], the desired robust properties can be specified by the proposed controller where the nominal responses can be specified by the optimal LQR controller and the effects of the total disturbance can be compensated by the compensation input based on the estimated total disturbance.This paper is structured as follows.The model of a hexarotor is described in Section 2. Section 3 presents the proposed robust optimal attitude control design.Then, the robust properties are analysed and are proven in Section 4. After this, the simulation results are presented and discussed.Finally, this paper is summarized in Section 6. Model of Hexarotor The hexarotor is a rigid body that consists of six rotors to generate the required forces and torques as shown in Figure 1.Let { , , } denote unit vectors along the respective body-fixed frame expressed in {} for the hexarotor airframe as presented in Figure 2. Let () = (() () ()) denote the attitude that represents roll (), pitch (), and yaw () angles.Then, the orientation of hexarotor is given by a rotation matrix in the special orthogonal group () ∈ SO(3) using -- Euler angles convention, expressed as ) , where c and s denote cosine and sine, respectively.Let Ω() = (Ω () Ω () Ω ()) ∈ R 3 denote the angular velocity expressed in {} and the standard attitude kinematics are given by where Ω × () represents the skew-symmetric matrix, such that Ω × () = Ω() × for vector cross product × and any vector ∈ R 3 , where As described in [31], the mathematical model of angular dynamics of hexarotor can be derived as where ∈ R 3×3 denote the inertia matrix, () = ( () () ()) ∈ R 3 denote the torque applied to the airframe in {} by the aerodynamics of the rotors, and () = ( () () ()) ∈ R 3 denote the external timevarying disturbance (e.g., wind disturbances).The hexarotor's mass distribution is assumed to be symmetrical with respect to coordinate frame and thus the products of inertia in are zero.Therefore, becomes a diagonal matrix that consists of moments of inertia, = diag( , , ). Let () ( = 1, 2, . . ., 6) denote the hover thrust force generated by individual rotor and it can be expressed as [31] where is the positive lift constant that can be obtained from static thrust tests and it depends on the chord length of the blade, the number of blades, the blade radius, and the air density. () ( = 1, 2, . . ., 6) is the angular velocity of the rotors.Based on [31], there exist the secondary aerodynamic forces on the translational dynamics when the rotor is not in hover.However, these effects can be ignored for attitude control design which depends on angular dynamics of hexarotor only. The reaction torque () ( = 1, 2, . . ., 6) generated by each hovering rotor can be modelled as [31] where is a positive torque constant that has relation with and it can be determined by static thrust tests as well. The total thrust force () for hovering is the sum of thrusts from each rotor: and its control input () to compensate the gravitational force can be defined as The torque can be expressed as where is the distance from the centre of the rotor to the centre of mass. Actually, the motion of hexarotor can be controlled directly using control input () and () because, in practice, the values of () and () will be combined through control mixer and then will be transformed to pulse width modulation values (PWM) (based on embedded flight controller specifications) with certain limit (upper and lower saturation values).Then, the PWM values will be distributed to each rotor by an electronic speed controller (ESC).Based on (4), (9), and (10), the attitude control input () is proportional to torque () such that −1 () = (), where vehicle parameter matrix ∈ R 3×3 can be defined as The vehicle parameter is a combination of nominal () and uncertain (Δ) parts as Finally, the hexarotor model in ( 4) can be rewritten as Based on (13), let us compute the tracking error model of hexarotor as where Ω () is the desired angular acceleration in {} and ∈ R 3 is the total disturbance which consists of multiple uncertainties with the following form: Let us define where 3 and 3 are a 3 × 3 identity matrix and a 3 × 3 zero matrix, respectively.From ( 14), the state equation is The proposed controller consists of the optimal LQR controller and the disturbance observer as depicted in Figure 3.The attitude control input () can be defined as where is the control input computed by the optimal LQR controller and is the compensation input derived based on the estimated total disturbance.The proposed method can be achieved in two simple steps.First, the optimal LQR controller is designed for the nominal system without considering the total disturbance Δ().After that, the disturbance observer is designed and integrated to the closed-loop system to estimate the total disturbance which consists of parametric uncertainties, external disturbances, nonlinear dynamics, and coupling.Then, the total disturbance is compensated from the system by the compensation input in (18). 3.1. Step 1: Optimal LQR Control Design.First, the optimal controller is designed based on the LQR control approach for the nominal system without considering the total disturbance Δ(): The quadratic cost functions J are minimized: where is a positive-definite and symmetric matrix and is a weighting matrix.Let us solve the Riccati equation to return the positive-definite matrices : Therefore, the linear-quadratic controller is the unique, optimal, full state feedback law: with Let us substitute ( 22) into (19); then, The closed-loop dynamics are guaranteed to be asymptotically stable if the state () is available for feedback, [ ] is stabilizable, and = > 0. 3.2. Step 2: Disturbance Observer.The system which consists of the total disturbance Δ() is considered by substituting (22) and ( 18) into (17); one can have where = − is Hurwitz.The performance of the optimal LQR controller in previous step will be degraded when the hexarotor system is subject to the total disturbance Δ() especially from external time-varying disturbances (e.g., wind disturbances in outdoor environments).In this section, the disturbance observer is designed based on [26,27] in order to estimate the total disturbance acting on the system and then eliminate it in (18).In practice, the actual value of total disturbance Δ() cannot be determined.From (17), Δ() can be derived as Equation ( 26) can be rewritten in terms of domain as where is the Laplace operator. Then, both sides of ( 27) are multiplied by a low-pass filter matrix () in order to estimate the total disturbance: where the low-pass filter matrix () is defined to be ( + ) ) . Let us define the compensation input dist () as However, the compensation input dist () in ( 30) is not suitable for practical implementation because it is in domain.Therefore, from ( 28) and ( 30), the compensation input dist () can be derived mathematically by introducing two new states 1 and 2 : where Remark 7. As can be seen, the designed control input () in ( 18) is achieved by the combination of the control input computed by the optimal LQR controller in (22) and the compensation input in (33).The proposed robust control design is linear time-invariant controller and can easily be implemented in practical applications. Robust Properties Analysis where and is a 9×1 vector with zeros except one on the th row.If positive constants , , and are sufficiently large, then can be made as small as desired because the gains of the low-pass filter () are gotten closer to 3×3 . Remark 10.Actually, the values of uncertainties bounds are difficult to obtain in real situations.However, the disturbance observer parameters , , and can be determined by an online tuning procedure: set from a small positive value (e.g., 0.5) and increase it in small step until satisfactory tracking performance is achieved.The similar tuning procedure is applied for and . Simulation Results Several simulation tests were carried out to evaluate the attitude tracking performances of the proposed robust optimal attitude control design in the presence of the total disturbance which includes parametric uncertainties, external timevarying disturbances, nonlinear dynamics, and coupling.The nominal parameters of hexarotor model are based on the actual hexarotor research platform as presented in Table 1. The nominal values of thrust coefficient and torque constant were obtained by the static thrust test.Figure 4 shows the result and experimental setup of the static thrust test.The details of the static thrust test for rotor identification can be found in [31,33].Based on Table 1, the parameter matrix = diag( / , / , / ).Simulation tests were conducted in MATLAB and Simulink environments.The dynamics model of the hexarotor used in this section consists of nonlinearities and coupling.In this section, the hexarotor is assumed to be attached on the spherical joint for attitude test only without translational motion (e.g., see [19]).The control input is assumed to be √ /6 in order to produce the thrust equal to the weight of the hexarotor in hovering conditions [34].In practice, will be produced by altitude controller. Three different cases are presented in this section.Firstly, the optimal LQR attitude controller is designed and evaluated without the effects of parametric uncertainties and external time-varying disturbances.After that, the total disturbance is considered and the optimal LQR attitude controller design is evaluated under the effects of the total disturbance.Finally, the proposed robust optimal attitude controller is evaluated under the effects of the total disturbance and its performances are compared with the optimal LQR controller.For each case, two different missions have been considered which are hovering and aggressive missions.In hovering mission, the hexarotor has to stabilize and the desired attitude is () = (0 ∘ 0 ∘ 0 ∘ ) .In aggressive mission, the large attitude reference is considered as where (), (), and () are periodic square waveforms. Case 1: Optimal LQR Attitude Controller without the Effects of Parametric Uncertainties and External Time- Varying Disturbances.The optimal LQR attitude controller is designed to achieve good attitude tracking performances for the nominal system.In this case, the total disturbance is small because it is only influenced by nonlinearities and coupling effects.Thus, the effects of the total disturbance can be ignored.However, the initial attitude in hovering mission is (0) = (−1 ∘ 1 ∘ 0 ∘ ) in order to show the transients when reaching the hover.The optimal LQR parameters and used in this case are presented in Table 2. Figures 5 and 7 show the attitude tracking responses of the optimal LQR controller for hovering and aggressive missions, respectively.The attitude control inputs for hovering and aggressive missions are presented in Figures 6 and 8, respectively.Based on the attitude tracking responses, the optimal LQR controller achieved excellent attitude tracking performances for both missions without the effects of parametric uncertainties and external time-varying disturbances.It is proven that the influence of the nonlinear dynamics and coupling could be restrained based on the accurate dynamics model of hexarotor.Based on LQR control theory [35], the closed-loop dynamics are guaranteed to be asymptotically stable if the state is available for feedback, [ ] is stabilizable, and = > 0. Case 2: Optimal LQR Attitude Controller under the Effects of the Total Disturbance.In this case, the effects of the total disturbance are considered which includes parametric uncertainties, external time-varying disturbances, nonlinear dynamics, and coupling.The uncertain part of parameter matrix Δ is assumed to be 90% of the nominal value which represents a large amount of parametric uncertainties and the external time-varying disturbance () is assumed to be Then, the performances of the designed optimal LQR attitude controller from previous case are evaluated under the effects of the total disturbance.The initial attitude for hovering mission in this case is (0) = (0 ∘ 0 ∘ 0 ∘ ) .Based on Figures 9 and 11, the optimal LQR controller is unable to sufficiently track the reference signals for both hovering and aggressive missions.Thus, the attitude tracking errors are increased.The attitude control inputs for hovering and aggressive missions are presented in Figures 10 and 12, respectively.Based on results, the optimal LQR controller is not robust against the total disturbance due to imperfections of the dynamics model.Actually, the model-based control approach like the optimal LQR controller needs an accurate model in order to achieve the desired tracking performances.However, in reality, an exact mathematical model of hexarotor cannot be obtained especially in outdoor flying environments because the model is only an approximation of the real system. Case 3: Proposed Robust Optimal Attitude Controller under the Effects of the Total Disturbance.In this case, the uncertain part of parameter matrix Δ and the external time-varying disturbance () are similar to those in Case 2. The disturbance observer was integrated into the existing closed-loop system to estimate the total disturbance.The disturbance observer parameters are presented in Table 2. Please refer to Remark 10 in Section 4 for online tuning procedure of disturbance observer parameters.It should be highlighted that the proposed robust optimal controller is a combination of the optimal LQR controller from previous case and the disturbance observer.First, the proposed robust optimal controller is evaluated in hovering mission.The initial attitude in hovering mission is (0) = (0 ∘ 0 ∘ 0 ∘ ) .Figures 13 and 14 show the attitude tracking responses and the attitude control inputs of the proposed robust optimal controller in hovering mission, respectively.As can be seen, the proposed robust control scheme achieved good steady-state tracking performances in the presence of the total disturbance.It is also proven that the attitude tracking errors are ultimately bounded in (2 × 10 −4 ) ∘ , (5 × 10 −5 ) ∘ , and (2 × 10 −6 ) ∘ for roll, pitch, and yaw angles, respectively.The total disturbance is successfully estimated by disturbance observer as presented in Figure 15. As can be seen, the estimated total disturbance Δ() = ( Δ () Δ () Δ ()) which is the major part of the total disturbance.In practical applications, the external time-varying disturbance would be the additional torque caused by wind disturbances.Then, the proposed robust optimal controller is evaluated in aggressive mission.The attitude tracking responses and the attitude control inputs of the proposed robust optimal controller in aggressive mission are presented in Figures 16 and 17, respectively.As can be seen, the proposed attitude controller achieved excellent dynamical tracking performances.The estimated total disturbance against the external time-varying disturbance is illustrated in Figure 18.As can be seen, there is a small glitch that occurred sometimes on the estimated total disturbance due to high coupling effects in aggressive mission.However, the total disturbance in the system has been successfully rejected.The attitude tracking errors are ultimately bounded in (2 × 10 −4 ) ∘ , (5 × 10 −5 ) ∘ , and (2 × 10 −6 ) ∘ for roll, pitch, and yaw angles, respectively. Compared to optimal LQR attitude controller as presented in Case 2 and previous model-based attitude controller design in [3][4][5][6][7][8][9][10][11], the proposed robust optimal controller is robust against multiple uncertainties and it does not require an accurate model in order to achieve the desired tracking performances.In addition, the proposed controller can improve the steady-state and dynamic attitude tracking performances by introducing the disturbance observer to estimate the total disturbance acting on the system.Compared to [16][17][18][19][20], the proposed method can specify the desired robust properties (e.g., the desired boundaries of attitude tracking errors).Based on the results in Case 3, the attitude tracking errors are ultimately bounded in specified boundaries for both missions ((2 × 10 −4 ) ∘ , (5 × 10 ) ∘ for roll, pitch, and yaw angles, resp.). Conclusion This paper has proposed a robust optimal attitude control design for multiple-input multiple-output (MIMO) uncertain hexarotor micro aerial vehicles (MAVs) in the presence of parametric uncertainties, external time-varying disturbances, nonlinear dynamics, and coupling.The parametric uncertainties, external time-varying disturbances, nonlinear dynamics, and coupling have been treated as the total disturbance in the proposed design.The proposed controller is designed by the combination of the optimal linear-quadratic regulator (LQR) controller and the disturbance observer.The total disturbance is estimated by the disturbance observer and is compensated from the system by the compensation input.Robust properties analysis proved that the state is ultimately bounded in specified boundaries.The simulation results demonstrated the robustness of the proposed controller for hovering and aggressive flight missions in the presence of the total disturbance, with excellent steady-state and dynamic attitude tracking performances.This research aims to integrate with the position controller for trajectory tracking. Figure 2 : Figure 2: The schematic of hexarotor.The hexarotor is attached on the spherical joint without translational motions. Figure 4 : Figure 4: (a) The result and (b) experimental setup of static thrust test. Figure 5 :Figure 6 : Figure 5: Case 1: hovering mission.(a) and (b) show the attitude tracking responses and the attitude tracking errors, respectively, of the optimal LQR controller.The red dashed line in (a) represents the attitude reference signal. Figure 7 :Figure 8 : Figure 7: Case 1: aggressive mission.(a) and (b) show the attitude tracking responses and the attitude tracking errors, respectively, of the optimal LQR controller.The red dashed line in (a) represents the attitude reference signal. Figure 9 :Figure 10 : Figure 9: Case 2: hovering mission.(a) and (b) show the attitude tracking responses and the attitude tracking errors, respectively, of the optimal LQR controller.The red dashed line in (a) represents the attitude reference signal. 𝑇 is almost similar to the externalFigure 11 : Figure 11: Case 2: aggressive mission.(a) and (b) show the attitude tracking responses and the attitude tracking errors, respectively, of the optimal LQR controller.The red dashed line in (a) represents the attitude reference signal. Figure 12 : Figure 12: Case 2: aggressive mission.The attitude control inputs of the optimal LQR controller. Figure 13 :Figure 14 : Figure 13: Case 3: hovering mission.(a) and (b) show the attitude tracking responses and the attitude tracking errors, respectively, of the proposed robust optimal controller.The red dashed line in (a) represents the attitude reference signal. Figure 15 : Figure 15: Case 3: hovering mission.The estimated total disturbance (the blue line) and the external time-varying disturbance (the red dashed line). Figure 16 : Figure 16: Case 3: aggressive mission.(a) and (b) show the attitude tracking responses and the attitude tracking errors, respectively, of the proposed robust optimal controller.The red dashed line in (a) represents reference signals. Figure 17 : Figure 17: Case 3: aggressive mission.The attitude control inputs of the proposed robust optimal controller. Figure 18 : Figure 18: Case 3: aggressive mission.The estimated total disturbance (the blue line) and the external time-varying disturbance (the red dashed line).	2018-12-07T06:26:42.597Z	2016-05-12T00:00:00.000	{ "year": 2016, "sha1": "1e67c0e5c49ffde207ee03359bb93d7e5efed4ad", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/mpe/2016/3154842.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "1e67c0e5c49ffde207ee03359bb93d7e5efed4ad", "s2fieldsofstudy": [ "Engineering", "Mathematics" ], "extfieldsofstudy": [ "Engineering" ] }
8354465	pes2o/s2orc	v3-fos-license	Flux tubes as the origin of Net Circular Polarization in Sunspot Penumbrae We employ a 3-dimensional magnetohydrostatic model of a horizontal flux tube, embedded in a magnetic surrounding atmosphere, to successfully reproduce the azimuthal and center-to-limb variations of the Net Circular Polarization observed in sunspot penumbrae. This success is partly due to the realistic modeling of the interaction between the flux tube and the surrounding magnetic field. A possible scenario that explains the magnetic field configuration of the sunspot penumbra is the so-called uncombed penumbral model (Solanki & Montavon 1993). In this model a horizontal flux tube that harbors the Evershed flow is embedded in a more vertical magnetic field. A similar configuration has been obtained by Heinemann et al. (2007), who carried out 3D MHD simulations of the penumbra and found that penumbral filaments are produced by bubbles of weak and horinzontal magnetic field fully embedded in a stronger and more vertical one. They also find field free gaps connected to the deeper convection zone which appear as bright regions in the emergent intensity. One of the most critical observations that any penumbral model must reproduce, is the Net Circular Polarization (Sánchez Almeida & Lites 1992). Different realizations of the uncombed penumbra have successfully explained these observations (Solanki & Montavon 1993;Martínez Pillet 2000;Schlichenmaier et al. 2002;Müller et al. , 2006. However, have pointed out that the models of the uncombed penumbra published so far, do not consider the effects of the external field wrapping around the flux tube. The goal of this work is to remove this point of inconsistency. As it will be shown, a uncombed model that consistently considers the perturbation in the external field introduced by a cylindrical flux tube, is also able to explain the Net Circular Polarization observed in the sunspot penumbra with surprising accuracy. flux tube model and reference frame Our embedded flux tube model is adopted from Borrero (2007). This model considers a cylindrical flux tube that carries the Evershed flow. The flux tube is located perpendicularly to the vertical z-axis and is embedded in an inclined potential magnetic field. This model prescribes the magnetic field configuration in the local reference frame (LRF): S = {e x , e y , e z }; see Fig. 1), being the flux tube's axis oriented along e x . Analytical expressions for B x (y, z), B y (y, z), and B z (y, z) are taken from Eqs. (33)-(34) in Borrero (2007). These equations take into account how the external field bends and surrounds the flux tube. Although the model is threedimensional, variations along the x-axis are neglected. The model is described by 6 parameters: B 0 and γ 0 (strength and inclination of the external magnetic field far away from the flux tube), R and z 0 (radius and central position of the flux tube), and finally B xt0 and v xt0 (component of the flux tube magnetic field along the flux tube's axis and magnitude of the Evershed flow). Figure 1 (top panel) illustrates the magnetic field lines in the plane perpendicular to the tube's axis. The velocity and magnetic field vectors enter the static momentum equation. This yields the density ρ(y, z), and gas pressure P g (y, z), that ensure force balance (see Borrero 2007 for details). Once gas pressure and density are known, the temperature T (y, z) is evaluated through the equation of state for ideal gases. A varying molecular weight is used to account for the partial ionization of the different atomic species. Once all the relevant quantities are known in the LRF, S, we project the magnetic field and velocity vectors on the observer's reference frame (ORF), S ′′ . This is accomplished by a rotation of angle Ψ along the vertical z-axis. This rotation ensures that the resulting x ′ -axis meets the line of symmetry of the sunspot. A second rotation of angle Θ along e y ′ is finally needed to direct the resulting z ′′ along the observer's line-of-sight. Mathematically, 1 Fig. 1.-Top panel: magnetic field lines tranversal to the flux tube's axis. Note that inside the flux tube the magnetic field is mostly aligned its axis and therefore it is not shown here. Bottom panel: inclination of the magnetic field in the observer's reference frame (i.e.: inclination respect to the line-of-sight). The white arrow represents a possible line-of-sight of an observer at Θ = 45 • looking at a penumbral flux tube located at Ψ = 45 • with respect to the line of symmetry in the center side. In this example, the intersection of the ray-path with the uppermost point in the yz plane occurs at (y 0 , zmax)= (−150, 200) [km]. This point is indicated by a black asterisk. The angle between the vertical z-axis (white dashed line) and the projected line-of-sight is β ≃ 35 • . Other ray-paths would be parallel to the one drawn but intersect at different y 0 's. where R y ′ (Θ) and R z (Ψ) are the corresponding rotation matrices. In this manner, we can locate the flux tube at any azimuthal position Ψ within the sunspot, and to place the sunspot at any heliocentric angle, Θ, on the solar disk. Thus, we can determine the line-of-sight velocity v los = v ]. An example of γ(y, z) is presented in Fig. 1 (bottom panel). Finally, our physical parameters are expressed as function of (y, z), but for our radiative transfer calculations we need to know them along the line-of-sight. To this end, we project the observer's line-of-sight onto the yz plane: U = sin Ψ sin Θe y − cos Θe z . With this vector we can construct the parametric equation of the line-of-sight in the yz plane: where (y 0 , z max ) is the uppermost intersection point of the ray-path with the yz plane. The slope-intercept form of the line-of-sight is: Note that, either at disk center (Θ = 0) or along to the line of symmetry of the sunspot (Ψ = 0, π/2), the ray-paths are given by y = y 0 . At any other position, the ray-paths enter the yz plane, forming an angle β = tan −1 (tan Θ sin Ψ) with the vertical direction. As expected, β = ±Θ perpendicularly to the line of symmetry ( Ψ = π/2, 3π/2). An example of the projection of the line-of-sight onto the yz plane is presented in Fig. 1. After these geometrical considerations, we are now ready to solve the radiative transfer equation and obtain theoretical Stokes profiles from our embedded flux tube model. We have employed three different numerical codes: SIR (Ruíz Cobo & Del Toro Iniesta 1992), DIA-MAG (Grossmann-Doerth 1994) and SPINOR (Frutiger 2000) and verified that the results are consistent among them. We first calculate the emerging polarization profiles for 128 rays that enter into the yz plane with different y 0 's (see Fig. 1). We then compute the Net Circular Polarization (NCP) as the wavelength integral of Stokes V . The total NCP is obtained as sum of the NCP produced by each individual ray-path (see example in Fig. 2; top panel). observations Five sunspots at five different positions on the solar disk were observed using the Tenerife Infrared Polarimeter (TIP; Martínez Pillet et al. 1999) at the Vacuum Tower Telescope (Izaña Observatory, Spain), and the Advanced Stokes Polarimeter (ASP; Elmore et al. 1992) at the Dunn Solar Telescope (Sacramento Peak, USA). The full Stokes vector of Fe I 15648.5Å (TIP) and Fe I 6302.5Å (ASP) were recorded . For each sunspot, radially averaged azimuthal variations of the NCP, N (Ψ), are computed. Here Ψ runs counter-clockwise, with Ψ = 0 referring to the line of symmetry on the center side of the penumbra. The determination of the position of the line of symmetry from the observations is a difficult task and can only be done to an accuracy of about ±10 • . Center-to-limb variation We have computed the NCP that emerges from our flux tube model for different heliocentric angles Θ. This is the so-called center-to-limb variation of the NCP. We have repeated this calculation for flux tubes located at the line of symmetry of the sunspot, on the center side (Ψ = 0) and on the limb side (Ψ = π). The NCP was evaluated for the same spectral lines of our observations (Sect. 3). Results are presented in Fig. 2 (bottom panel). The parameters used for the flux tube model are: B 0 = B xt0 = 1000 Gauss, γ 0 = 60 • , v xt0 = 6 km s −1 , R = 75 Km, z 0 = 0 km. These values are consistent with result from spectropolarimetric observations of the penumbral fine structure. In addition, we have used the hot umbral model by Collados et al. (1994) to represent the thermodynamic parameters of the atmosphere surrounding the flux tube. Martínez Pillet (2000) has presented the observed center-to-limb variation of the NCP in Fe I 6302.5Å for a large number of sunspots. Our predictions (Fig. 2; bottom panel; blue lines) agree very well with his findings. Our theoretical N (Θ) curve for 6302.5Å does not cross zero at cos Θ = [0.8, 1], however. This is due to our model simplifications: flux tube is always perpendicular to the vertical z−axis, and the external atmosphere does not harbor any flows. For the near infrared neutral iron line at 15648.5Å we are not aware of any similar work to that of Martínez Pil-let. However, our theoretical curve is in very good agreement with other theoretical predictions that use a simpler uncombed scenario (see Müller et al. 2002Müller et al. , 2006. The reason is that, along the line of symmetry (Ψ = 0, π), raypaths are not inclined on the yz plane regardless of the heliocentric angle (Eq. 3). Azimuthal variations In this section we compare the observed N (Ψ), for different sunspots at different heliocentric angles Θ, with the theoretical predictions from the embedded flux tube model. This is done individually for each sunspot. Theoretical curves have been obtained using the same model parameters as in Sect. 4.1. Results are presented in Figure 3. It can be seen that our model is able to reproduce many features of the observed azimuthal variations of the NCP. This achievement is especially remarkable if we consider that all we have done is changing the spectral line and heliocentric angle (model parameters were kept constant). Particularly interesting is the fact that we can also predict the existence of secondary maxima/minima in the N (Ψ) curves. This is clearly the case of Fe I 15648.5Å , where more simple uncombed models, predict only the existence of 2 maxima and 2 minima at all heliocentric angles. The improved agreement between observations and predictions is to be ascribed to our more realistic modeling of the external magnetic field bending and wrapping around the horizontal flux tube. This is a crucial ingredient for Fe I 15648.5Å , where gradients in azimuthal angle of the magnetic field play a major role (Landolfi & Landi degl'Innocenti 1996;Müller et al. 2002). An important detail is that, in our model, the Evershed flow is channeled along the horizontal flux tube. This means that the line of sight velocity, v los = v ′′ z = −v xt0 cos Ψ sin Θ, vanishes always perpendicularly to the line of symmetry (Ψ = π/2, 3π/2). Thus, gradients in v los do not exist there, yielding always zero Net Circular Polarization. In agreement with Müller (2001), decreasing the magnitude of the Evershed flow decreases the amount of NCP roughly linearly. In the absence of a complete parameter study, this effect alone cannot be used to rule out weaker horizontal flows, however. Borrero (2007) expressed concerns about flux tubes with circular cross sections having very smooth variations in γ. He pointed out that those variations might be too small to generate enough NCP (Sánchez Almeida & Lites 1992). Results presented in this work prove those concerns to be unfounded. conclusions We have employed the model of Borrero (2007) to predict the behavior of the Net Circular Polarization in the sunspot penumbra. This model finds the equilibrium configuration of a horizontal flux tube with circular cross section, that carries the Evershed flow, and is embedded in a atmosphere with a potential magnetic field pointing towards a different direction. Energy transfer is neglected and therefore we cannot address how the penumbra is heated. This model consistently considers how the external magnetic field opens and bends in order to accommodate the horizontal flux tube. This generalizes the work of Solanki & Montavon (1993), Martínez Pillet (2000), Müller et al. (2002; and Schlichenmaier et al. (2002), by considering the 3D geometry of the problem. We have compared our predictions with the observed NCP in two neutral iron lines and in five different sunspots. The agreement between theory and observations is remarkable, improving previous determinations based on simpler realizations of the uncombed penumbral model. Other models for the penumbral fine structure (Sánchez Almeida 2005; should also try to explain these observations. In the future, we will attempt to reproduce also the full polarization profiles of these spectral lines (cf. Bellot Rubio et al. 2004). We wish to thank Daniel Cabrera Solana for kindly providing some of the observations used in this work.	2007-07-27T15:48:31.000Z	2007-07-27T00:00:00.000	{ "year": 2007, "sha1": "626ff34e0acceb238f86de2d69360673261c43a5", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0707.4145", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "626ff34e0acceb238f86de2d69360673261c43a5", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
64646686	pes2o/s2orc	v3-fos-license	Class Characteristic Classification of Test Fired Cartridge Cases: A Digital Image Decision Tree Approach to Kennington’s Matrix for Initial Stages of Criminal Investigation When a gun is fired, ammunition components are transferred to a crime scene. The ammunition components alone are a source of information about the gun fired. One piece of information about the gun is the broad class of caliber, such as a 9 mm, for semi-automatic pistols. But what is more important in a no-gun crime scene for criminal investigations “What was the make/ model of 9 mm gun used?” While ammunition components such as bullets are currently in an electronic General Rifling Characteristic (GRC) database maintained by the FBI for this exact purpose of possible gun type determination, the list of brands generated is often quite lengthy. There currently exists no equivalent detailed electronic database for spent cartridge cases. Thus, a database and non-cumbersome standardized procedure for determining the brand of gun which fired a cartridge case based on detailed class characteristics is much needed in order for investigators and examiners to have images and quantitative measurements to support conclusions such as “what make/model is most likely.” An attention to keeping up to date with more detailed class characteristics which were specific to a particular model and make of firearm as it entered the market was clearly evident in the early 1990s. Nordhoff solely dedicated to a new model of .25 ACP Lorcin L25 Pistols [1]. The company Lorcin itself had just been established Abstract Introduction When a gun is fired, ammunition components are transferred to a crime scene. The ammunition components alone are a source of information about the gun fired. One piece of information about the gun is the broad class of caliber, such as a 9 mm, for semi-automatic pistols. But what is more important in a no-gun crime scene for criminal investigations "What was the make/ model of 9 mm gun used?" While ammunition components such as bullets are currently in an electronic General Rifling Characteristic (GRC) database maintained by the FBI for this exact purpose of possible gun type determination, the list of brands generated is often quite lengthy. There currently exists no equivalent detailed electronic database for spent cartridge cases. Thus, a database and non-cumbersome standardized procedure for determining the brand of gun which fired a cartridge case based on detailed class characteristics is much needed in order for investigators and examiners to have images and quantitative measurements to support conclusions such as "what make/model is most likely." An attention to keeping up to date with more detailed class characteristics which were specific to a particular model and make of firearm as it entered the market was clearly evident in the early 1990s. Nordhoff solely dedicated to a new model of .25 ACP Lorcin L25 Pistols [1]. The company Lorcin itself had just been established less than a year prior and was marketing inexpensive firearms targeted towards lower income individuals. The designs of the firearms were similar to corresponding models made by another earlier manufacturer Raven Arms. Specifically, both the L25 and the MP-25 were both 7-shot pocket pistols designed to be easily concealed. Nordhoff published the similarities and important differences in the class characteristics of these similar firearms which would likely become "Saturday night special" firearms distributed through pawn shops and eventually be used in the commission of a crime [1]. In response to Nordhoff, Thompson published a similar article in 1991 on class characteristics, but for the 10 mm Wyoming Arms Parker [2]. One reason for a 10 mm handgun to make its way in the commission of a crime can be seen given the historic move of the FBI towards .40 Smith & Wesson (S&W) in 1990. The .40 S&W was the replacement for the higher recoil 10 mm cartridge and delivered the desired terminal ballistics in a cartridge which could be chambered in smaller frame handguns. Later in 1991, Gieszl argued the need for examiners to update their class characteristic files for the new Model 83 and 85 handguns chambered in .380 ACP manufactured by Argentine arms maker Bersa [3]. Once again, the 83 and 85 were examples of affordable and concealable firearms which would be popular and potentially end up being used in crimes. The significance of selecting firearms based on market models and subsequently the likelihood in casework is to provide for an examiner an update his/her lab's personal reference files of the detailed class characteristics. As a result, the firearm could then be easily referred to in future case work involving no-gun cases. Finally in 1992, Carr re-examined the class characteristics of the Lorcin L25 for the unique way it marked the shoulder of bullets due to the lead or gap between the firearm's chamber and button rifled barrel [4]. Again, in a no-gun case, knowledge of these class characteristics would allow the investigator and the examiner to narrow down the make and model of firearm. The Kennington Matrix System Perhaps the greatest advancement for class characteristic prediction of firearms during this time was Robert Kennington's publication of his book "The Matrix; 9 mm Parabellum: An Empirical Study of Type Determination." Kennington and also his lab had been using physical cards for class characteristics since the 1950s. The cards were organized into a matrix by manufacturer and type of bullet, chamber marks, extractor mark shape, ejector mark, firing pin impression, and breech face marks. In some instances, Kennington was able to narrow the possibility of manufacturers down to one with the caveat there could exist a manufacturer which was not in the database [5]. Practically, the police detective who is armed with the hypothesized manufacturer at the time of interrogating a suspect is more likely to get a meaningful confession, particularly if the suspect believes his accomplice has already confessed. This confession could than lead to other evidence such as the actual firearm itself to be used for test firing as well as additional incriminating evidence such as the buried body which is needed to secure a guilty verdict if a case were to go to trial. However, the format of using cards to update the database has proved to be not only inconvenient but also outdated because new firearms entered the market almost annually. Therefore, there exists a need for an electronic version which still captures the essence of the original method but in a more quantitative format for easier searching and greater objectivity. The last major effort of earlier type determination on class characteristics in the literature was perhaps a book review of Kennington's previously discussed works on 9 mm and .380 ACP done by Dutton, of the Tasmania Police in 2003 [6]. According to Dutton, Kennington's decade old work contained not only a great deal of technical information but was still relevant to the current time. Specifically, Dutton considered the potential workflow of how shooting crimes should be investigated and how labs encountering 9 mm and .380 ACP on a regular basis ought to devote time now for implementation of Kennington's method in order to save time later in the future [6]. It is such encouragement which serves and inspires the basis for this research project. Current Lab Databases and Systems One electronic class characteristic database for a fired bullet would be the General Rifling Characteristic (GRC) database maintained by the FBI. The GRC database is for the number of and numerical widths of lands and grooves which were engraved on a bullet when the bullet engaged the rifling of the barrel it was fired through. However, a detailed class characteristics database for cartridge cases in an electronic format to facilitate easier information sharing amongst examiners is long overdue as GRC databases are often insufficient for no-gun cases. As Oberg observed for a 9 mm caliber right twisted bullet with seven lands and grooves and a cartridge case with hemispherical firing pin impression, two extractor marks, and one ejector mark: the initial search of the GRC database returned zero hits [7] Nevertheless, it was additional research which was found to be more useful using the online Association of Firearms and Tool mark Examiner (AFTE) forum which suggested the firearm may have been made by SCCY. Oberg then had to reach out to the manufacturer SCCY in order to deduce the type of firearm as being a model CPX-1 with class characteristic 4 o'clock extractor and 7-8 o'clock ejector [7]. Another electronic system which is commonly misunderstood by non-firearm examiners is the Integrated Ballistics Identification System® (IBIS®) system. Past public policy has also been slightly misguided with respect to using cartridge case databases powered by computer algorithms such as IBIS® in an attempt to preliminarily identify or individualize a particular gun as opposed to a broader class of make/model. While IBIS® is useful for linking different shootings to one another and to an already seized firearm (where a one-to-one comparison by a trained human examiner is still needed thereafter for identification conclusions for court). IBIS® will not deliver a list of manufacturers. The related state governments perhaps misunderstood its capabilities when they had respectively passed costly legislature to mandate every new gun legally sold to have test fired cartridge cases entered into an IBIS® powered database. Thus, there currently exists no electronic database of fired cartridge cases for generating investigative leads in the common situation where a gun has yet to be recovered. Meanwhile, there are many gun manufacturers introducing new models of guns every year, making it difficult to update such a database unless a cost-effective, non-cumbersome standardized procedure is developed. A Model Digital Image Database Purposive sampling was employed to select firearms, in this case, the 9 mm semiautomatic pistols, with three major considerations: first, the 9 mm is the most common or mode of firearms used in crimes nationwide; second, the availability of the firearm in local indoor shooting ranges; and lastly, the personal experience and expertise of the author. As a result, the following seven models of popular recoil action operated 9 mm models were selected The seven models of 9 mm pistols were test fired in an indoor shooting range using the standard factory Winchester "white box" 115 grain with Full Metal Jacket ammunition. Glock firearms such as the 17 and 19 chambered in 9 mm which produced a rectangular firing pin aperture mark were not studied due to the widespread knowledge of this signature class characteristic. Glocks such as the Glock 43 and the latest Generation 5 Glock which do not produce this signature rectangular firing pin aperture mark are an object of future study and comparison. Additionally, one additional blowback operated Ruger Police Carbine 9 was test fired for an anticipated study and comparison with the popular blowback operated Hi-Point C9 handgun and 995TS carbine in the near future. Ten test fired cartridge cases from each firearm were randomly selected and analyzed using a method-based to what was discussed by Warren and Pitts [8]. While Warren and Pitts placed each cartridge case image in a 4X4 grid in a Microsoft PowerPoint slide in order to quantify the location of an ejector mark in a meaningful manner, this method was considered to be too imprecise and does not address the original challenge referenced by Kennington in 1992 [5]. This challenge is the need for collaborative sharing. Collaborative sharing is not only important for practitioners since new firearms are constantly being manufactured, but also for the broader legal and academic communities, since continued confusion and criticism of the discipline is still prevalent and rooted in a lack of understanding and transparency over the existence of personal databases for known test fired cartridge cases. Rather than using a 4 X 4 grid for the entire cartridge case, a 24 X 24 grid was used just focusing on the small pistol 4.45 mm primer with the 24 grid units corresponding to or covering 4.45 mm primer area. To put this in perspective, each one-half grid unit corresponded to 0.0927 mm or less than 100 um. The cropping of only the primer area was done because ejector marks are often not present and the center of mass of irregularly shaped ejector marks is more difficult to determine as compared to a firing pin impression which is always present and symmetric. However, the original uncropped images are still retained within the same folder and a slide show can still be used not only for one-to-one comparisons of ejector mark shapes as opposed to using an algorithm [9] which may not be able to handle a multiclass problem as well as the human eye, but also to assess the visual variability within an individual firearm as well as within a make/model. Next, when firing pin drag marks were present, they were used to orient the cartridge case so the drag mark pointed up towards 12 o'clock or (0,1) in an x-y coordinate system. Similarly, when firing pin drag marks were absent, the extractor marks were used to orient the cartridge case so the extractor mark pointed towards 3 o'clock or (1,0). The center of the grid is the origin (0,0) of the primer. The center of the firing pin impression was determined by placing the mouse over the center of the firing pin impression, reducing the transparency of the image until the background grid lines were visible. The coordinate was then recorded as the number of grid units away from the center in the horizontal x-axis direction (negative corresponding to left, positive corresponding to right) followed by the number of grid units away from the center in the vertical y-axis direction (negative corresponding to low, positive corresponding to high). Independent of rotation, the numerical distance between the center of the firing pin and the center of the primer was determined using the Pythagorean Theorem. The Google Drive/Slides/Sheets Platform While Warren and Pitts chose to use Microsoft Power point, this was found to be less useful than using Google Slides since the ultimate goal is to facilitate sharing. Google Slides is Google's version of Power point and exists within an individual or organization's Google Drive, Google's cloud-based file storage and sharing platform. Although Microsoft does offer their version, One Drive, Google Drive was used since it is the default cloud storage system used by the author's institution of study: West Virginia University. Within Google Slides, a template for measurements was created by selecting a 4:3 aspect ratio for the slide and adding 33 evenly spaced lines in the horizontal direction and 25 evenly spaced lines in the vertical direction for the generation of 32 by 24 boxes (4:3 ratio), where only the center 24 by 24 boxes were used (to correspond to a circular primer area). The two lines composing the x and y axes were highlighted in red (Figure 1). On this particular cartridge case, the Smith & Wesson M&P9 Shield's firing pin struck at (1.0) square or grid units. One Slide presentation was made for each individual firearm. Each of the ten tests fired primers represented one slide in each Slide presentation. The coordinates of each primer were entered into a Google Sheets spreadsheet (Google's equivalent to Excel) corresponding to each individual firearm in order for the distances to be computed based on the scale of 24 square units corresponding to 4.45 mm. The resulting plots of firing pin strikes were then added to each firearm's slide presentation. The coordinates from firearms of the same class (make/model) were than pooled into another spreadsheet corresponding to the class. The Classification of Digital Images The logical progression which followed was to create various Google Drive folders corresponding to the various makes or manufacturer. While within each manufacturer folder contains the model folders, within each model folder contains the individual firearm folders labeled by serial number. Finally, a Decision Making folder for hypothetical unknown cartridge cases was created based on the previous folders of ground truth known's ( Figure 2). For example, within the Decision Making folder is the first folder "FP drag present?" which then contains two folders: "Present" and "Not present." For a firearm which does not have a tilting barrel, there would not typically be any drag marks present. Most firearms without a tilting barrel are blowback action operated, as opposed to recoil action. The only instance for a blowback operated firearm to leave a drag mark would be if the firing pin served a dual purpose as an ejector (such as on the Hi-Point C9 where a lateral drag mark may be occasionally observed). Following this proposed hypothetical decision tree, within ""Not present" is a folder entitled "FPI shape?" wherein lies only one folder currently: "Circular," as opposed to the more elongated "Elliptical" Glock-type firing pin impressions. Within this folder lies "Type of circular FPI?" wherein there lies four folders corresponding to "Smooth," "Irregular," "Concentric," or "Can't decide, light strike" (Figure 2). Within "Smooth" contains a folder "Size of FPA?" wherein contains two folders: "Large" or "Not large." And within "Large" is the folder corresponding to the Beretta 92FS with the characteristic large firing pin aperture mark of approximately 17 grid units (3.15 mm) which has been described previously as "primer blowback" or "pillow-like ( Figure 3)." The images and the resulting variability present can then be used for a one to one comparison with the questioned cartridge cases to guide the decision maker in an objective manner akin to the sketches within the rolodex cards of Kennington's matrix system. With respect to ejector marks, rather than giving a subjective name or classification such as "oriental fan shaped," a one to one comparison can be performed ( Figure 3). Discussion There are many features which can be considered for the classification of class characteristics present on the surface of a primer. Although the optimal sequence and discriminating power of features is an object of future study, in no particular order are: A example summary of obvious feature differences between the different firearms studied can be seen in Table 1. Conclusion When firing pin drag marks are fully present, the Sig Sauer P226 may potentially be mistaken for the Springfield XD9, if no ejector marks can be compared, however its firing pin did strike further from the center (Figure 4). Future statistical analyses are being developed to attempt to classify cartridge cases by treating the firing pin strike x-y coordinates both first and foremost as a uni-variate normally distributed distance from the center of the primer independent of cartridge case orientation/rotation as well as bi-variate normally distributed x and y coordinates where the subjective state of striking to the "right and low," for example, should be supported and quantified by x-y coordinates where X_firing_pin > 0 and Y_firing_pin < 0 and classifying based upon the resulting means and variance-covariance of each firearm class. Besides the need for further statistical analyses, it is interesting to note Springfield's newer XD-S 9 mm has an entirely different firing pin aperture than the older XD9. The XD-S has an aperture mark similar to the signature rectangular aperture of the Glock, but more rounded and with less apparent primer shear marks, as noted by Warren and Pitts as another direction for future study. When firing pin drag marks are not present, the CZ 75 and Sig Sauer P226 can potentially be mistaken for the popular blowback operated Hi-Point C9 (Figure 4). With respect to the Smith & Wesson M&P9, a direction of further study with respect to the potentially mistaken for Glock 43 as identified by Warren & Sheets (2017), it was observed to produce a round "point of toe" shaped ejector mark roughly equidistant between the primer and the rim, although this mark was sometimes obscured by the head stamp. A potential difference may be found with respect to the position of the Glock 43 ejector mark distribution as being more towards the rim of the cartridge case than the Smith & Wesson M&P9. Additionally, the Smith & Wesson M&P9 has a smaller firing pin aperture mark of approximately 11 square units (2.0 mm) at the widest point as opposed to the Glock 43's 13 square units (2.4 mm). The similarity between the Ruger SR9 to the Smith & Wesson M&P9 was also noted with the key difference being the SR9 having a common circular aperture mark instead of the "light bulb" shaped aperture of the M&P9. One final observation is the larger firing pin aperture mark of the Glock 43 is reminiscent of the Kel-Tec PF9 in size (also approximately 13 square units), however the 43 had a characteristic difference in shape ("light bulb" shaped aperture) from the PF9's circular shaped aperture (similar to the Beretta 92FS with "primer flow back"). Acknowledgement The author would like to express sincere appreciation for his Master's thesis proposal committee chair Dr. Morris and committee members Mr. Carroll and Dr. Jelsema for their insightful guidance and continued mentoring as the thesis is being executed at the time of this writing.	2019-02-17T14:20:27.769Z	2017-11-30T00:00:00.000	{ "year": 2017, "sha1": "26d840094e3b430d6f0ec455b1f2d665e3f1816e", "oa_license": "CCBY", "oa_url": "https://juniperpublishers.com/jfsci/pdf/JFSCI.MS.ID.555693.pdf", "oa_status": "HYBRID", "pdf_src": "MergedPDFExtraction", "pdf_hash": "f207e1a1aa197a1d4b0cca7bf2a135d12732d856", "s2fieldsofstudy": [ "Law" ], "extfieldsofstudy": [ "Computer Science" ] }
221464091	pes2o/s2orc	v3-fos-license	Phytoplankton community structure and water quality assessment in an ecological restoration area of Baiyangdian Lake, China Shihoudian Lake is one of the ecological restoration engineering pilot sites of Baiyangdian Lake, China. To evaluate the phytoplankton characteristics and eutrophication status in Shihoudian Lake, we investigated the community structure of phytoplankton, including the species composition, density, biomass dominance, biodiversity and water quality parameters, in autumn 2018 and spring and summer 2019. The relationships between the community structure and the main environmental factors were analysed using a multivariate statistical method. A total of 143 species of phytoplankton were identified, belonging to 53 genera and eight phyla, and Cyanophyta and Prochlorophyta were the most dominant phyla. Both the density and the biomass were the highest in the summer. A redundancy analysis showed that total phosphorus and chemical oxygen demand were the primary influencing factors of the community distribution of Cyanophyta. Evaluation of the comprehensive diversity index and water quality index revealed that the water of Shihoudian Lake was lightly to moderately polluted, providing scientific evidence for eco-environmental protection and remediation. Introduction Freshwater ecosystems provide important benefits for humans, including providing drinking water, aquatic products and entertainment venues (Strayer and Dudgeon 2010). In recent decades, many lakes have become eutrophic, and some, such as lake Taihu (China's third largest lake) (Li et al. 2014b) and lake Erie (USA) (Michalak et al. 2013), have even suffered cyanobacterial blooms. Such blooms cause a variety of environmental problems, including reductions in fish yields, deterioration of water quality (Chen et al. 2019), loss of submerged macrophytes (Li et al. 2014b) and an overall decline in biological diversity. Phytoplankton are essential primary producers (Becker et al. 2010) in water bodies, and changes in phytoplankton species and numbers could directly influence water ecosystem structure and function (Lepistö et al. 2004). Thus, phytoplankton not only represent the basis of mass cycling and energy flow in the whole aquatic ecosystem (Wang et al. 2014a;Wang and Wang 2014) but are also an important indicator of the eutrophic status of water (Kolar et al. 2005). A study of the community structure of phytoplankton in Taiping Lake, Anhui, by Xiong et al. (2016) provided scientific evidence for water eco-environmental protection in this water body. In addition, eutrophication in the Chagan Lake Wetland was evaluated through a multivariate analysis of the relationship between phytoplankton and environmental factors (Li et al. 2014a). Similarly, the status of living organisms in lakes and other water bodies has been evaluated according to the phytoplankton community structure (Jun et al. 2019). Therefore, it is important to research the phytoplankton community, environmental factors and their role in the ecosystems to provide a theoretical basis for lake ecological restoration and management. Development of phytoplankton populations is dependent on the concentration of nutrients and other ecological factors such as light, temperature, composition and quantity of organic matter, currents and grazing. Outbreaks of cyanobacterial blooms occur when eutrophic water bodies are exposed to the appropriate water temperature, air temperature, flow rate, radiation and other external conditions (Heisler et al. 2008). Thus, it is important to identify the changes in phytoplankton communities and the key environmental factors impacting changes in the Baiyangdian Lake. With the construction of the Xiongan New Area, the eutrophication of Baiyangdian Lake has drawn great attention from researchers (Tang et al. 2019;Yang et al. 2020). A demonstration project for phytoplankton resource investigation and water ecological remediation was launched in 2018. Shihoudian Lake was one of five engineering pilot sites used in this project, wherein fishing and the construction of habitats were the primary ecological remediation technologies. Thus, the first step in conducting ecological remediation was to evaluate the phytoplankton characteristics and pollution status. The aims of this work were to determine the relationships between environmental factors and phytoplankton communities and to identify predominant environmental factors of phytoplankton communities. The density, biomass and dominant species of phytoplankton at multiple sites in Shihoudian Lake were investigated in autumn 2018 and spring and summer 2019. The physicochemical factors of the water were also monitored. The community structure characteristics and trophic level of phytoplankton were systematically analysed to provide basic data for the further development of ecological environment remediation and fish breeding in Shihoudian Lake. These factors will also be helpful for scientific management and protection of lakes in North China. Study site Baiyangdian Lake is a large natural lake on the North China Plain and is located at 38° 44′-38° 59′ N and 115° 45′-116° 26′ E, with an average water depth of 2-3 m and an approximate area of 366 km 2 (Wang et al. 2014b). It is a well-known water body in North China and named "A pearl of North China". For years, the ecological system of Baiyangdian Lake has become increasingly fragile due to human activities, and severe destruction of its biological resources has occurred. According to a study conducted by Li et al. (2018), Baiyangdian Lake has been in a state of eutrophication since 1999. Five sampling sites, labelled A, B, C, D and E, were established in Shihoudian Lake (Fig. 1). Sampling was carried out six times, including in the autumn of 2018 (October and November), the spring of 2019 (April and May) and the summer of 2019 (June and July). No sampling was performed in winter due to the presence of ice on the lake surface up to a depth of 0.5 m. Sample collection and treatment In total, five water samples of 1 L were collected for phytoplankton analyses by mixing water from the surface, a depth of 0.5 m, a depth of 1 m and 0.5 m above the bottom in open waters. Samples were preserved with 1% Lugol's iodine solution and concentrated to 30 mL after Fig. 1 The sampling distribution map of Shihoudian Lake sedimentation for 48 h. An Olympus CX31 optical microscope (Olympus, Tokyo, Japan) was used for plankton species identification. For each taxon, a minimum of 20 cells were detected, and the geometric shape closest to the cell shape was used to calculate the mean biovolume, which was then transformed into the biomass (expressed as mg/L wet weight) based on an assumed density of 1 g/cm 3 (Zhang and Huang 1991;Hillebrand et al. 1999). The data of eight physicochemical environmental factors in water were also measured and collected at the five sampling sites. Dissolved oxygen (DO) and the pH were determined using a portable multimeter (YSI Pro Plus; YSI Incorporated, USA). Water samples were collected in 5-L polypropylene buckets and preserved in the field and the laboratory until analysis. Ammonia nitrogen (NH 3 -N), total nitrogen (TN), nitrite nitrogen (NO 2 -N), nitrate nitrogen (NO 3 -N), total phosphorus (TP) and chemical oxygen demand (COD Mn ) were determined using the Nessler test method, alkaline potassium persulfate digestion-UV spectrophotometric method, N-(1-naphthalene)-diaminoethane spectrophotometry, UV spectrophotometry method, ammonium molybdate tetrahydrate spectrophotometry method and potassium dichromate method, respectively (Jiang et al. 2014;Amri et al. 2017). Index calculation The dominant species of phytoplankton were identified by calculating the dominance index (Y) for each species. where Ni is the abundance of the ith species, N is the abundance of all species and fi is the frequency of occurrence of the ith species. The dominant species had a value of Y > 0.02 (Lin et al. 2011). The indices of the diversity of plankton and fish included the following (Shannon 1948;Margalef 1958). The Shannon-Weaver diversity index was calculated with the following equation: H′ values of 0-1, 1-2, 2-3 and > 3 corresponded to heavy, moderate, light and no pollution. Simpson's diversity index (D′) was calculated according to the following equation: Pielou's evenness index was calculated with the following equation: where Ni is the abundance of the ith species, N is the abundance of all species and S is the species. J values of 0-0.3, 0.3-0.5 and > 0.5 corresponded to heavy, moderate and light or no pollution. Data analysis The statistical analysis and data plotting were conducted with Excel and SPSS 13.0. Redundancy analysis (RDA) was carried out to analyse the relationship between phytoplankton and environmental factors using Canoco 5.0 software. The length of the first axis was used to identify the analysis category as follows: > 4: canonical correspondence analysis (CCA), < 3: RDA and 3-4: either of the two (Muylaert et al. 2000;Beyene et al. 2009). Physicochemical factors of the water The phytoplankton community changed greatly in Shihoudian Lake due to natural and man-made interference. In 2018, 250 acres of cage net and fishing facilities were cleared in Shihoudian Lake, which led to an improvement in water quality. However, due to the enclosed aquaculture for many years, a large amount of residual diet and manure was deposited, which influenced the water quality of the lake. The physicochemical index values for Shihoudian Lake across the five sampling sites and six sampling dates are provided in Fig. 2. The following values were observed: COD Mn of the water, 2.60-9.4 mg/L; NH 3 -N, 0.15-1.24 mg/L; TP, 0.01-0.08 mg/L; TN, 0.44-2.35 mg/L; NO 3 -N, 0.11-0.35 mg/L; NO 2 -N, 0.0003-0.014 mg/L; DO, 6.93-11.43 mg/L; pH 8.2-9.1. According to the water quality evaluation standards for groundwater (GB 3838-2002), the overall, the status of Shihoudian Lake was between categories IV and V. Phytoplankton community composition A total of 143 phytoplankton species were collected across the three seasons (Table 1), representing eight phyla. (4) (5) J = H � ∕ ln S Chlorophyta had the highest species richness of the total phytoplankton (66 species; 46.2%), followed by Bacillariophyta (28 species; 19.6%). With the gradual increase in water temperature, a simultaneous increase occurred in light intensity and duration during the summer. In addition, with the gradual increase in nutritional salt, a concurrent increase in phytoplankton number occurred (Lehman 2000;Chuai et al. 2012). The number of phytoplankton species in different seasons exhibited an order of summer > autumn > spring, with numbers of 102 and 72 in the summer and spring, respectively. Green algae dominated in all three seasons, with the highest percentage in autumn (56.0%). The phytoplankton community was dominated by blue algae-green algae throughout the year in Shihoudian Lake. Dominant species had a dominance index value of Y > 0.02. According to the phytoplankton density and distribution (Table 2), 13 dominant species belonging to three phyla were identified in this study. The dominant species were members of Cyanophyta, with the highest dominance observed for Phormidium and Oscillatoria. Dominance of Oscillatoria, Phormidium, Anabaena and Microcystis species indicates water eutrophication. Xanthophyta species indicate clean water and were found occasionally, but they were not the dominant species. In all three seasons, Cyanophyta were dominant species, while Prochlorophyta were dominant species in spring and autumn. Cryptophyta and Bacillariophyta were dominant in the spring. This proportion of dominant taxa to total phytoplankton abundance was similar to that of Taihu Lake during a summer cyanobacteria bloom. Although no algal blooms were previously recorded for the study lake, the high proportion of Cyanophyta was also similar to that of another eutrophic lake (Jiang et al. 2014). Wang et al. (2013) showed that dominant taxa of Chlorophyta and Cyanophyta indicate that a lake is eutrophic to some extent. The density and biomass of phytoplankton The average density and the biomass level of phytoplankton in the three seasons are shown in Tables 3 and 4. The seasonal variation in the average density of phytoplankton was in the range of 341.75 × 10 4 to 1752.61 × 10 4 ind./L with a medium value of 927.49 × 10 4 ind./L. The average density in different seasons exhibited the following order: summer > spring > autumn. The density composition of Cyanophyta was highest, followed by that of Bacillariophyta and Prochlorophyta. The seasonal variation in the average biomass of phytoplankton was in the range of 1.74-6.73 mg/L with a medium value of 3.54 mg/L. The average biomass in different seasons exhibited the following order: summer > spring > autumn. The biomass composition of Cyanophyta was highest, followed by that of Bacillariophyta and Prochlorophyta. Among them, both the density and the biomass of Cyanophyta were highest in all seasons, indicating eutrophication of the water (Ke et al. 2009;Zhang and Zang 2015). The diversity index of phytoplankton The seasonal variation of the determined biodiversity index of phytoplankton is presented in Table 5. The Shannon-Wiener diversity index in the three seasons was in the range of 1.31-2.194 with an annual average of 1.865. The highest and lowest index values occurred in spring and autumn, respectively. The Simpson abundance index in the three seasons was in the range of 0.595-0.777 with an annual average of 0.714. The highest and lowest index values occurred in spring and autumn, respectively. The Pielou evenness index in the three seasons was in the range of 0.316-0.504 with an annual average of 0.425. The highest and lowest index values occurred in spring and autumn, respectively. Finally, the Margalef abundance index in the three seasons was in the range of 4.093-4.959 with an annual average of 4.402. The highest and lowest values occurred in summer and autumn, respectively. In a normal environment, the diversity index is high. When the environment is polluted, the density index decreases (Gao et al. 2019). Shihoudian Lake is a typical lake in the Baiyangdian Lake region, around which there is a large population, with well-developed tourism. Thus, the water was polluted due to the gradual acceptance of wastewater in the river basin, and aquatic living resources were severely damaged. Judging from the relationship between the diversity index and the level of water pollution (Negro et al. 2000), the lake exhibited a state of light to moderate pollution. The relationship between the phytoplankton community and environmental factors The evolution of the phytoplankton community was comprehensively influenced by the environmental factors of this water body. In addition to the effect of water temperature on phytoplankton, nutritional salt was also a dominant factor that influenced the phytoplankton community (Muylaert et al. 2000) because nutrition is the most basic factor that affects the growth of phytoplankton (Nydick et al. 2004). RDA preliminarily demonstrated a correlation between phytoplankton in the ecological remediation area and the main environmental factors (Fig. 3, Table 2). The length of the first axis was 2.0 (< 4). Thus, it was appropriate to choose the linear model of RDA, which showed that the former two axes of RDA1 and RDA2 were significantly different (P < 0.01). The characteristic values of these two axes were 0.164 and 0.09936, respectively. The explanation degree reached 62.15%, indicating that the two sequencing axes could efficiently demonstrate the mutual relationship between phytoplankton in Shihoudian Lake and different environmental factors. The abbreviations of environmental factors and the codes for phytoplankton are listed in Tables 2 and Fig. 2. Oscillatoria positively correlated with NO 2 -N and DO. Kruskopf and Plessis (2006) proposed that nitrogen had the greatest influence on Oscillatoria growth, followed by ferric iron and phosphorus, which was similar to the present study. Low and high pH values would inhibit the enzyme activity in algal cells, influencing algal metabolism, leading to a decrease in growth and proliferation (Melack 1981). In this study, Chroomonas acuta Uterm correlated positively with NO 3 -N. Reynolds (2006) proposed an optimum N-to-P ratio of 16:1 for the growth of phytoplankton. When the ratio was larger than 16:1, phytoplankton growth was limited mainly by P, while when the ratio was smaller than 16:1, it was limited mainly by N. In this work, Raphidiopsis sinensia and Microschizophyllum correlated positively with TP, NH 3 -N and COD Mn , with N and P ratios greater than 16. Thus, the growth of blue algae was mainly limited by P in Shihoudian Lake. The environmental factors that dramatically affected the phytoplankton in Baiyangdian Lake were different in various areas in the water body and during different periods (Shen and Liu 2008;Zhang et al. 2010;Jin et al. 2017). In total, the environmental factors that mainly affected blue algae in Shihoudian Lake were in descending order: total P and COD Mn > molecular nitrogen, pH and DO. The phytoplankton community structure of various types of lakes exhibits significant differences (Lepistö et al. 2004;Lv et al. 2013;Deyab et al. 2019). Baiyangdian Lake is a typical aquatic macrophyte-dominated lake in northern China (Yang et al. 2020) that is distinguished from other lakes. The dominant species of cyanobacteria in this survey were Oscillatoria sp. and Phormidium sp., with dominance indexes of 0.55 and 0.63, respectively. This study provides a reference for the monitoring and evaluation of water quality of lakes in northern China and similar lakes worldwide at the same latitude, as well as a basis for the formulation of specific measures for ecological remediation of Baiyangdian Lake. Although the water quality of Baiyangdian Lake has been improved, changes in the dominant species require a long time (Zhao et al. 2019). Therefore, both short-term remediation and long-term maintenance are key factors to ensure the remediation target. Conclusion This study analysed the trophic states, species numbers, community structures and biodiversity of phytoplankton in Baiyangdian Lake. The species richness, abundance, diversity index and evenness index of phytoplankton showed the lake exhibited a state of light to moderate pollution. The phytoplankton abundance was highest in summer, Cyanophyta were the dominant tax of plankton. TP and COD Mn were the main environment factors influencing the species number and diversity of phytoplankton based on the redundancy analysis (RDA) results. It provides a reference for the formulation of specific measures for ecological remediation of Baiyangdian Lake.	2020-09-03T13:52:11.661Z	2020-09-03T00:00:00.000	{ "year": 2020, "sha1": "ec86ff83d97eb560e7de0e22df1c376ecaf95797", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s13762-020-02907-6.pdf", "oa_status": "HYBRID", "pdf_src": "Springer", "pdf_hash": "ec86ff83d97eb560e7de0e22df1c376ecaf95797", "s2fieldsofstudy": [ "Environmental Science" ], "extfieldsofstudy": [ "Environmental Science" ] }
13471574	pes2o/s2orc	v3-fos-license	Effects of indexes of consciousness (IoC1 and IoC2) monitoring on remifentanil dosage in modified radical mastectomy: a randomized trial Background This study investigated the effects of indexes of consciousness (IoC1 and IoC2) monitoring on remifentanil dosage. Methods In this randomized, single-blinded, prospective study, 120 patients undergoing unilateral modified radical mastectomy were randomly assigned to the treatment group (T group, n = 60) or control group (C group, n = 60). In the T group, patients received both IoC1 (sedation) and IoC2 (analgesia) monitoring, and remifentanil dosages were adjusted by anesthetists according to IoC2. In the C group, remifentanil dosages were adjusted based on the anesthetists’ judgment according to the patients’ vital signs. Remifentanil dose, adjustment frequency, infusion duration, intraoperative adverse events, and quality of anesthetic recovery were compared between the two groups. The primary outcome was the dose of remifentanil. Results Compared with the C group, mean remifentanil dosage was significantly higher in the T group (3.8 ± 1.9 versus 3.2 ± 1.2 μg kg-1 h-1, P < 0.05) during the anesthetic period, as was the adjustment frequency of target-controlled infusion (2.9 ± 1.9 versus 2.0 ± 1.2 times/surgery, P < 0.05), but there was no difference in infusion duration. Voluntary eye opening, extubation time, and recovery score were not significantly different between the two groups (P > 0.05). Total adverse events were significantly reduced in the T group (P < 0.05). Conclusions IoC1-targeted propofol dosing does not seem to be significantly different to hemodynamic-based monitoring, whereas IoC2 monitoring can increase remifentanil dosage during modified radical mastectomy, but the anesthetic process is more controllable and total adverse events are reduced, which improves the controllability of anesthesia. Trial registration Trial registration number: ChiCTR-TRC-13004101, registered on 27 November 2013. Background Depth of anesthesia is commonly assessed in clinical practice by the patient's clinical signs and symptoms such as blood pressure, heart rate variability, and body movement, but these measures are difficult to convert into a quantitative standard measure. In addition, some technologies are available for objective monitoring of intraoperative pain, but they suffer from limitations and disadvantages [1] and body movements can be used as a surrogate for pain [2]. In recent years, the index of consciousness (IoC) has emerged as a new technique for monitoring depth of anesthesia, which not only objectively measures the patient's awareness level [3] but also reflects analgesic status [2]. At present, many studies have focused on IoC1 monitoring for sedative depth [4,5], but only a few studies have focused on IoC2 monitoring for analgesic depth [6]. We hypothesized that IoC2 monitoring would help control the depth of anesthesia. In this study, we applied IoC1 and IoC2 to the use of a sedative agent, propofol, and most importantly, an analgesic agent, remifentanil. Furthermore, we evaluated the effectiveness of IoC monitoring for anesthetic depth (IoC1 and IoC2) versus commonly used vital sign monitoring based on factors such as blood pressure and heart rate. Study design and patients This study was a randomized single-blind prospective trial. It was registered with http://www.chictr.org.cn/ index.aspx (ChiCTR-TRC-13004101). This study was approved by the Medical Ethics Committee of Liaocheng People's Hospital (approval number 2013XJS-023). In total, 120 patients who were undergoing elective unilateral modified radical mastectomy under total intravenous anesthesia (TIVA) from 20 January 2014 to 1 June 2014 were consecutively enrolled at the Liaocheng People's Hospital. Inclusion criteria were: (1) American Society of Anesthesiologists (ASA) class I or II; (2) age 18-65 years old; and (3) body mass index (BMI) 18-30 kg/m 2 . Exclusion criteria were: (1) pregnancy; (2) allergy to the agents used in the study; (3) hypertension; (4) hypotension; (5) tachycardia; or (6) bradycardia. Signed informed consent was obtained from all participants. They were randomized 1:1 to the trial group (T group, n = 60) or the control group (C group, n = 60). Randomization method: 120 opaque and sealed envelopes marked from 1 to 120 were prepared by the researchers, each containing one card written with a random number generated using SPSS 17.0 (IBM, Armonk, NY, USA). If the random number was odd, the patient was allocated to the T group; otherwise the patient was allocated to the C group. Randomization was implemented by a designated individual who did not participate in the subsequent inclusion of patients. The single-blind method was applied to the patients. The T group was monitored using IoC for depth of anesthesia monitoring while the C group was not (Fig. 1). Fig. 1 Consort patients flow diagram A patient would be excluded after recruitment if the surgical method was changed peri-operatively or if the anesthetics were finally not used according to the study protocol, for any reason. Other conditions affecting this trial (e.g., airway obstruction, secondary anesthesia given before the patient completely regained consciousness) also led to patient exclusion. Anesthesia After admission to the operating room, the patient was administered 8 ml/kg of Ringer's solution with an intraoperative maintenance dose of 4 ml kg -1 h -1 . Baseline blood pressure and heart rate were acquired 15 min after admission to the operating room. Both the T and C groups were induced using the Marsh pharmacokinetic model-based target-controlled infusion of propofol. Based on an initial plasma target concentration of 4.0 μg/ml, fentanyl was intravenously injected at 3 μg/kg and cisatracurium at 0.2 mg/kg. Tracheal intubation was performed for mechanical ventilation when satisfactory muscle relaxation was achieved (about 3 min) according to train-of-four (TOF) stimulation evaluation, followed by connection to an anesthetic machine (Drägerwerk AG & Co. KGaA, Lübeck, Germany) for volumecontrolled ventilation with a tidal volume of 8 ml/kg, a respiratory rate of 12/min, a respiratory ratio of 1:2, and a pressure of end-tidal carbon dioxide (P ET CO 2 ) of 35-45 mmHg. At 45 min of surgery, 0.2 mg/kg of cisatracurium was administered for muscle relaxation. Anesthesia monitoring For both groups, non-invasive blood pressure, electrocardiogram (ECG), peripheral oxygen saturation, and P ET CO 2 were routinely monitored. The T group received IoC1 monitoring for depth of sedation and IoC2 monitoring for depth of analgesia (Angel-6000D Multiparameter Anesthesia Monitor, Shenzhen Weihaokang Medical Technology Co., Ltd, Guangdong, China). For the generation of IoC [2,7], the recorded electroencephalogram (EEG) signals are divided into linear and non-linear modules and partitioned using a symbolic dynamics approach. Each part is then labeled by a symbol to convert time series to symbol sequences. Finally, IoC1 is acquired using an adaptive neuro fuzzy inference system based on β ratio and burst suppression rate [8] and IoC2 is derived. The index of depth of sedation, IoC1, ranges from 0 to 99 and is controlled to be within 40-60 during the operative period, with IoC1 > 60 indicating insufficient use of sedative agents while IoC1 < 40 indicates excessive sedation. The index of depth of analgesic, IoC2, ranges from 0 to 99 and is controlled to be within 30-50 during the operative period, with IoC2 > 50 indicating insufficient use of analgesic agents and IoC2 < 30 indicating excessive analgesic effects. Target-controlled infusion is a common, convenient, and accurate administration approach for intravenous anesthesia. In this study, target-controlled infusion was used to record the infusion duration and total dosage of propofol and remifentanil accurately. Previous studies have shown good consistency between IoC and bispectral index [9][10][11][12][13][14]. During anesthesia, anesthetists adjusted the dosages of propofol and remifentanil according to the changes in IoC1 and IoC2. IoC1, a sedative index, was used as the guide for the adjustment of propofol target concentration, which was increased by 0.5 μg/ml per adjustment when IoC1 > 60 and was increased by 1 μg/ ml when body movements were observed. Target concentration was decreased by 0.5 μg/ml per adjustment when IoC1 < 40, with a maintenance value between 40 and 60. IoC2, an analgesic index, was used as the guide for the adjustment of remifentanil target concentration (based on the Minto remifentanil pharmacokinetic parameter set), in which the target concentration was increased by 1 ng/ml per adjustment when IoC2 > 50 but was decreased by 1 ng/ml per adjustment when IoC2 < 30, with the maintenance value between 30 and 50. The C Group was not monitored using IoC, and the doses of propofol and remifentanil were adjusted by the anesthetists according to vital signs such as blood pressure and heart rate so as to control the fluctuation of blood pressure and heart rate within 20 % of baseline values. Meantime, adverse events, such as hypertension, hypotension, tachycardia, and bradycardia, were recorded. The patient's quality of anesthetic recovery was recorded at the completion of anesthesia. Hypertension was defined as intraoperative systolic pressure >160 mmHg, hypotension as intraoperative systolic pressure <90 mmHg, tachycardia as heart rate >90 bpm, and bradycardia as heart rate <45 bpm. Voluntary eye opening time was defined as the interval from stopping the infusion of narcotic drugs to showing voluntary eye movements when calling the patient's name in a normal voice. The OAA/S score was assessed at the moment of extubation. Intraoperative awareness (using modified Brice questionnaires) was assessed after the patient sobered up. Outcomes The primary outcome was the dose of remifentanil. The secondary outcomes were remifentanil adjustment frequency, infusion duration, intraoperative adverse events, and quality of anesthetic recovery. Statistical analysis The sample size was calculated using a significance level α = 0.05 and test power 1 -β = 0.80. Based on a preliminary trial performed with 20 patients (unpublished data), the mean dosage of remifentanil was 3.0 ± 1.1 μg kg -1 h -1 in the T group and a significant difference in dosage was defined as a fluctuation ≥20 % compared with the C group. Based on the formula provided by Chow et al. [15] for the calculation of clinical trial sample size, 46 patients were required in each group. However, a total of 120 patients were enrolled in consideration of a dropout rate of 25 %. All data were analyzed using SPSS 17.0 (IBM, Armonk, NY, USA). Continuous data are shown as mean ± standard deviation (SD). Between-group comparisons were performed using the independent sample t test or the rank sum test. Categorical data were compared using the chi-square test. A P value <0.05 was considered statistically significant. Results A total of 120 patients with breast cancer were enrolled in January to June 2014 and randomly assigned to the T group (n = 60) or C group (n = 60). Thirteen patients were excluded. Six patients were excluded from the T group due to a change of the surgical method (n = 3), airway obstruction at extubation (n = 1), or venous pathway obstruction (n = 2). Seven patients were excluded from the C group for non-compliance to trial protocol (n = 3), change of the surgical method (n = 3), or secondary anesthesia due to a small vascular rupture before extubation (n = 1). Therefore, 54 and 53 patients were included in the final analyses for the T and C groups, respectively (Fig. 1). The two groups were not significantly different in age, height, weight, BMI, systolic pressure, diastolic pressure, and heart rate (all P > 0.05) ( Table 1). As shown in Table 2, remifentanil infusion duration was not significantly different between the two groups (P > 0.05), but the adjustment frequency of remifentanil target concentration and mean dosage were different (P < 0.05). Comparing the use of propofol and quality of anesthetic recovery, the groups were not significantly different either in adjustment frequency or in mean dosage, with similar voluntary eye opening, extubation time, and awakening score (all P > 0.05). Discussion This study investigated the effects of IoC1 and IoC2 monitoring on remifentanil dosage. Results showed that compared with the C group, the mean remifentanil dose was significantly higher in the T group during the anesthetic period, as were the adjustment frequency of target-controlled infusion, but there was no difference in infusion duration. Voluntary eye opening, extubation time, and recovery score were not significantly different between the two groups. Total adverse events were significantly reduced in the T group. Some studies have shown that pain can induce changes in EEG power [16][17][18]. Jensen et al. [2] confirmed that IoC1 (qCON) can reliably predict the disappearance of eyelash reflex (or, the disappearance of awareness) during TIVA using propofol and remifentanil. Moreover, with similar concentrations of anesthetics, IoC2 (qNOX) can predict the occurrence of body movements to nociceptive stimuli. In the present study, nociceptive stimuli mainly occurred during anesthesia or surgical procedures such as tracheal intubation/extubation, skin incision, resection of mammary tissues, and axillary lymph node dissection. This study revealed a slight delay (around 1.5-2 min) in IoC2 reduction compared with IoC1 during anesthesia induction, possibly due to a slower action (1 min) of remifentanil versus propofol (30-40 s) or due to the computational errors in the calculation of IoC2 based on IoC1, or both. Like EEG curves, IoC1 and IoC2 curves constantly change during the maintenance of anesthesia, and they are not smooth. This demonstrates that the so-called depth of anesthesia is essentially a state of the central nervous system that is affected by the interactions between the irritations from nociceptive stimuli and the inhibitory effects of anesthetic agents. In other words, it is a functional state of the central nervous system occurring when surgical stimulation dynamically balances against the control effects of general anesthetics, indicating that IoC1 and IoC2 will still slightly fluctuate due to surgical stimuli although the effect compartment concentrations of propofol and remifentanil or depth of anesthesia is relatively stable. Based on the characteristics of IoC1 and IoC2 and based on our clinical experience, the doses of propofol and remifentanil should be changed when both IoC1 and IoC2 exceed or are lower than their reference ranges for over 2 min, or when both indexes exceed more than 20 within 1 min, to avoid frequent adjustments of infusion speed. Nevertheless, trials should look specifically at the most optimal protocols for changes in anesthetic doses using IoC1 and IoC2. In the C group, the doses of propofol and remifentanil were adjusted based on the patient's baseline blood pressure and heart rate to make them fluctuate within 80-120 % of baseline values during surgery. Although the adjustment frequency and mean dose of remifentanil were higher in the T group than in the C group, the T group showed more stable hemodynamics, and smaller frequencies of hypertension, hypotension, and adverse events. In the T group, a relatively high incidence of bradycardia may have resulted from the side effects of slowing down the heart rate by remifentanil, but as an ultra-short-acting opioid analgesic, remifentanil does not affect the patient's awakening. During maintenance of anesthesia, the effect compartment concentration of propofol was kept within 2.5-6.0 μg/ml in both groups, but remifentanil showed a huge variation among patients, with the effect compartment concentrations being within 0-7 ng/ml. In TIVA, the sedative agent (propofol) and analgesics (fentanyl and remifentanil) do not show muscle relaxation effects, and there is a need for administration of muscle relaxants. During surgery, the muscle relaxant cisatracurium was administered at 45 min with 0.2 mg/kg. In the T group, seven patients showed body movements before muscle relaxants were administered, although their hemodynamic indexes reached the lower limits, and interestingly, three patients showed body movements even with a low yet normal index of depth of anesthesia, indicating that the reliability of IoC monitoring for depth of anesthesia in predicting body movements should be further studied in the future. In the present study, five patients who underwent preoperative chemotherapy showed higher IoC1 and IoC2 than the reference ranges for about 75 % of the time (among whom some showed light anesthesia with IoC1 and IoC2 reaching 73-88, which almost indicated regained consciousness), although their effect compartment concentrations of propofol and remifentanil were 3.5-5.5 μg/ml and 2-5 ng/ml, respectively, and their hemodynamic indexes were within the reference ranges. According to previous studies, preoperative chemotherapy can damage liver and renal functions and the nervous system [19,20], and chemotherapy drugs can boost the effects of and reduce the use of anesthetic drugs. However, in this study, the doses of anesthetics were higher in the patients with breast cancer who underwent preoperative chemotherapy. They also showed faster metabolism of muscle relaxants and faster recovery of spontaneous respiration, inconsistent with previous studies. Since only a few patients (17 in the C group and 12 in the T group) with preoperative chemotherapy were included in this study, the above inconsistency is not explainable and thus, should be further validated by other studies. Unlike bispectral index monitoring, the monitor used in this study does not require special electrodes, which reduces medical expenditures and promotes clinical propagation and application. Normally, the EEG amplitude is within 0-200 μV, about 1/400 of cardiac electrical activities; hence, compared with ECG, it is more easily affected by widely used high-frequency electrical equipment such as an electrocoagulation electrotome. We chose patients undergoing mastectomy because the electrotome used for surgery is low in power and distant from the site of data collection and as a result, the effects of interference are smaller. Nonetheless, this disadvantage may still affect its further clinical application and some improvements should be made on surgical site selection, materials for production of the monitor, and filtering clutter, among others. This study suffers from some limitations. The sample size was small. We only studied the application of IoC monitoring to TIVA and thus its effectiveness with inhaled anesthesia is unclear. In addition, only female patients were included in our study, but the possible differences of pharmacokinetics with male patients should be considered. Fentanyl was used for intubation, but the same dose was used in both groups; therefore, it should not influence the comparison between the two groups. Importantly, propofol administration was not standardized and was based on IoC1, therefore introducing variability that could mask the real effects of IoC2 monitoring. In addition, standard monitoring parameters, such as bispectral index, should be included in future studies to correlate the parameters with IoC2. Finally, some confounding factors that may affect our results have not been controlled and additional studies are necessary to examine the impact of neoadjuvant chemotherapy on IoC2. Conclusions In summary, EEG anesthesia depth index (IoC) monitoring during TIVA is safe and effective for patients with breast cancer. IoC1-targeted propofol dosing does not seem to be significantly different to hemodynamic-based monitoring, whereas the application of IoC2 might be used to guide the use of remifentanil. It might reduce the occurrence of adverse events and keep hemodynamics more stable, but its reliability in predicting body movements as well as the anti-interference ability should be further improved.	2016-05-12T22:15:10.714Z	2016-03-29T00:00:00.000	{ "year": 2016, "sha1": "0c74a60524688ee25649e10e88ea567df9058bc5", "oa_license": "CCBY", "oa_url": "https://trialsjournal.biomedcentral.com/track/pdf/10.1186/s13063-016-1298-0", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "92ad2656932073663f6bcf8df2091ba52aebaff4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
244154175	pes2o/s2orc	v3-fos-license	Chronic Disease Management Programs Based On Caring Theory With Blood Pressure Reduction Background: Hypertension cases to overcome the patient's hypertension condition would be well or prosperous condition and the patient could prevent complications and control blood pressure. Activities in Chronic Disease Management Program (Prolanis) include medical or educational consultations, home visits, reminders, club activities. Purpose: This study aimed to analyze the application of the prolanis program based on the caring theory by reducing blood pressure in hypertensive patients. Methods: The design of this research is pre-experimental with cross sectional study approach. The population in this study were all patients with hypertension who took part in management program activities in the public health center (PHC), a number of 40 people, with the sampling technique used was accidental sampling and the total sample was 28 people. The data collection method used a questionnaire. The data analysis of this research is to use the Paired t test. Results: The result of this study is that there is a relationship between the application of a chronis disease management programs based on caring theory with blood pressure reduction with an average systolic blood pressure before the intervention of 142 26.15 and after the intervention of 138 1.21 with p= 0.000 which means that the module intervention can decrease systolic and diastolic blood pressure. While for diastolic blood pressure, the results of the study showed that the mean blood pressure before the intervention was 85 10.36 and after the intervention was 85 7.61 with p= 0.000. Conclusion: It can be concluded that there is a possitive effect between giving prolanis module based on caring theory with systolic and diastolic blood pressure. BACKGROUND Hypertension is caused by several factors that increase the risk of hypertension, namely lifestyle, fast food, obesity, smoking and alcohol. Problems that usually arise in clients with hypertension are activity intolerance disorders, pain (headaches), high risk of injury, and lack of knowledge related to lack of information. If not managed properly, hypertension can cause complications such as stroke, coronary heart disease, heart failure, chronic kidney disease, kidney failure (M. Christopher 2016). The high prevalence of chronic and degenerative diseases, one of which is a health problem is hypertension. Hypertension can occur in everyone, both young and old. The government through Health Assurance launched a Chronic Disease Management Program (Prolanis) purposed at patients with chronic disease such as hypertension cases to maintain stability the patient's hypertension condition (BPJS 2014). Activities in Chronic Disease Management Program (Prolanis) include medical or educational consultations, home visits, control blood pressure, and club activities. WHO 2013 data found that 79% of people are at risk of experiencing hypertension, and 67% of people in the world are positive for hypertension with relatively high blood pressure (WHO 2013). In Indonesia, hypertension is a case that is often experienced, especially in old age. From the public health survey data, it was found that hypertension still dominates disease cases in Indonesia, with 59% incidence. From the monthly report data of the East Java Provincial Health Office, it was found that hypertension cases were included in the top 10 diseases with 65% of cases. The government through health assurance seeks one program to overcome these problems through the Chronic Disease Management Program (Prolanis) (BPJS 2014). The aim of the chronic disease management program activities is to encourage participants with chronic diseases, especially hypertension to achieve optimal quality of life with an indicator that 75% of registered participants who visit first level facilities have good results on specific examinations of Type 2 Diabetes Mellitus and Hypertension according to related clinical guidelines so that can prevent disease complications (BPJS 2014). The targets in Chronic Disease Management Program (Prolanis) are all health assurance participants with chronic diseases such as Type 2 Diabetes Mellitus and Hypertension (BPJS 2014). With the running of the program will be able to overcome cases of hypertension experienced by patients, on the contrary, if the program does not run, the cases of hypertension will be resolved longer because the program can be used as screening for cases of hypertension and as a medium for evaluating the extent to which cases of hypertension are experienced by the community. It needs the role of various parties, both from health workers who should strive for the chronic disease management program to run so that it can overcome cases of patient hypertension, using promotive, preventive, curative, and rehabilitative methods. In this program, health workers can do promotive by providing counseling about the disease experienced, preventive by providing counseling about prevention that clients must do such as implementing a low salt and high potassium diet by not consuming salty foods and often consuming fruit such as oranges. Curative action can be done in collaboration in providing therapy. And rehabilitative can be done by providing support to patients to recover, as well as restoring the patient's condition to the state before being sick (Rachmawati, Prihhastuti-Puspitasari, and Zairina 2020). Patients should be more active in participating in the program so that they can overcome hypertension, which is seen from normal blood pressure. This study aim to analyzed the effect of Chronic Disease Management Program (Prolanis) based on caring theory to reduced blood pressure in hypertensive patients in Public Health Center Malang City. METHODS The research design in this research is pre experiment with cross sectional approach (Nursalam 2011). The population was all people with hypertension who participated in the Prolanis activities which were carried out every month with a total of 40 people. The sampling technique used in this study was accidental sampling with a sample size of 28 people. From respondents who are willing to be given a Prolanis module based on the caring theory that can be used as a guide for hypertensive sufferers who participate in these activities. Data collection in Februari-Maret 2020 at Pandanwangi Public Health Center Malang using a questionnaire instrument. Analysis of the data in this study used Paired t test. The protocol used in this study was approved by the Ethical Commission of Faculty of Medicine and Health Science Maulana Malik Ibrahim University. RESULTS This research was conducted in the working area of the Pandanwangi Health Center starting from February 2 nd to March 12 th 2020. The management of hypertension patients (Prolanis) at the Pandanwangi Public Health Center in Malang City was in accordance with the operational standards of the Puskesmas when new patients were found with blood pressure reaching 140/90 mmHg. to make lifestyle changes, both eating patterns, increasing physical activity, losing weight, limiting and even stopping smoking, and stress management for one month. Furthermore, the patient was asked to come back for control to the Public Health Center, if the blood pressure was found to be at a fixed value or increased, then proceed to the treatment program. If the blood pressure falls, then lifestyle arrangements are continued at home and asked to return in the following month. The results of general data analysis that researchers got in this study based on gender, age, education, occupation, history of hypertension, and history of kidney disease are as follows: Results describe the major findings of the study. It should be clear, concise and can be reported on texts or graphics. Please provide some introduction for the information presented on tables or images. Based on table 1, it can be seen that most of the respondents in this study were female, namely a total of 20 respondents (71%) with almost all of them being 46-55 years old as many as 26 people (93%) and most of the education levels were junior high school as many as 10 people (36 types almost half of them are housewives as many as 15 people (54%). History of hypertension Most of them have as many as 16 people (57%) and none of them suffer from kidney disease Specific research data will describe the respondent's data including, pre-test and post-test blood pressure analysis. Based on table 2, it was found that the pretest systolic blood pressure had a mean of 14226.15 and the post-test decreased to 1381.21, with a value of p=0.000. Diastole blood pressure in the results of the study showed that the pretest diastolic blood pressure had a mean of 850.36 and the post test 857.61, with a value of p = 0.000. DISCUSSION From the results of statistical tests, it shows that H0 is rejected, meaning that there is an effect of Chronic Disease Management Program (Prolanis) on systolic blood pressure and diastolic blood pressure in patients with hypertension. The results showed that the data showed that most of the respondents' education level was junior high school. However, the approach provided with simple and practical health information is easier for respondents with hypertension to follow and work on. The Chronic Disease Management Program (Prolanis) that is given directly to the respondent is applicable, for example simple activities that involve the respondent directly are easier to catch and remember by the respondent and then easy to implement. This activity also involves the active role of village health cadres who are very helpful in getting directly involved and motivating respondents. This fact is in accordance with the theory of Huffman (2007) that the practice of health education and promotion with the aim of improving individual health and promoting the achievement of health goals, which effectively stimulates behavioral change in an organized manner through a supportive interaction between nurses and hypertension respondents. (Notoatmodjo 2014). It is also in line with research which states that Chronic Disease Management Program (Prolanis) can influence patient behavior in managing their disease (Rachmawati, Prihhastuti-Puspitasari, and Zairina 2020). Prolanis can motivate behavior change as stated there are three factors in achieving behavior change, namely the individual's readiness to change behavior in order to avoid a disease or minimize health risks, the encouragement in the individual's environment that makes him change behavior, and behavior itself (Priyoto 2014). During the research process, most of the Intervention module filling had been filled in correctly according to the column, were orderly, neat, routine, and filled out consciously and did not feel burdened. Some respondents who are not good at writing took the initiative to ask their family for help in writing about their diet, activities and blood pressure that they do every day. Respondents themselves also felt that it was helped by filling in this booklet because they could automatically adjust the diet and what activities were carried out. This prolanis module is given with the aim of increasing the patient's understanding and confidence in their disease that the dangers of hypertension are very dangerous and even cause death if it is not followed up with changes in physical activity behavior. Increase the confidence and enthusiasm of respondents that there is still time and the ability to make changes. The activities carried out include the selection of recommended physical activities and regular supervision in carrying out these physical activities by involving the family. Another factor that affects blood pressure is gender. Most of the sex of the respondents were postmenopausal women. As mentioned, several factors supporting the occurrence of hypertension are the female sex with post menopausal age (Singh, Shankar, and Singh 2017). In research which analyzed factors related to hypertension, it was found that more than half of the research respondents were women with most of them being over the age of 20 years, which is the age of menopause (Alloubani, Saleh, and Abdelhafiz 2018). It was also reported that the percentage of incidence of hypertension increases in women over 49 years. Because the permeability of major blood capillaries declines with age until the seventh decade, systolic blood pressure, while diastolic blood pressure rises until the fifth and sixth decades then persists or tends to decrease. With increasing age, several physiological changes occur, such as increased peripheral resistance and catecholamine activity, decreased sensitivity to blood pressure regulation, especially the baroreceptor reflex, and a reduced in the role of the kidneys, as measured by renal blood flow and glomerular filtration rate. As a result of the kidneys' failure to appropriately eliminate the salt load, salt and water resistance occurs, resulting in an increase in plasma volume. Furthermore, if the glomerular cells of the apartus in the kidney secrete the hormone renin, which activates angiotensinogen in plasma to become angiostensin I, which subsequently goes through the pulmona, the kidney's filtration rate would decrease, the granular cells of the apartus in the kidney will secrete the hormone renin which activates angiotensinogen in plasma to become angiostensin I which then passes through the pulmonary circulation and is converted by Angiotensin Converting Enzyme (ACE) to angiotensinogen II which is a strong vasoconstrictor. Angiotensin II stimulates the production of aldosterone from the adrenal cortex, causing a rise in salt retention and a rise in plasma osmolality, which is then countered by a rise in water absorption. This will cause an increase in cardiac output which will then increase arterial blood pressure (Sherwood 2011). The hormone estrogen, which has a role in increasing levels of High Density Lipoprotein, protects women who have not gone through menopause (HDL). High HDL cholesterol levels are indeed a protective factor in the prevention of atherosclerosis. The presence of immunity in premenopausal women is assumed to be due to estrogen's protective influence. Women in premenopause begin to lose the hormone estrogen, which has been protecting blood vessels from damage, progressively. This process continues as the estrogen hormone naturally increases in quantity with a woman's age, which usually begins in women between the ages of 45 and 55. Menopause happens to women over 50 years old in a variety of ways, including physical, hormonal, and mental changes. Fatigue, anxiousness, headaches, insomnia, sadness, irritability, joint and muscle pain, dizziness, and heart palpitations are some of the symptoms. Unstable emotions can also cause sleep disturbances (Association 2013). Psychological difficulties of the respondents that generated stress, such as the presence of a sick family member, a disaster, urgent economic demands, employment challenges, and problems with children, were some of the things discovered throughout the study that may be utilized as causes. This condition manifested evident near the end of the study, resulting in a increase in the blood pressure of some respondents. Peripheral vascular resistance, cardiac output, and parasympathetic central nervous system activity generally rise in stress response. Busyness, infection, trauma, obesity, old age, psychological disorders, medicines, disease, surgery, and medical therapy are just some of the stressors that can cause stress. The sympathetic nerves are activated in response to stress (nerves that work when we are active) (Priyoto 2014). Increase in blood pressure is caused by increased sympathetic nerve activity. Stress can cause the adrenal glands to release adrenal hormones and the heart to beat faster and stronger, resulting in an increase in blood pressure.This can happen because most of the respondents' jobs are housewives who carry out the same routine every day, do the same tasks, and focus on family and existing family problems without any distraction of entertainment with a 24-hour workload (James, PA, S Oparil, BL Carter, J Handler WC Cushman 2014). When family problems arise, it will become the focus of thought for the respondent. This can be a stressor and cause stress for the respondent which can result in an increase in the respondent's blood pressure. CONCLUSION Based on the results of the research that has been done, it can be concluded that: There is a difference in systolic blood pressure before and after intervention with systolic blood pressure 14226 and after 1381.21 (p=000). There is a difference in systolic blood pressure before and after intervention with dyastolic blood pressure 8510.36 and after 857.61(p=000). So it can help the community to increase activities in the Chronic Disease Management Program (Prolanis) and module intervention can be used by nurse to nursing interventions to improve behavior change, maintain blood pressure stability and regularly control their blood pressure.	2021-11-17T16:30:29.678Z	2021-10-01T00:00:00.000	{ "year": 2021, "sha1": "eb316d48a5428d9f6f341822cb94720c074610bd", "oa_license": "CCBYNCSA", "oa_url": "http://thejnp.org/index.php/jnp/article/download/153/116", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "ed4899bfe5de6c3c8b2ff7a321052c9af15341b0", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
6923254	pes2o/s2orc	v3-fos-license	Time-reversal and super-resolving phase measurements We demonstrate phase super-resolution in the absence of entangled states. The key insight is to use the inherent time-reversal symmetry of quantum mechanics: our theory shows that it is possible to \emph{measure}, as opposed to prepare, entangled states. Our approach is robust, requiring only photons that exhibit classical interference: we experimentally demonstrate high-visibility phase super-resolution with three, four, and six photons using a standard laser and photon counters. Our six-photon experiment demonstrates the best phase super-resolution yet reported with high visibility and resolution. Common wisdom holds that entangled states are a necessary resource for many protocols in quantum information. An example is quantum metrology, which promises super-precise measurement, surpassing that possible with classical states of light and matter [1,2]. In the last 20 years quantum metrology schemes have been proposed for improved optical [3][4][5][6][7][8] and matter-wave [9] interferometry, atomic spectroscopy [10], and lithography [11][12][13]. The entangled states in these schemes give rise to phase super-resolution, where the interference oscillation occurs over a phase N-times smaller than one cycle of classical light [14,15] and phase super-sensitivity, a reduction of phase uncertainty. Many quantum metrology schemes are based on pathentangled number states. The canonical example is the noon-state [1], a two-mode state with either N particles in one mode and 0 in the other or vice-versa, i.e., (\|N0 +\|0N )/ √ 2. A deterministic optical source of path-entangled states is yet to be realised, requiring optical nonlinearities many orders of magnitude larger than those currently possible. However, entangled states can be made non-deterministically using single-photon sources, linear optics, and photon-resolving detectors [16]: leading to a flurry of proposals to generate pathentangled states [17][18][19][20]. While phase super-resolution with two-photons has been demonstrated often since 1990 [21][22][23][24], phase super-resolution was experimentally demonstrated for 3-photon [14] and 4-photon [15] states only recently. As efficient photon sources and photonnumber resolving detectors do not yet exist, all demonstrations to date necessarily used multiphoton coincidence post-selection [25]. Problematically, current photon sources are extremely dim and true photon-number resolving detectors are expensive and uncommon. In this paper we introduce a time-reversal technique that eliminates the need for exotic sources and detectors, achieving high-visibility phase super-resolution with a standard laser and photon detectors. port interferometer, U multi [26]. With probability η p , no photons are found in modes 3 to N, heralding the noonstate in modes 1 and 2, (\|N0 12 + \|0N 12 )/ √ 2. A relative phase shift, φ, between modes 1 & 2 introduces a Nφ shift between the terms in the state-phase super-resolution. Maximum fringe visibility will be achieved when the system is measured in a state ψ N \| which has equal overlap, κ N =\| ψ N \|N0 \| 2 =\| ψ N \|0N \| 2 , with both components of the noon-state. Fig. 1 shows two possible measurements, i) yields κ i N =1/2 N [19], ii) yields κ ii N =κ i N / √ 2πN in the optimum case \|α\| 2 =N/2. The probability of detecting a final state Ψ f \|= ψ N \| 12 0...0\| 3...N after propagating through the multiport and phase shifter, U φ , is P =\| Ψ f \|U φ U multi \|Ψ i \| 2 =η p κ N (1+cos Nφ). This probability exhibits phase super-resolution since the fringes complete N oscillations over a single cycle of 2π. Probabilities in quantum mechanics are invariant under time reversal [27][28][29], i.e., if we swap the input and measured states and suitably time-reverse the operation of the multiport, as shown in Fig. 1 In the time-reversed picture, the interferometer no longer plays the role of probabilistic noon-state generator, but rather constitutes a probabilistic noon-state detector : since the probability, P , is invariant under time reversal, phase super-resolution is also invariant. Experimentally, detecting noon-states is much easier than creating them: time-reversing turns the difficult generation of N . In c) the indicated HWP's are set to 22.5 • to form 1/2 beamsplitters with the beam displacers, the third is set to 17.6 • so that 2/3 of the light intensity takes the upper path; the angle of the tilted HWP sets β, its optic axis is at 0 • . In d), the beamsplitter for modes 3, 4 is a pellicle; for modes 5, 6 it is a microscope slide set at a small angle of incidence, ∼10 • , to avoid polarisation effects. Reflectivities are not ideal, the rates are equalised with lossy coupling. U 1 to U 3 , are realised using waveplates: the orientation and tilt of each waveplate was adjusted so that one detector reaches an interference minimum every 22.5 • for N=4; every 15 • for N=6. single photons into straightforward detection of N photons in coincidence, and turns the problematic detection of the vacuum into vacuum inputs which are automatically available with perfect fidelity. Successful detection of a noon-state is signalled by the coincident detection of a single photon at each output detector; this is the time-reverse of the coincident creation of a single photon at each input. This time-reversal technique is a simple example of a more general measurement technique introduced by Pregnell and Pegg [29,30]. This theory assumes that the input photons are indistinguishable. A significant advantage of our approach is that it is robust: phase super-resolution occurs even when the photons are distinguishable. Creating noon states relies on non-classical interference, which requires indistinguishable photons. In our time-reversed scheme we require only that the inputs display classical interference: this is not affected by the degree of distinguishability between the photons, or the photon arrival statistics. Experimentally, there is a trade-off between temporal distinguishability and counting rate: photons become distinguishable as the coincidence window time is increased above the input light coherence time, but this increases the counting rate. We run in the high counting rate limit to achieve the best statistics, limited only by saturation effects in our coincidence-counting electronics. In our experiments the two bright inputs to the multiport, modes 1 & 2, are the vertical and horizontal polarisation modes of the one spatial mode from a laser. We use an attenuated He:Ne laser (Uniphase 1135P) and set the polarisation with a half-wave plate (HWP) followed by a quarter-wave plate (QWP) at an angle of 45 • . Changing the angle of the HWP by φ/4 changes the relative phase between the modes by φ, while ensuring the vertical & horizontal modes are the same amplitude. In classical interferometry, this yields one oscillation for 0<φ<2π. Multiports can be symmetric (every input mode is converted into an equal superposition of N output modes [19]) or asymmetric (not every input satisfies this condition [18]). Scaling up symmetric multiports beyond N=2 can be done either with a polynomial number of nested standard interferometers [26], which would be arduous to phase lock, or a single N×N fused fibre except that it is not known how to control the large set of internal phases [19]. Fortunately symmetric multiports are not required for phase super-resolution: an asymmetric multiport suffices for even-N. Fig. 2 shows our symmetric N=3, and asymmetric N=4, 6 multiports (the N=4 multiport was independently proposed in [31]): all designs are passively stable and do not require active phase-locking. In fig. 2c) the output modes are sent to three pinhole photon counting detectors, D1-D3, where the small aperture is a single-mode fibre without a coupling lens; in Fig. 2d) each output mode is first passed through a polarising beamsplitter and then detected. The singles rate is the number of photons per second detected by an individual detector: for N=3 the maximum was 5×10 4 Hz; for N=4, 6 the maximum singles rate was 1.3×10 5 Hz. The N singles rates are recorded individually. For N=3, the N-fold coincidence rate is measured using two ORTEC 567 Time-to-Amplitude Converter/Single Channel Analyzer (TAC/SCA) modules each with a 1.5µs coincidencewindow; for N=4, 6 coincidence counting was performed using up to 3 TAC/SCAs and an ORTEC CO4020 Quad Logic Unit. For N=4 (6) all pulse length inputs to the Quad were set to 1.5µs (5µs) as were the coincidencewindows on the TAC/SCA. In all cases, due to a restricted number of recording channels, the singles were measured immediately after a coincidence run. To avoid saturation in the coincidence electronics, the mean number of photons per coincidence-window must be ≤1: for N=3, 4, and 6 it was up to 0.07, 0.15, and 0.48. Fig. 3 shows the coincidence and singles rates for the N=3 symmetric, and the N=4, 6 asymmetric experiments of Fig. 2c) & d). Fig. 3a)-c) shows the three-, four-, and six-fold coincidence rates as a function of the phase, φ, with three, four, and six distinct oscillations within a single phase cycle. This is in contrast to the fringes observed in the singles rates, Fig. 3d)-f), which undergo only a single oscillation over the same range. This is the experimental signature of phase super-resolution. We emphasize that this was achieved without production of a path-entangled state, which would have had the signature of flat singles rates over an optical cycle [15,24]. As discussed above, time-reversed phase superresolution does not rely on non-classical interference: the coincidence rate is determined entirely by the product of the singles rates. Consider an N×N multiport set up so that the detection probability in the k th output mode is P k ∝ 1+cos(φ+2πk/N+ϕ), where ϕ is a constant phase offset. The N-fold coincidence probability is then simply the product of the single mode probabilities, i.e., P 11...1 ∝ 1+cos (Nφ+∆(N, ϕ)) which clearly exhibits N oscillations per cycle, where ∆ is the offset. Applying this to Figs. 3a)-c), we respectively fit a product of 3, 4, and 6 sinusoidal fringes, s i =c i v i sin(φ+δ i )+c i where v i is the visibility, and c i & δ i are amplitude and phase offsets, of the i th fringe. The resulting fits-the solid lines in Figs. 3a)-c)-are very good, with reduced χ 2 of 1.6, 6, and 1.7, respectively. (The high value in the N=4 case is most likely due to observed amplitude instability of the D3 signal during the course of the coincidence measurement.) The coincidence fringes for all three experiments differ from a pure sinusoid due to small variations in the underlying singles rates, and become more pronounced for larger values of N . The solid lines in Fig. 3d)-f) are the individual sinusoidal fringes, s i , scaled by a constant factor that matches the amplitude to the data but does not alter the visibility or phase of each fringe. Again the agreement is very good. The N =6 case matches the largest phase super-resolution reported to date, obtained in an ion-trap system [32], but has significantly better visibility. Our results clearly show that path-entangled states are not required for phase super-resolution [33]. Previous optical demonstrations used nonclassical light sources, which are notoriously dim, limiting the three-and fourfold coincidence rates to 5 Hz [14] and 0.1 Hz [15] respectively [34]. We significantly improve on this, achieving phase super-resolution with a six -photon coincidence rate of about 2.7 Hz; furthermore, owing to the high-visibility singles and extremely stable construction of the multiports, our fringe patterns all exhibited high visibility. Fitting a single sinusoid, and without any background subtraction, the fringe visibilities for the N=3, 4, and 6 cases are respectively 81±3%, 76±2%, and 90±2%-well exceeding previously reported raw visibilities of 42±3% for N=3 [14] and ∼61% for N=4 [15]. An alternative technique for realising phase super-resolution sums multiple occurrences of a fringe pattern narrowed by nonlinear detection, either spatial [36] or temporal [37]. This suffers from exceedingly low visibility: when the number of exposures equals the number of fringes, V =2(N !) 2 /(2N )!, for N=6 this predicts V ∼0.2%. Phase super-sensitivity occurs when there is a reduction of the phase uncertainty as compared to that possible with classical resources. Unlike phase superresolution, phase super-sensitivity cannot be determined solely from the fringe pattern: careful accounting is needed to determine the resource consumption required to achieve the measured signal. For small variations in phase around a given value, the phase uncertainty is ∆φ=∆A/ d A dφ , where A and ∆A are an observable and its associated uncertainty [3]. All other things being equal, the slope in the denominator is increased by phase super-resolution, reducing ∆φ. Phase super-sensitivity is achieved when ∆φ is less than the classical limit, where η allows for non-ideal efficiency in using N tot resources to estimate ∆φ. Phase super-resolution produces normalized fringes of the form (1-V cos Nφ)/2, where V is the fringe visibility, and the slope is d A /dφ = 1 2 N V sin Nφ. Beating the classical limit requires, Consider A to be a projector with measurement outcomes bounded by 0 and 1; the worst case is ∆A=1/2. At the point of minimum phase uncertainty, Eq. 2 reduces to, By this criterion, although several experiments have demonstrated phase super-resolution, there has been no unambiguous demonstration of phase super-sensitivity. The best known preparation efficiency in nondeterministic optical schemes is η=2N!/N N [17][18][19]. In the ideal limit, Eq. 3 gives 2N!/N N−1 >1, which is true only for N=2, 3. Phase super-sensitivity cannot be achieved in any described nondeterministic scheme for N≥4 [38]. An alternative version of phase super-sensitivity arises if the important physical resource is the number of photons passed through the sample, not the total number of photons consumed. Phase super-sensitivity can then be achieved for all N since in principle time-forward schemes can be heralded with perfect efficiency [17][18][19]. In a recent work using trapped ions, phase super-resolution was observed for 4, 5, and 6 ions with respective visibilities of 69.8±0.3%, 52.7±0.3%, and 41.9±0.4% [32]. Determining if phase super-sensitivity was achieved requires knowledge of the uncertainty for each data point, which can not be determined from the published data. In the worst case, Eq. 3 shows that phase super-sensitivity was achieved if the overall efficiencies were respectively 51.3±0.4%, 72.0±0.8%, and 95±2% for 4, 5, and 6 ions. We have used a time-reversal analysis to show that it is not necessary to produce path-entangled states to achieve phase super-resolution. We show that phase super-resolution is possible even in the absence of nonclassical interference; and derive the necessary conditions to claim phase super-sensitivity from phase superresolution. Using standard laser sources we obtain highvisibility and high-contrast phase super-resolution of up to 6 oscillations per cycle in a six-photon experiment. The improvement in phase resolution is homologous to that achieved in a standard path interferometer driven at a wavelength of 105.5 nm-one-sixth the wavelength of our He:Ne laser. Inverting the roles of state production and measurement is an application of a more general time-reversal analysis technique [29,30]: given the dramatic improvement demonstrated here, it remains an interesting open question as to which other quantum technologies will benefit from this technique. This work supported in part by the Australian Research Council and the DTO-funded U.S. Army Research Office Contract W911NF-05-0397. We thank T. C. Ralph and G. J. Milburn for discussions, and H. Wiseman, D. T. Pegg and P. Meredith for helpful MS comments.	2017-04-01T13:48:25.335Z	2005-11-22T00:00:00.000	{ "year": 2005, "sha1": "d0ab6e480d009686433c05a755b7622001f60f23", "oa_license": null, "oa_url": "https://research-repository.griffith.edu.au/bitstream/10072/15624/1/43226_1.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "8ff0174380ac2482046981e87a16ada7b1e113af", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics", "Medicine" ] }
195066602	pes2o/s2orc	v3-fos-license	Capacitive Bio-Inspired Flow Sensing Cupula Submersible robotics have improved in efficiency and versatility by incorporating features found in aquatic life, ranging from thunniform kinematics to shark skin textures. To fully realize these benefits, sensor systems must be incorporated to aid in object detection and navigation through complex flows. Again, inspiration can be taken from biology, drawing on the lateral line sensor systems and neuromast structures found on fish. To maintain a truly soft-bodied robot, a man-made flow sensor must be developed that is entirely complaint, introducing no rigidity to the artificial “skin.” We present a capacitive cupula inspired by superficial neuromasts. Fabricated via lost wax methods and vacuum injection, our 5 mm tall device exhibits a sensitivity of 0.5 pF/mm (capacitance versus tip deflection) and consists of room temperature liquid metal plates embedded in a soft silicone body. In contrast to existing capacitive examples, our sensor incorporates the transducers into the cupula itself rather than at its base. We present a kinematic theory and energy-based approach to approximate capacitance versus flow, resulting in equations that are verified with a combination of experiments and COMSOL simulations. Introduction Recent work in bio-mimetics has shown the advantages taking inspiration from fish for the purposes of creating aquatic robots [1,2]. In particular, body shape [1], thunniform swimming techniques [1,3], and skin texture [4] have been shown to improve efficiency and increase maneuverability. However, behavior [5] is also a key factor in leveraging these advantages. The ability for fish to sense and react to flow enables them to orient efficiently (rheotaxis) [6], detect objects [7], and navigate turbulent flow more efficiently [8]. This concept has been applied to some robots by employing rigid commercial pressure sensors [9,10], but truly biomimetic robots require sensors that are compliant, introducing no rigidity to an already soft bio-mimetic robot. In this work, we develop a capacitive device inspired by superficial cupula structures in fish. In an effort to remain more robust and bio-mimetic, the sensor is created as an all-soft-matter device, consisting of soft silicone rubber and liquid metal. The fabrication methods are unique to this application, employing sacrificial lost wax methods and vacuum filling of channels. The biological inspiration for this work is derived from the lateral line sensing networks located on fish. Sensors are positioned both on the creature's surface-called superficial neuromasts-and in recessed channels-called canal neuromasts [11,12]. These two subsystems are considered to have different sensing purposes, such as velocity for superficial versus acceleration sensing for canal (though this is a simplification that ignores factors such as resonance [12]). Our sensor format is most similar to the superficial neuromast, which tends to bend under flow rather than slide like a rigid body as in the case of canal neuromasts [11]. [11,13,14]. Inset-detailed image of the hair bundle, highlighting the fiber links between cilia; (b) artificial capacitive flow sensor and components. The biological sensor itself consists of several sensory hair cells that extend microscopic hair (cilia) bundles into the base of a gel cupula [11,13], as shown in Figure 1. These bundles usually include one tall kinocilium and a "staircase" of shorter stereocilia. The cilia are attached to one another with filament "links." Of particular interest are the kinocilial link and the tip links, as shown in the inset of Figure 1. Under flow, the structure deforms and an electrochemical reaction is produced by relative motion between cilia [13,15]. Specifically, as the hairs move, the links are slackened or pulled taut, activating ion channels/gates at the connection points. While the stereocilia and tip links [15] exist primarily for activating gates, the kinocilia and kinocilial links [13] as well as the gel cupula structure [16,17] have been shown to increase overall sensitivity. For this work, resistive and capacitive sensing are particularly attractive due to their prevalence in soft robotics. Soft resistive sensors typically consist of rubber embedded with conductive particles and have been developed with applications in medical monitoring [31], motion tracking [31], bioinspired unmanned air vehicles [32], and prosthetics [33]. Following Ohm's law, deformation results in either increased or decreased conductance due to changes in micro and macro geometry. In contrast to traditional, non-hyperelastic strain gauges whose material (i.e., constantan alloy foil) can generally be assumed to maintain constant conductivity, the separation and contact of the filler material within conductive elastomers invalidates this assumption and often results in greater [11,13,14]. Inset-detailed image of the hair bundle, highlighting the fiber links between cilia; (b) artificial capacitive flow sensor and components. For this work, resistive and capacitive sensing are particularly attractive due to their prevalence in soft robotics. Soft resistive sensors typically consist of rubber embedded with conductive particles and have been developed with applications in medical monitoring [31], motion tracking [31], bio-inspired unmanned air vehicles [32], and prosthetics [33]. Following Ohm's law, deformation results in either increased or decreased conductance due to changes in micro and macro geometry. In contrast to traditional, non-hyperelastic strain gauges whose material (i.e., constantan alloy foil) can generally be assumed to maintain constant conductivity, the separation and contact of the filler material within conductive elastomers invalidates this assumption and often results in greater sensitivity. These conductive rubbers, however, are vulnerable to hysteresis, viscoelastic responses, and drift under environmental influences, such as humidity or temperature [31][32][33]. (Note that temperature sensitivity for our device is discussed in Appendix A.) For this reason, low toxicity room temperature liquid alloys (gallium-based) offer a promising alternative and have been widely applied in resistive sensing for robotics [34], sensing skins [35], and motion tracking [36]. A simple capacitive pressure sensor consists of two compliant electrodes separated by a soft dielectric. Deforming the electrode surface area or changing the dielectric thickness results in measurable changes in capacitance. This value can be measured accurately even when the electrodes and interconnects have low and/or inconsistent conductivities. As a result, the resistive hysteresis and drifting behavior of conductive rubbers that make some piezoresistive sensing applications infeasible are not a major limitation for measuring capacitance, allowing the use of such materials in capacitive sensors [31,33]. Regardless, liquid metals have been applied to comb capacitor stretch sensors [37] and grid-based capacitive skins [38][39][40], particularly where viscoelasticity, total loss of conductivity, or spotty performance is a concern (e.g., during hyperelastic stretch). A further benefit over resistive sensing is the ease of multiplexing and the ability to use of grid formations without ghosting when multiple nodes (i.e., multi-touch) are affected (mutual capacitance sensing) [41]. This makes capacitive sensing particularly useful for systems where networks of many sensors may be required. It should be noted, however, that capacitive sensors are often slower in response time when compared to piezoresistive, likely due to the time constant associated with charging and discharging. We have found that the sampling rates of capacitance to digital converters are typically below 1 kHz, whereas analog to digital converters (which can be used for resistive sensing) exhibit frequencies several orders of magnitude higher. In some cases, it may be appropriate to use circuits from condenser (capacitor) microphones, which rely on impedance changes during vibrations and generally include an impedance converter [42] to output a voltage that can be read with an analog to digital converter. This sort of dynamic sensing approach, while capable of sampling high frequency vibrations, is less useful for the work presented here, where a static deformation during flow is observed. Given the benefits described above, capacitive sensing was chosen for our bio-mimetic device. In contrast to existing examples [21][22][23]28], the sensing plates are located in the cupula itself rather than at its base. In some regards, this more closely resembles biological structures, which consist of hair/cilia encased by gel cupulas. In nature, hairs perform the mechanoelectrical transducing, whereas capacitive plates perform that function in our artificial version at a much larger scale, as seen in Figure 1a,b. Furthermore, although several existing works exist for bio-inspired capacitive flow sensors, to the best of the authors' knowledge, only one example (seal whisker-inspired [28]) exists that includes testing in water rather than gas flow. This is likely due to difficulties associated with MEMS devices in aqueous environments, such as fragility and corrosion [43]. Our sensor incorporates liquid metal embedded in silicone, making it robust to water and direct handling. Design and Operating Principle The sensor is comprised of three liquid metal plates embedded into the structure of a rubber cupula ( Figure 2a). Thus, two parallel plate capacitors are formed, where one central plate, providing the excitation (Exc) for capacitance measurement, is shared for each. In contrast to microtubules [11] that are generally rigid, these capacitive plates are liquid and are not meant to contribute to the stiffness of the structure. whereas capacitive plates perform that function in our artificial version at a much larger scale, as seen in Figure 1a,b. Furthermore, although several existing works exist for bio-inspired capacitive flow sensors, to the best of the authors' knowledge, only one example (seal whisker-inspired [28]) exists that includes testing in water rather than gas flow. This is likely due to difficulties associated with MEMS devices in aqueous environments, such as fragility and corrosion [43]. Our sensor incorporates liquid metal embedded in silicone, making it robust to water and direct handling. Embedded liquid metal geometry Silicone body Ch- Exc Embedded liquid metal 1 cm 1 cm 1 cm Device overview. (a) rendered device image with cupula cutout for clarity (bottom) and a rendering of the internal liquid metal geometry (top). Electrical excitation signals are provided through the channel labeled "Exc" while capacitance is monitored on the receiving ends of "Ch+" (capacitance C+) and "Ch-" (capacitance C-); (b) the device geometry and behavior under no load. . Electrical excitation signals are provided through the channel labeled "Exc" while capacitance is monitored on the receiving ends of "Ch+" (capacitance C + ) and "Ch−" (capacitance C − ); (b) the device geometry and behavior under no load. Here, undeformed capacitance values are C + = C − = C 0 ; (c) the device geometry and behavior under the influence of fluid flow; (d) photo of the fabricated device (mounted on a 3D printed holder); (e) photo of the cupula being stretched by tweezers to demonstrate its compliance. In our device, one capacitor is referred to as the "+" channel (Ch+ with a capacitance of C + ) and one is referred to as the "−" channel (Ch− with a capacitance of C − ). Data are collected based on the difference between the "+" and "−" channels such that, ideally, a sensor under no load should output a capacitance difference value of 0. This is shown in Figure 2b, where the capacitance values are both equal to C 0 . When the cupula deflects away from the "+" channel side, the "+" channel increases in capacitance due to stretch while the negative channel decreases due to compression, resulting in a differential increase (Figure 2c). When deflecting away from the "−" channel, the opposite occurs and the capacitance difference decreases. While a single capacitor placed off-center (similar to a unimorph) is adequate for sensing bi-directional flow, a pair of sensing elements (like a bimorph) has the added benefits of increased sensitivity as well as differential removal of noise (limitations are discussed below and in Appendix A). Furthermore, the pair keeps the system mechanically and electrically symmetric, making responses easier to model and characterize. Figure 2d shows a photo of the completed device after being mounted to a 3D printed holder. The design dimensions ( Figure 3) are 5 mm in height (H), 5 mm in width (W), and 1.75 mm in thickness (T). The liquid metal plates and insulating elastomer are 0.25 mm in thickness (G) such that T = 7G. Each capacitor comprises a 3 mm height (A) by 3 mm width (B) overlapping area that extends into the cupula. Note that the center plate extends slightly to aid visually during fabrication. This is ignored for the analytic approximation. Furthermore, since the liquid metal is practically incompressible, capable of flowing, and under stress, it realistically can contribute to the mechanics of the system, causing behavior such as buckling. However, for simplicity, the deformation of the liquid metal is assumed to contribute nothing to the system during the analytic calculations-an approximation that is shown to be reasonable by COMSOL agreement. The sensitivity of ΔC to tip deflection, y m , is doubled for our design due to the presence two differential capacitors. From this theory, we expect that a tip deflection should result in a linear response from the capacitance. Deformation under Flow To determine the behavior of the cupula under flow, we take an energy minimization approach that has been valuable in the past for predicting beam deformation [45] and buckled beam snap-through [46]. The system consists of elastic energy from the deforming rubber and work performed by the drag associated water flowing past the structure. The elastic energy [44,46] is a Kinematics and Capacitance The behavior of the sensor can be predicted based on kinematics ( Figure 3). In classical beam theory, the neutral axis deflection of a cantilever beam under a tip load follows a third order polynomial [44]. While a fourth order polynomial can be used to describe deformation under a uniform distributed load [44] and is more reflective of the cupula in flow, we found that it contributed little to improving accuracy. Furthermore, when compared to the third order approximation, the fourth order increases the complexity of the solutions, which are primarily meant for understanding the mechanics and for scaling. Thus, we approximate that the cupula deflects into a configuration prescribed by where y m is the max deflection, H is the cupula height, and x is the coordinate along the length (height). This approximation satisfies the essential boundary conditions of zero displacement (y(x) = 0) and zero slope (y (x) = 0) at x = 0. Note that we are following a Lagrange notation for derivatives, where y (x) is the first derivative with respect to x and y"(x) is the second derivative. A third natural boundary condition is satisfied where the moment is 0 at the free end of the cupula due to lack of traction. This is true when the curvature, which can be approximated as y"(x) [44] for small deflections, is 0. Thus, we enforce that y"(x) = 0 at x = H. Given the above geometry, the stretch at some distance, η, from the neutral axis can be approximated as λ ≈ 1 + L (η). The axial strain along the length of the cupula, can approximated as L ≈ ηy"(x). The average stretch experienced by one capacitor (plate height A and dielectric center offset η = G) can then be calculated asλ Note that the parallel plates' capacitance is calculated as C PP = 0 AB/G, where 0 is the permittivity of free space, is the dielectric constant, A is the plate height, B is the plate width, and G is the dielectric thickness. Under uniaxial loading, the capacitance as a function of stretch can be approximated as C ≈ C 0 λ, where C 0 is the value under no deformation. This approximation assumes that the rubber and liquid metal are incompressible with a Poisson's ratio of 0.5. Thus, the change in capacitance (for a single capacitor) as a function of max deflection is simply ∆C = C 0ˆ L (whereˆ L is the average axial strain experienced by the capacitors), or The sensitivity of ∆C to tip deflection, y m , is doubled for our design due to the presence two differential capacitors. From this theory, we expect that a tip deflection should result in a linear response from the capacitance. Deformation under Flow To determine the behavior of the cupula under flow, we take an energy minimization approach that has been valuable in the past for predicting beam deformation [45] and buckled beam snap-through [46]. The system consists of elastic energy from the deforming rubber and work performed by the drag associated water flowing past the structure. The elastic energy [44,46] is a function of the flexural rigidity and the deformation (cupula curvature). In this case, flexural rigidity is the product of the area moment, I, and the elastomer's Young's modulus, E. Thus, the elastic energy is calculated as The energy is calculated with two integrals-one for the lower half of the structure which includes liquid metal and an area moment I 1 and one for the upper half which is solid rubber and has an area moment I 2 . The curvature, κ, can again be approximated as y"(x). We approximate the distributed drag (along the length of the cupula) from the flowing water with the well-known equation w D = 0.5C D ρWu 2 [20,47], where C D is the drag coefficient (~1.05), ρ is the water density, W is the cupula width, and u is the average flow velocity. The influence of the boundary layer (no-slip condition) is ignored for simplicity, though it is considered in the COMSOL modelling. From here, the total work done by the fluid can be calculated with the integral The use of a constant drag coefficient at Reynold's numbers below 10,000, which is applicable to our experiments, is a somewhat crude approximation [47,48]. Nevertheless, we apply it in order to acquire a simple closed-form solution. The total energy of the system, U = U e − W f , is then described simply as the sum of Equations (4) and (5). At this point, the cupula deformation and the system energy is defined by one free parameter: y m . The tip deflection for a series of inputs can then be determined by minimizing the energy as a function of y m (using dU/dy m = 0). Thus, we calculate the change in capacitance as a function of flow for a single capacitor in our device by determining the y m that minimizes U and substituting it into Equation (3): Note that this, once again, must be doubled to account for differential capacitors. This theory provides some design variables to follow and indicates that we expect a parabolic response from capacitance as a function of flow velocity. Fabrication A wide variety of liquid metal patterning methods have been proposed and demonstrated in recent years for stretchable electronics [49]. Many techniques, such as stencil lithography [39], laser patterning [50,51], microcontact printing [37], direct write printing [35], and selective wetting [52,53], are primarily 2D in structure. After application on a soft substrate, the circuits can be sealed with layers of elastomer. Some 3D features can be created with 3D printing [54], but more defined structures and higher aspect ratios require freeze printing [55] or freeze casting [56]. These techniques, however, require customized equipment or additional fabrication steps. Instead, we focus on the methods of lost wax mold casting and vacuum injection. In most examples of casting, elastomer parts are peeled out of reusable molds that can be created with 3D printers [34] or lithography [40]. Multiple molds, sometimes with multiple assembly parts [38], must be used, and several rubber segments may require alignment and bonding to achieve complex geometries [34]. However, overhanging structures and delicate features, as in the case of our proposed design, can be extremely challenging to demold and align. As a result, we employ a sacrificial mold in a lost wax approach. For a single sensor, only one mold is required, though it is dissolved in the fabrication process to release the device. Options for sacrificial lost wax materials include fugitive inks that can be melted [57], poly(lactic acid) that can be vaporized [58], and poly(acrylonitrile-co-butadiene-co-styrene) that can be dissolved in acetone [59]. To avoid custom equipment, excessive temperatures, and harsh solvents, we instead utilize a commercial 3D printer (3Z Pro, SolidScape, Inc., Merrimack, NH, USA) that produces molds from wax-like materials. These materials can melted at low temperatures and dissolved in mild solvents. SolidScape printers and similar components have been applied to the fabrication of scaffolds [60] and microfluidic valves [61,62]. Here, it allows a cupula flow sensor with embedded capacitive sensors and channels for liquid metal wiring to be fabricated in a single casting process. Fabrication steps are shown in Figure 4. The resolution of the printer (~250 µm) required the device to be of substantial size. In order for a liquid metal parallel plate capacitor of 0.75 mm thickness (including liquid metal plate, dielectric, liquid metal plate) to be substantially deformed, the cupula had to be several millimeters tall. 3D printed parts include both structure and support wax-like materials. The support material was dissolved at a lower temperature of~60 • C in a bath of non-polar solvent (BIOACT VSO, Vantage Specialty Chemicals, Inc., Gurnee, USA) for several hours. After removing from the bath, heavy drops of BIOACT were soaked up with a Kim Wipe, and the mold was left out overnight to dry. Before casting elastomer, it is useful to inspect the mold under a microscope to check for debris, particularly between fine features such as the capacitive plates. A strip of paper or a thin wire was used to remove dust and particles as necessary. Next, the uncured polymer can be mixed and casted into the mold. In our case, the silicone elastomer Ecoflex 0030 (Smooth-On, Inc., Macungie, USA) was used for its low Young's modulus. After degassing in a desiccation chamber until bubbles cease to rise, the samples were left out overnight to cure (higher temperatures increase curing rate but risk deforming or melting the wax-like mold). In most examples of casting, elastomer parts are peeled out of reusable molds that can be created with 3D printers [34] or lithography [40]. Multiple molds, sometimes with multiple assembly parts [38], must be used, and several rubber segments may require alignment and bonding to achieve complex geometries [34]. However, overhanging structures and delicate features, as in the case of our proposed design, can be extremely challenging to demold and align. As a result, we employ a sacrificial mold in a lost wax approach. For a single sensor, only one mold is required, though it is dissolved in the fabrication process to release the device. Options for sacrificial lost wax materials include fugitive inks that can be melted [57], poly(lactic acid) that can be vaporized [58], and poly(acrylonitrile-co-butadiene-co-styrene) that can be dissolved in acetone [59]. To avoid custom equipment, excessive temperatures, and harsh solvents, we instead utilize a commercial 3D printer (3Z Pro, SolidScape, Inc., Merrimack, NH, USA) that produces molds from wax-like materials. These materials can melted at low temperatures and dissolved in mild solvents. SolidScape printers and similar components have been applied to the fabrication of scaffolds [60] and microfluidic valves [61,62]. Here, it allows a cupula flow sensor with embedded capacitive sensors and channels for liquid metal wiring to be fabricated in a single casting process. Fabrication steps are shown in Figure 4. The resolution of the printer (~250 μm) required the device to be of substantial size. In order for a liquid metal parallel plate capacitor of 0.75 mm thickness (including liquid metal plate, dielectric, liquid metal plate) to be substantially deformed, the cupula had to be several millimeters tall. 3D printed parts include both structure and support wax-like materials. The support material was dissolved at a lower temperature of ~60 °C in a bath of non-polar solvent (BIOACT VSO, Vantage Specialty Chemicals, Inc., Gurnee, USA) for several hours. After removing from the bath, heavy drops of BIOACT were soaked up with a Kim Wipe, and the mold was left out overnight to dry. Before casting elastomer, it is useful to inspect the mold under a microscope to check for debris, particularly between fine features such as the capacitive plates. A strip of paper or a thin wire was used to remove dust and particles as necessary. Next, the uncured polymer can be mixed and casted into the mold. In our case, the silicone elastomer Ecoflex 0030 (Smooth-On, Inc., Macungie, USA) was used for its low Young's modulus. After degassing in a desiccation chamber until bubbles cease to rise, the The SolidScape structure material is soluble in polar solvents and melts at a higher temperature of about 100 • C. To remove the sensor from the mold, the bulk of the material was softened and scraped away after sitting in an oven set at 100 • C for at least 10 min. The sample was then placed in a small beaker of nearly boiling water until the mold sufficiently melted and dissolved. Isopropyl alcohol was used to clean the surfaces and was injected into the channels with a syringe to remove trapped particles. In many cases, the sample had to be cycled between the hot water bath and alcohol cleaning before all of the mold was removed. The oven set at 100 • C was used to dry the Ecoflex part. Next, three wires (22 AWG) were stripped on both sides. One end was inserted into the bottom side of the sensor to interface with the liquid metal. The opposite end had a 3-terminal female pin connector soldered for interfacing with external electronics. Sil-Poxy (Smooth-On, Inc.) was used to seal the region between the wire and the Ecoflex. Sil-Poxy was also used to seal the inlets on one side of the sensor, leaving a single inlet for each channel/capacitive plate. For most liquid metal injection techniques, an inlet and an outlet is required since air must be evacuated [34,38,40], rendering "dead-ends" difficult to successfully fill. One solution to this is to use vacuum filling techniques [63]. We took a similar approach by applying a vacuum with a syringe, and injecting with a second syringe. This is achieved by interfacing between the sensor, the vacuum syringe (30 mL), and the liquid metal syringe (5 mL) with a three way valve. First, the valve was positioned to connect the sensor and the vacuum syringe. Note that a sufficiently large dispensing needle is required to adequately seal with the channel. While pulling a vacuum with the syringe, the valve was rotated to connect the sensor and the liquid metal (eutectic gallium-indium from Sigma-Aldrich, St. Louis, USA) syringe. At this point, the vacuum syringe was released, and liquid metal was carefully injected. Manual injection with a syringe rather than atmospheric pressure, as demonstrated in the literature [63], allows collapsed membranes (common for very soft polymers) to be forced apart by applying additional positive pressure. Cycling this vacuum and injection process can help with the removal of small trapped bubbles. After injection, the last inlet was sealed with Sil-Poxy and the device was adhered (also with Sil-Poxy) to a 3D-printed (PolyJet, Stratasys, Ltd., Rehovot, Israel) holder to facilitate testing. The interface between the wires and the sensor were sealed with additional Ecoflex 0030 and a cap of epoxy (Devcon 14250, ITW Performance Polymers, Danvers, MA, USA). We found that this sealing was especially important for flow testing, where low pressures during high flow rates within the channel would cause air seep into the device from outside, causing drift and eventual cupula inflation. In some cases, the 3-terminal connectors were removed, re-soldered, and resealed with epoxy to ensure no air could leak through the connector and along the wires. Experimental Setups and Methods To evaluate the performance of the sensor, two experiments were performed. The first examined the response to direct manipulation with an ADMET tensile testing machine (eXpert 2611, ADMET, Inc., Norwood, MA, USA), and the second observed response to water flow in a channel (more details shown in Appendix D). The goal of directly manipulating the cupula was to characterize the capacitive change as a function of cupula tip deflection. While the deformation is not identical to that under liquid flow, this method avoids complexities introduced by vibrations and imprecise flow control, and it is simpler to compare to theory based solely on kinematics. Naturally, the experimentation under flow is a truer evaluation of intended sensor performance. In every case, capacitance values were recorded with an AD7746 evaluation board in conjunction with an Arduino Due. The AD7746 was used at an update rate of 16.1 Hz and with a measured resolution of about 100 aF, though the chip is capable of 90.9 Hz frequency with lower resolution or 4 aF resolution at lower frequencies. No filtering, aside from that internal to the chip, was applied. While the evaluation board includes means to output to a USB and includes software, using the Arduino Due for communication enabled more accurate timing, including trigger functionality. Data was sent through a serial port and recorded with a MATLAB 2017b script. Cupula Displacement Testing For cupula displacement testing, the sensor was mounted to one wall of a box (10 cm × 10 cm × 10 cm), and a notched end effector was attached to the translating portion of the ADMET tensile testing machine. The notch interfaced with the cupula tip. The motion profile featured vertical motions of ±1.25 mm at a quasi-static rate of 0.1 mm/s. This translated to about 1 mm of vertical tip deformation (~2.1% sensor strain). Larger deformations (~1.5 mm vertical deformation and~3.2% sensor strain) are discussed in the supporting information. To accurately record tip deflection as ground truth, the procedure was recorded by a triggered video camera (Phantom Miro M310, Vision Research, Wayne, PA, USA) whose footage was later analyzed with Tracker Video Analysis and Modeling Tool (copyright © 2018 Douglas Brown), with a resolution of about 20 µm. The Arduino was likewise triggered to begin recording capacitance at the start of the ADMET procedure. The side walls of the box were constructed of acrylic to allow for viewing both when the sensor was in air and when submersed in water, during which the water was connected to ground through a metallic bolt. Flow Testing For flow testing, a 7.5 mm × 7.5 mm × 304.8 mm channel was constructed out of acrylic (for viewing through the side), including a mount for the sensor to be inserted (see Figure 5) 229 mm downstream. 3D printed flow-transitions on the ends carried flow from brass tube fittings to the channel to prevent separation of the fully developed flow. The brass fittings were grounded (on both ends of the setup) during testing to prevent interference from other electrical sources and charge buildup during flow. A pump (Unilift KP250, Grundfos, Bjerringbro, Denmark) was used to send water from a 6 m × 6 m × 4 m tank through the system. This large tank was used to avoid temperature changes, which can cause sensor drift (see supporting information for more information). 3D printed flow-transitions on the ends carried flow from brass tube fittings to the channel to prevent separation of the fully developed flow. The brass fittings were grounded (on both ends of the setup) during testing to prevent interference from other electrical sources and charge buildup during flow. A pump (Unilift KP250, Grundfos, Bjerringbro, Denmark) was used to send water from a 6 m × 6 m × 4 m tank through the system. This large tank was used to avoid temperature changes, which can cause sensor drift (see supporting information for more information). The flow rate was manipulated with an Alicat Liquid Flow Controller (LCR-10LPM-D/5V, Alicat Scientific, Tucson, USA). In particular, rates were increased from 0 L/min to 1 L/min by 0.2 L/min intervals and decreased back to 0 L/min by the same. In practice, a flow rate of 0.01 L/min was used instead of 0 L/min to prevent the controller from entirely shutting its valve-a procedure which resulted The flow rate was manipulated with an Alicat Liquid Flow Controller (LCR-10LPM-D/5V, Alicat Scientific, Tucson, USA). In particular, rates were increased from 0 L/min to 1 L/min by 0.2 L/min intervals and decreased back to 0 L/min by the same. In practice, a flow rate of 0.01 L/min was used instead of 0 L/min to prevent the controller from entirely shutting its valve-a procedure which resulted in large pressure fluctuations within the channel. No triggering mechanism is included with the flow controller, so capacitive data collection was started manually. Two scenarios were recorded. For quick sensor evaluation, each interval was set to 20 s, and 5 cycles were completed. This was repeated for the sensor at 0 • (its front facing upstream), 45 • , 90 • (oriented "incorrectly" in the flow), 135 • , and 180 • (backwards from the 0 • case). For experiment at the 0 • positioning, video was recorded (manually started) with a camcorder (Vixia HF11, Canon, Inc., Tokyo, Japan) and again analyzed as ground truth with Tracker at a resolution of around 20 µm. Furthermore, a test was performed at 0 • with 5 min intervals and 3 cycles to observe longer term behavior. COMSOL Setup Finite element flow-structure interaction (FSI) simulations using COMSOL Multiphysics 5.3a are performed to model the displacement of the cupula tip under different flow conditions. These simulations provide a bridge between the experimentally measured tip displacements and the analytical model, enhancing our understanding of the conditions under which both are relevant. The cross-section of the cupula (5 × 5 mm 2 ) is 44% of that of the present flow channel (7.5 × 7.5 mm 2 ), resulting in a significant obstruction to the flow profile upon encountering the cupula. The flow is expected to accelerate around the cupula increasing the force experienced by it, and thereby its deformation. In its intended application on an underwater vehicle, this is not expected to occur, as the flow will be unbounded on the outside. The analytic expression derived earlier is closer to the latter, assumes that the deformation is a result of the form drag experienced by the cupula under uniform flow, which is closer to external flow conditions. Therefore, two simulations are performed, one replicating the present experiments with the same cross-section as the flow channel (denoted as FSI-1) and the other with the externally unbounded flow (denoted as FSI-2), presumably more representative of the analytical solution. The simulation setup is shown in Figure 6. The cupula geometry is matched exactly to that of its fabrication design, i.e., three liquid metal inclusions in an Ecoflex 0030 housing of the same shape. The elastic modulus of the Ecoflex in both simulations is taken to be 125 kPa, based on test measurements and following [64]. The simulation domain ( Figure 6) is rectangular with the cupula fixed at its bottom center. The streamwise, wall-normal and spanwise directions are represented by the y-, xand z-axis respectively. The flow inlet and outlet are marked accordingly and the flow-structure interactions between the cupula and the water flow (outside) as well as liquid metal (inside) are modeled separately. The volumetric flow rates vary from 0.1 to 1 L/min in increments of 0.1 L/min, following the experimental conditions for both the simulations. behavior. COMSOL Setup Finite element flow-structure interaction (FSI) simulations using COMSOL Multiphysics 5.3a are performed to model the displacement of the cupula tip under different flow conditions. These simulations provide a bridge between the experimentally measured tip displacements and the analytical model, enhancing our understanding of the conditions under which both are relevant. The cross-section of the cupula (5 × 5 mm 2 ) is 44% of that of the present flow channel (7.5 × 7.5 mm 2 ), resulting in a significant obstruction to the flow profile upon encountering the cupula. The flow is expected to accelerate around the cupula increasing the force experienced by it, and thereby its deformation. In its intended application on an underwater vehicle, this is not expected to occur, as the flow will be unbounded on the outside. The analytic expression derived earlier is closer to the latter, assumes that the deformation is a result of the form drag experienced by the cupula under uniform flow, which is closer to external flow conditions. Therefore, two simulations are performed, one replicating the present experiments with the same cross-section as the flow channel (denoted as FSI-1) and the other with the externally unbounded flow (denoted as FSI-2), presumably more representative of the analytical solution. The simulation setup is shown in Figure 6. The cupula geometry is matched exactly to that of its fabrication design, i.e., three liquid metal inclusions in an Ecoflex 0030 housing of the same shape. The elastic modulus of the Ecoflex in both simulations is taken to be 125 kPa, based on test measurements and following [64]. The simulation domain ( Figure 6) is rectangular with the cupula fixed at its bottom center. The streamwise, wall-normal and spanwise directions are represented by the y-, x-and z-axis respectively. The flow inlet and outlet are marked accordingly and the flow-structure interactions between the cupula and the water flow (outside) as well as liquid metal (inside) are modeled separately. The volumetric flow rates The size of the FSI-2 domain is 11.25 × 11.25 × 30 mm 3 , slightly larger than that of FSI-1 to ensure that the entire wake of the cupula is captured in both dimensions. Owing to the increased size and an absence of any other length scale operating on this 'external flow', the inlet flow condition is assumed fully developed and turbulent (k-ω model). A no-slip flow boundary condition is applied on the bottom wall, whereas the side and top boundaries are considered open, representative of an externally unbounded flow as discussed previously. Both simulations employ a zero-pressure condition for the outflow. A physics based tetrahedral mesh is used, with element sizes ranging from 0.30 to 9.0 mm and 0.45 to 3.0 mm for the FSI-1 and FSI-2 cases, respectively. Furthermore, to evaluate the effect of the elastic modulus on the tip displacement, FSI-1 simulations are also performed for moduli of 60 kPa (a lower value that has also been reported for Ecoflex 0030 [65]) and 1 MPa (an approximation for typical 10:1 polydimethylsiloxane). Results and Discussion The following results are presented from a single sensor, tested both in direct tip deflection and in water flow. Complete data from the single device, including six trials of small tip deflection, nine trials of large tip deflection, and a total of 13 flow trials at varying sensor orientations, are presented in the Appendices B and C. Here, we present the primary results and outcomes. In all tests, due to fabrication imperfections and asymmetry in the device, the differential capacitance measured across the device did not fall on 0 pF. Instead, the "+" channel demonstrated a C 0 of~1.91 pF while the "−" channel showed~1.131 pF. This was determined by taking the difference between measurements on the sensor and measurements on devices fabricated without capacitive plates extending into the cupula. Note that some error is expected due to differences in parasitic capacitances between devices. Nevertheless, we can extract an approximate dielectric constant for Ecoflex 0030 of about 4.77, which is in reasonable agreement (given geometric tolerances) with literature values that ranges from 2.8 [66] to 4.4 [67] depending on factors such as testing input signal frequency [67]. Capacitance vs. Deflection The results from a representative direct displacement test are reported in Figure 7, where capacitive change is plotted as a function of tip deflection while the device was immersed in water. A plot with all six trials' identical runs is shown in Appendix B. In agreement with the theory, the trend is linear with a sensitivity of about 0.0512 pF/mm. In fact, Equation (3), assuming design dimensions and the average C 0 of 1.521 pF, predicts a sensitivity of 0.0639 pF/mm (falling more in line with flow results). The theoretical value will be off due to an error in the calculation of C 0 , as discussed above. However, the theory appears to overestimate sensitivity, likely due to simplifying assumptions on geometry and lack of consideration of mechanical effects such as membrane buckling, which can be seen in the insets of Figure 7. unbounded flow as discussed previously. Both simulations employ a zero-pressure condition for the outflow. A physics based tetrahedral mesh is used, with element sizes ranging from 0.30 to 9.0 mm and 0.45 to 3.0 mm for the FSI-1 and FSI-2 cases, respectively. Furthermore, to evaluate the effect of the elastic modulus on the tip displacement, FSI-1 simulations are also performed for moduli of 60 kPa (a lower value that has also been reported for Ecoflex 0030 [65]) and 1 MPa (an approximation for typical 10:1 polydimethylsiloxane). Results and Discussion The following results are presented from a single sensor, tested both in direct tip deflection and in water flow. Complete data from the single device, including six trials of small tip deflection, nine trials of large tip deflection, and a total of 13 flow trials at varying sensor orientations, are presented in the Appendices B and C. Here, we present the primary results and outcomes. In all tests, due to fabrication imperfections and asymmetry in the device, the differential capacitance measured across the device did not fall on 0 pF. Instead, the "+" channel demonstrated a C0 of ~1.91 pF while the "-" channel showed ~1.131 pF. This was determined by taking the difference between measurements on the sensor and measurements on devices fabricated without capacitive plates extending into the cupula. Note that some error is expected due to differences in parasitic capacitances between devices. Nevertheless, we can extract an approximate dielectric constant for Ecoflex 0030 of about 4.77, which is in reasonable agreement (given geometric tolerances) with literature values that ranges from 2.8 [66] to 4.4 [67] depending on factors such as testing input signal frequency [67]. During ADMET testing, hysteresis varied slightly from test to test. For a tip deflection of 0.4 mm in the data plotted in Figure 7, capacitance change increased by 0.53 fF (~3%) from the upstroke to the downstroke. During another test, the increase was 1.9 fF (~10%). Spread of data points indicates that our sensor can resolve displacements of about 40 µm in terms of accuracy while being able to detect dynamic changes on the order of microns or smaller, though future dynamic testing is required to verify this. Some hysteresis and drift could likely be caused by factors such as viscoelasticity and movement of liquid metal within the device. It should be noted that large deformations (~1.5 mm deflection) resulted in increased nonlinear behavior and increased hysteresis (see Appendix B). This was well beyond what we encountered during flow experiments and requires additional study for further understanding. Capacitive change versus deflection while in air was found to be nearly identical to the aqueous case (see Appendix B). For a single device, capacitance versus tip deflection sensitivities varied from 0.0511 to 0.0543 pF/mm. In particular, values increased by about 0.003 pF/mm between two separate tip deflection test sessions while varying by less than 0.0005 pF/mm within each. It is likely that handling the device between experimentations resulted in differing overall sensor geometry as the liquid metal and soft elastomer body would settle into various minimum energy states. Trapped air due to imperfect fabrication could also contribute. Figure 7 also reports point clouds for the relationship between deflection and capacitance during the 0 • flow experiment when video was recorded. Here, we see that the sensitivity is 0.0632 pF/mm. Falling closer to the mathematical prediction, this suggests that the flow may have resulted in deformations that more closely followed those prescribed in the theoretical kinematics. However, the fluid-structure interactions are more complex than what was assumed in the theory, and so other factors such as pressure differences on either side of the cupula could be playing a role. The spread of the point clouds is due to several factors. First, the flow controller (and its internal flow sensor) is imperfect, resulting in horizontal spread. Since the flow controller and the bio-inspired sensor could not be perfectly synchronized together, some error due to temporal offset was present in Figure 7. Second, there were vibrations that could not be appropriately resolved without higher sampling rates and proper triggering. Finally, there is some sensor hysteresis and drift, as mentioned above, due to viscoelasticity and liquid metal flow. Capacitance vs. Flow Capacitive output is compared to flow speed as a function of time in Figure 8a, where a 20 s interval testing session is shown. The inset magnifies a particular set of times, highlighting how well the sensor follows the data output from the flow controller. To achieve the capacitive change as a function of flow in Figure 8b, 5-s intervals of data are taken from the middle of each 20 s step. Standard deviation of the flow rate is minimized to avoid large changes during the controller's output. These 5-s intervals are indicated by the grey boxes in Figure 8a. Figure 8b is an average of several trials (5 for 0 • and 2 for the remaining angles), all of which are reported in Appendix C. Sensor orientation was varied as indicated by the angles illustrated in Figure 8c. The first observation is that the response is nonlinear in shape, as indicated by the theory discussed above, which predicts quadratic. However, assuming an Ecoflex 0030 Young's modulus of 125 kPa [64], the sensitivity is underestimated by 86% when compared to the 0 • average at 1 L/min. This is largely due to the impact of fluid-structure interactions around the cupula, which occupies a large percentage of the channel's cross-section. During its intended application in a much larger free-stream (unbounded flow), the theory should more accurately predict behavior. To verify this claim, we performed COMSOL simulations, as discussed below. The asymmetry of the device is again shown in the capacitive change versus flow plot, where 180 • does not perfectly mirror 0 • . However, we do see the expected behavior, where sensitivity decreases to nearly zero at 90 • . In this case, any deformation to the device should nearly equally impact both sides of the device, resulting in a capacitive change of zero. Furthermore, the device is physically less likely to deflect at 90 • due to the higher area moment in relation to the fluid flow. Especially at higher flow rates, large vibrations are apparent in the sensor output due to dynamic flow-structure interactions. Note that the Reynold's number at 1 L/min is 2300, indicating a transitioning flow for the channel (ignoring the interaction with the sensor). Furthermore, the Strouhal number for a flat plate is 0.15-0.2 [68,69], indicating a vortex shedding frequency of around 12 Hz and below for our system. With the low (16.1 Hz) capacitance sampling rate, a thorough study of vibrations was not in the scope of this study. Instead, these features simply contribute to the vertical error bars in Figure 8b. However, with the AD7746 (at a max frequency of 90.9 Hz), frequency studies are feasible and of interest for future work. discussed above, which predicts quadratic. However, assuming an Ecoflex 0030 Young's modulus of 125 kPa [64], the sensitivity is underestimated by 86% when compared to the 0° average at 1 L/min. This is largely due to the impact of fluid-structure interactions around the cupula, which occupies a large percentage of the channel's cross-section. During its intended application in a much larger freestream (unbounded flow), the theory should more accurately predict behavior. To verify this claim, we performed COMSOL simulations, as discussed below. The asymmetry of the device is again shown in the capacitive change versus flow plot, where 180⁰ does not perfectly mirror 0°. However, we do see the expected behavior, where sensitivity decreases to nearly zero at 90°. In this case, any deformation to the device should nearly equally impact both sides of the device, resulting in a capacitive change of zero. Furthermore, the device is physically less likely to deflect at 90° due to the higher area moment in relation to the fluid flow. Especially at higher flow rates, large vibrations are apparent in the sensor output due to dynamic flow-structure interactions. Note that the Reynold's number at 1 L/min is 2300, indicating a transitioning flow for the channel (ignoring the interaction with the sensor). Furthermore, the Strouhal number for a flat plate is 0.15-0.2 [68,69], indicating a vortex shedding frequency of around 12 Hz and below for our system. With the low (16.1 Hz) capacitance sampling rate, a thorough study of vibrations was not in the scope of this study. Instead, these features simply contribute to the Drift during flow testing was best reflected by the long-term (~3.5 h) test, where 0 L/min values increased by about 0.7 fF. The data is plotted in Appendix C. Sensitivity did change from test to test. After substantial testing and handling (including squeezing and stretching the sample), the final experiment showed a sensitivity decrease of about 15.4% from previous trials. Note that this final experiment is included in the data presented in Figure 8b and is the flow data plotted in Figures 7 and 8a. Since the sensor response to flow is nonlinear, it is more useful to look at minimum resolvable flow rather than an overall resolution. For our setup, the initial 0.2 L/min (0.06 m/s within the channel) step was clearly identifiable based on the error bars and the long term testing. This could be described as the threshold accuracy. As with displacement testing, much smaller deviations in flow can be detected, though not accurately classified as a specific rate. This is demonstrated by the Figure 8a inset, which shows sensor changes to very small changes in flowrate. Figure 9 shows snapshots of simulation results to elucidate salient features. Contour plots of the flow velocity magnitude overlaid with velocity vectors for the streamwise-wall-normal and streamwise-spanwise planes are shown for (a) FSI-1 and (b) FSI-2. Surface contours of the total displacement of the cupula are also shown. Note the difference between color-map scales. It is evident that the flow and displacements are substantially different from each other. The effects of the walls in FSI-1 are manifested in the form of the accelerated region (~0.7 m/s) on the sides of the cupula in comparison to the mean velocity (~0.3 m/s). This imposes a pressure gradient across the cupula, resulting in a maximum displacement of~500 µm at the tip. The wake of the cupula extends more than 15 mm downstream. In comparison, the velocity profiles for FSI-2 are uniform. The maximum velocity magnitude is equal to the mean velocity at the inlet (~0.3 m/s), and the wake of the cupula is substantially more homogenized 15 mm downstream. Consequently, this results in a much lower maximum displacement of~80 µm at the cupula tip. COMSOL Results vertical error bars in Figure 8b. However, with the AD7746 (at a max frequency of 90.9 Hz), frequency studies are feasible and of interest for future work. Drift during flow testing was best reflected by the long-term (~3.5 hr) test, where 0 L/min values increased by about 0.7 fF. The data is plotted in Appendix C. Sensitivity did change from test to test. After substantial testing and handling (including squeezing and stretching the sample), the final experiment showed a sensitivity decrease of about 15.4% from previous trials. Note that this final experiment is included in the data presented in Figure 8b and is the flow data plotted in Figure 7 and Figure 8a. Since the sensor response to flow is nonlinear, it is more useful to look at minimum resolvable flow rather than an overall resolution. For our setup, the initial 0.2 L/min (0.06 m/s within the channel) step was clearly identifiable based on the error bars and the long term testing. This could be described as the threshold accuracy. As with displacement testing, much smaller deviations in flow can be detected, though not accurately classified as a specific rate. This is demonstrated by the Figure 8a inset, which shows sensor changes to very small changes in flowrate. The maximum tip displacements over the cupula surface are plotted w.r.t. the flowrate for FSI-1 in Figure 10a for all the elastic moduli. These are overlaid on a scatter plot of the measured tip displacements in the flow channel. Similarly, Figure 10b displays a comparison between the maximum tip displacements obtained from FSI-2 simulations and those predicted by the analytical model. As evident from Figure 10a, the agreement between the FSI-1 results at E = 125 kPa and the experiments is very good and the deviation of the results from the other two cases confirms that, for Ecoflex 0030, E is closest to 125 kPa. There is also a good agreement between the analytical model and the FSI-2 results. This also confirms the parabolic nature of the displacement predicted by the model. Even though the analytical model does not account for the boundary layer and variations in the velocity profile along the wall-normal direction, it provides a reasonable estimate of the maximum tip displacement. Consequently, the deviation between the model prediction and FSI-2 increases as we move closer to the bottom wall (not shown). Figure 9 shows snapshots of simulation results to elucidate salient features. Contour plots of the flow velocity magnitude overlaid with velocity vectors for the streamwise-wall-normal and streamwise-spanwise planes are shown for (a) FSI-1 and (b) FSI-2. Surface contours of the total displacement of the cupula are also shown. Note the difference between color-map scales. It is evident that the flow and displacements are substantially different from each other. The effects of the walls in FSI-1 are manifested in the form of the accelerated region (~0.7 m/s) on the sides of the cupula in comparison to the mean velocity (~0.3 m/s). This imposes a pressure gradient across the cupula, resulting in a maximum displacement of ~500 μm at the tip. The wake of the cupula extends more than 15 mm downstream. In comparison, the velocity profiles for FSI-2 are uniform. The maximum velocity magnitude is equal to the mean velocity at the inlet (~0.3 m/s), and the wake of the cupula is substantially more homogenized 15 mm downstream. Consequently, this results in a much lower maximum displacement of ~ 80 μm at the cupula tip. Table 1 shows how our device compares to several natural and artificial aqueous sensors listed in literature. The parameters include sensing type, cupula material, cupula height, aspect ratio (height over width facing flow) and minimum sensed flow rates demonstrated for both steady (DC) and alternating (AC) flows. Note that minimum values for DC are typically much higher than those for AC. This is likely due to a mix of equipment limitations for steady flows, resonance, and circumvention of drift during alternating flows. When tracking lateral line nerve responses of an African clawed frog, brief flow application with a speaker coil [70] and sinusoidal inputs [71] resulted in detection at velocities around 30 µm/s. However, rheotaxis experiments [6] indicate that, when exposed to a steady flow, some fish do not respond until 5 to 30 mm/s of flow velocity is reached. Likewise, artificial flow sensors demonstrate particularly low thresholds (as low as 2.5 µm/s [16]) for AC signals, whereas the lowest DC flow rates range from 25 mm/s [19] to 100 mm/s [28]. The device sensor presented here falls in the same range of DC values, though future testing is required for a quantitative minimum AC rate. Note that the sensitivity to flow and the minimum threshold can be manipulated using the parameters, such as Young's modulus, in Equation (7). In terms of size, it is not as large as the only other capacitive example that is demonstrated in water (40 mm "seal whisker" [28]), but it is large compared to many devices (often MEMS-based) that are as small as 700 µm [19]. Regardless, natural cupula, which are often around 100 µm or less in scale [16,70,72], tend to be smaller than the artificial devices. Conclusions We have presented the design, theory, fabrication, and testing of an all-soft-matter bio-inspired flow sensor. Unlike existing capacitive flow sensors, ours includes liquid metal plates embedded within a silicone cupula. The three-dimensional liquid metal paths were created with a combination of lost-wax molding and vacuum injection. Direct cupula manipulation and flow experiments demonstrate repeatable behavior with little hysteresis within single testing sessions and the ability to accurately determine flow rates. The theory does a satisfactory job of predicting capacitance change versus tip deflection and of reflecting flow experiment trends. Despite the fairly reliable performance within individual sessions, some variability showed up between tests after handling. Given the likely cause of liquid metal flow and deformable elastomer body, a simple design change may decrease deviations. In particular, adding structure, such as a reinforcing grid of supports, within the capacitive plates may force the cupula to remain in a defined geometry rather than allowing multiple minimum energy configurations. Furthermore, the entire structure can be scaled down-a procedure that Equation (7) suggests will linearly decrease sensitivity. This future work will also require new fabrication techniques or a printer with a better resolution, but miniaturization will bring our device closer to examples in nature and will allow us to create a device capable of monitoring boundary layer behavior. Additionally, smaller sensors will enable greater packing density for designing networks. The theory presented above provides reasonable closed-form equations for evaluating device behavior and guiding future designs. For further understanding of fluid-structure interaction and complex deformations such as membrane buckling, COMSOL finite element simulation was used, verifying the analytic solutions as sound approximations for the physical phenomena. As the device is modified and miniaturized, these models will prove invaluable for optimizing dimensions and materials, particularly as we look at vibrations and possible frequency filtering. Moving testing methods forward, time-resolved particle image velocimetry (PIV) and capacitance measurements are required for analysis of the cupula under various flow rates. With these capabilities, additional information on turbulence and flow structure can be extracted. Furthermore, vortex-induced vibrations from non-turbulent flow may be tracked, as demonstrated in piezoelectric [14] and IPMC [17] devices in steady flows. Additional future studies may include exploring the interactions between sensors when placed in a lateral line and testing the response when placed on a deformable substrate. In general, the sensors should be spaced to avoid one device being in the wake of another, which could result in complex behavior. Extrapolating from the COMSOL simulations, the wake is about four cupula body lengths, or 2 cm. Future work might draw from wake interaction studies regarding wind turbines [73]. When placed on a deformable substrate, such as an artificial skin, we expect potentially complex interactions involving changing parasitic capacitances as well as changing flow dynamics. As of now, we cannot make a strong prediction, but it will be a focus as the device finds an application. The device presented here is uniquely positioned to be applied on a bio-inspired underwater robot. Since the sensor itself includes no rigid components, its application will not reduce the compliance of a robotic "skin." Additionally, the capacitive design will allow for multiplexing in grid formations for efficient gathering of flow data in a network. With some further design and fabrication refinement, the work presented here will contribute to the navigation and sensing capabilities of swimming robots. increased regardless of orientation. This indicates that the effect is mostly due to the larger dielectric constant of water. After this initial shift in capacitance, it continues to drift for hours as the elastomer dielectric constant and geometry change, presumably due to water absorption. Bubbles ( Figure A1b) can cause changes in capacitance as they deflect the cupula and represent a volume of lower dielectric constant in comparison to surrounding water. In fact, capacitance is used as a method for detecting [74] and measuring [75] bubbles. Temperature ( Figure A1c) also results in changing dielectric constants and geometry. Furthermore, trapped bubbles within the liquid metal may expand and contract, causing larger value deviation. The changes in capacitance are at least somewhat reversible. Finally, hydrostatic pressure ( Figure A1d) causes a small amount of deviation, limited by the high bulk moduli of silicone and liquid metal. A change of about 150 cm of water in pressure head resulted in about 1 fF of drift. During experimental testing, steps were taken to avoid the above factors. The sensor generally had at least ~20 minutes to settle prior to taking data. Bubbles were avoided by using a pump rather than an air pressure pot (which resulted in dissolved gas and bubble formation). The temperature was kept constant by pumping fluid from a large tank of water, and steady hydrostatic pressure was maintained by keeping a fixed channel outlet height. Note that the above factors also influence the sensor's sensitivity. However, this has not yet been explored. Appendix B-Additional Direct Deflection Data Capacitance change versus small tip deflection for all six trials (two sessions of three tests-one session in shades of grey and one session in shades of blue) are shown in Figure B1a. One can see that the sets of sessions differ slightly, but trials are very repeatable within each. At larger deflections ( Figure B1b, which shows nine trials in three sets of three trials), we see additional hysteresis behavior. One trial includes a decrease (instead of increase as in the lower deflection cases) of 10.8 fF (~24%) from upstroke to downstroke at 1 mm. Nonlinear behavior is expected due to the higher strain values and greater buckling present in such large deformations. However, in some cases (red plot points, for example), one can see steps in the curve. This indicates the possible presence of a stick- Figure A1. Plots demonstrating various causes of drift. (a) drift while sitting in air at 20 • C for one hour, followed by immersion in water for over two ours at 21 • C; (b) changes in capacitance due to air bubble placement; (c) drift in water due to temperature change; (d) capacitance change when lifting and lowering the channel outlet (referenced to sensor height). During experimental testing, steps were taken to avoid the above factors. The sensor generally had at least~20 min to settle prior to taking data. Bubbles were avoided by using a pump rather than an air pressure pot (which resulted in dissolved gas and bubble formation). The temperature was kept constant by pumping fluid from a large tank of water, and steady hydrostatic pressure was maintained by keeping a fixed channel outlet height. Note that the above factors also influence the sensor's sensitivity. However, this has not yet been explored. Appendix B. Additional Direct Deflection Data Capacitance change versus small tip deflection for all six trials (two sessions of three tests-one session in shades of grey and one session in shades of blue) are shown in Figure A2a. One can see that the sets of sessions differ slightly, but trials are very repeatable within each. At larger deflections ( Figure A2b, which shows nine trials in three sets of three trials), we see additional hysteresis behavior. One trial includes a decrease (instead of increase as in the lower deflection cases) of 10.8 fF (~24%) from upstroke to downstroke at 1 mm. Nonlinear behavior is expected due to the higher strain values and greater buckling present in such large deformations. However, in some cases (red plot points, for example), one can see steps in the curve. This indicates the possible presence of a stick-slip behavior. Thus, some hysteresis seen here could be due to the testing method rather than the sensor itself. This plot also again demonstrates how the sensor is repeatable within testing sessions. The shades of grey, blue, and red represent three different testing sessions, each with three trials. Prior to the red session, the sensor was handled heavily, evidently impacting the sensitivity. The plot in Figure B1c demonstrates how a smaller deflection trial (black) compares to a larger deflection trial (red). Furthermore, lines and numbers are included for the larger deflection trial, indicating the direction of motion temporally. Finally, Figure B1d shows a comparison between deflection tests in water (black shades) and in air (blue shades). The difference between the two are negligible, especially compared to possible differences due to device deformation between sessions in general explored. Figure S3 shows flow data from individual trials (rather than an overall average). Note that there are two trials for each angle (45°, 90°, 135°, and 180°) in black and red except for 0°, which has 5. The final 0° trial (which was video-taped for position data) is plotted and labeled in green. This trial demonstrates a lower sensitivity, as discussed in the main text, likely due to substantial handling and shifting of the liquid metal/elastomer minimum energy physical configuration. The plot in Figure A2c demonstrates how a smaller deflection trial (black) compares to a larger deflection trial (red). Furthermore, lines and numbers are included for the larger deflection trial, indicating the direction of motion temporally. Finally, Figure A2d shows a comparison between deflection tests in water (black shades) and in air (blue shades). The difference between the two are negligible, especially compared to possible differences due to device deformation between sessions in general explored. Figure A3 shows flow data from individual trials (rather than an overall average). Note that there are two trials for each angle (45 • , 90 • , 135 • , and 180 • ) in black and red except for 0 • , which has 5. The final 0 • trial (which was video-taped for position data) is plotted and labeled in green. This trial demonstrates a lower sensitivity, as discussed in the main text, likely due to substantial handling and shifting of the liquid metal/elastomer minimum energy physical configuration. The final 0° test is labeled since it was videotaped and was performed after substantial handling; (b) comparison of data based on arbitrary 10 second intervals and specified 5-second intervals that lower standard deviation. Appendix C. Additional Flow Data Average flow data was also compiled based on an arbitrary 10 second interval taken in the middle of each 20 second flow rate step. This is plotted in red on the right plot. The data based on 5second intervals that minimized flow rate standard deviation is plotted in black. This demonstrates that the capacitance versus flow rate is consistent regardless of the time intervals taken, assuming that it is adequately positioned after transient flow change effects and prior to the next step. Figure C2 demonstrates how stable the sensor is over about three hours of continuous testing. Figure D1 shows the experimental setups for direct deflection tests and channel flow. Average flow data was also compiled based on an arbitrary 10 s interval taken in the middle of each 20 s flow rate step. This is plotted in red on the right plot. The data based on 5-s intervals that minimized flow rate standard deviation is plotted in black. This demonstrates that the capacitance versus flow rate is consistent regardless of the time intervals taken, assuming that it is adequately positioned after transient flow change effects and prior to the next step. Figure A4 demonstrates how stable the sensor is over about three hours of continuous testing. The final 0° test is labeled since it was videotaped and was performed after substantial handling; (b) comparison of data based on arbitrary 10 second intervals and specified 5-second intervals that lower standard deviation. Appendix D -Experimental Setup Images Average flow data was also compiled based on an arbitrary 10 second interval taken in the middle of each 20 second flow rate step. This is plotted in red on the right plot. The data based on 5second intervals that minimized flow rate standard deviation is plotted in black. This demonstrates that the capacitance versus flow rate is consistent regardless of the time intervals taken, assuming that it is adequately positioned after transient flow change effects and prior to the next step. Figure C2 demonstrates how stable the sensor is over about three hours of continuous testing. Figure D1 shows the experimental setups for direct deflection tests and channel flow. Figure A5 shows the experimental setups for direct deflection tests and channel flow.	2019-06-20T13:11:19.886Z	2019-06-01T00:00:00.000	{ "year": 2019, "sha1": "d042e2847c7ee2fb4fbb17ba6bd2fb6b076d2ea8", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1424-8220/19/11/2639/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "d042e2847c7ee2fb4fbb17ba6bd2fb6b076d2ea8", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Materials Science", "Medicine", "Computer Science" ] }
249354191	pes2o/s2orc	v3-fos-license	Diagnostic accuracy and inter-reader reliability of the MRI Liver Imaging Reporting and Data System (version 2018) risk stratification and management system Background Hepatocellular carcinoma (HCC) can be diagnosed non-invasively, provided certain imaging criteria are met. However, the recent Liver Imaging Reporting and Data System (LI-RADS) version 2018 has not been widely validated. Objectives This study aimed to evaluate the diagnostic accuracy and reader reliability of the LI-RADS version 2018 lexicon amongst fellowship trained radiologists compared with an expert consensus reference standard. Method This retrospective study was conducted between 2018 and 2020. A total of 50 contrast enhanced liver magnetic resonance imaging (MRI) studies evaluating focal liver observations in patients with cirrhosis, hepatitis B virus (HBV) or prior HCC were acquired. The standard of reference was a consensus review by three fellowship-trained radiologists. Diagnostic accuracy including sensitivity, specificity, positive predictive value (PPV), negative predictive values (NPV) and area under the curve (AUC) values were calculated per LI-RADS category for each reader. Kappa statistics were used to measure reader agreement. Results Readers demonstrated excellent specificities (88% – 100%) and NPVs (85% – 100%) across all LI-RADS categories. Sensitivities were variable, ranging from 67% to 83% for LI-RADS 1, 29% to 43% for LI-RADS 2, 100% for LI-RADS 3, 70% to 80% for LI-RADS 4 and 80% to 84% for LI-RADS 5. Readers showed excellent accuracy for differentiating benign and malignant liver lesions with AUC values > 0.90. Overall inter-reader agreement was ‘good’ (kappa = 0.76, p < 0.001). Pairwise inter-reader agreement was ‘very good’ (kappa ≥ 0.90, p < 0.001). Conclusion The LI-RADS version 2018 demonstrates excellent specificity, NPV and AUC values for risk stratification of liver observations by radiologists. Liver Imaging Reporting and Data System can reliably differentiate benign from malignant lesions when used in conjunction with corresponding LI-RADS management recommendations. Introduction Hepatocellular carcinoma (HCC) is the sixth most common malignancy and the second most common cause of malignancy-related mortality worldwide. 1 Unlike most other cancers, HCC can be confidently diagnosed non-invasively on imaging without mandatory pathology confirmation provided strict imaging criteria are met. 2 The Liver Imaging Reporting and Data System (LI-RADS), first released in 2011 by the American College of Radiology (ACR), is an imaging reporting algorithm designed to standardise radiology reporting of HCC in high-risk patients in terms of screening, surveillance, diagnosis and treatment response assessment. 1,3 The LI-RADS categories have the ability to accurately stratify the probability of HCC and overall malignancy without potential risks of biopsy, including inadequate sampling, haemorrhage and biopsy tract seeding. 4,5 Accordingly, there has been increasing reliance upon imaging and radiologists for both early and accurate diagnosis of HCC, using a universal reporting language. 6 the current LI-RADS version 2018 lexicon amongst board certified fellowship trained body imaging radiologists as compared with an expert consensus reference standard. Research methods and design Patient selection The University of Alberta Hospital Picture Archiving and Communication System (PACS) was reviewed for all cases with contrast-enhanced MRI studies evaluating the liver, performed between 01 January 2018 and 31 March 2020. Cases with observations found in patients with a high-risk feature for HCC including cirrhosis, chronic hepatitis B viral infection or current or prior HCC and at least 1 year of crosssectional imaging follow-up were selected for inclusion. Observations in patients under the age of 18 years, those with absence of high-risk factors and those with cirrhosis caused by non-hepatitis aetiologies were excluded as per the ACR CT/MRI LI-RADS v2018 core guidelines. 7 For the purposes of this study, only LI-RADS categories 1 to 5 were considered, with cases involving other malignancy (LR-M), tumour in vein (LR-TIV) and treatment response (LR-TR) categories also excluded. The 50 cases included in the study consisted of those lesions with typical representative features for each of the LI-RADS categories. All MRI studies were of good technical quality and in line with the ACR recommendations. 7 A total of 50 non-consecutive cases were selected by consensus from a Steering Committee of two authors with 6-and 13-years experience. Cases were chosen to represent a mix of classic imaging features and equivocal and challenging features in order to reflect a range of cases, which may be seen in a routine tertiary hospital setting. Only a single lesion per case was considered. When multiple lesions were present on a single case, only the lesion with the highest suspicion score was considered and annotated for review. Liver MRI protocol All liver MRI examinations included in this study were performed by using 1.5-T MRI scanners (GE Healthcare, Milwaukee, Wis; HD, GE Healthcare). Pre-contrast sequences included axial DWI (b values: 0, 50, 150 and 500) with ADC images, axial T2-weighted images with single-shot fast spin echo (FSE) technique, gradient echo (GRE) T1-weighted outphase and in-phase axial images and axial pre-contrast breath hold fat saturated spoiled-GRE images. Fat saturated post-contrast dynamic images were acquired in late arterial (30-40 s), portal (60-90 s), late portal (120-150 s) and delayed phases (180-210 s and at 300+ s) with breath-hold spoiled-GRE 3D technique in the axial and coronal planes. Where necessary, subtracted images were obtained from the dynamic sequences in order to aid lesion interpretation. Gadobutrol (Gadovist; Bayer Healthcare Pharmaceuticals, Whippany, NJ, United States) was the contrast agent used in all cases. A weight-based dose bolus of gadolinium contrast (0.1 mmol/kg body weight) was injected intravenously, followed by a normal saline flush (20 mL). Contrast material injection was given via a peripheral vein at 5 cc/s. Image processing All cases were randomised using the Microsoft Excel randomisation function and identifiers were removed from each MRI examination. The cases were subsequently networked to the PACS workstation (IMPAX 6 AGFA Healthcare) under an allocated post-randomisation case number. When one or more comparison studies were available, the most relevant comparison was selected and was similarly de-identified and stored on the workstation under the same case identifier. Liver lesion imaging atlas Single images that best depicted each observation (n = 50) were captured and stored in their respective case folders on the intuitional PACS (IMPAX 6 AGFA Healthcare) in order to guide the readers and to allow for a targeted assessment of individual observations. The image that most clearly showed each observation was chosen, regardless of the MRI sequence type or post-contrast phase. Each de-identified case folder contained the representative image that depicted the targeted observation including the size of the observation to be used, the MRI sequences pertaining to the targeted observation and comparison studies, where available. No patient identifiers were included in the case folders. Image review All 50 lesions were reviewed and assigned a LI-RADS category by consensus reading of three fellowship-trained body imaging radiologists (G.L., M.P.W., F.M.). Expert consensus for any given score was considered if all three radiologists agreed on the same score. Any disagreement was resolved by consensus re-review of the imaging and discussion. The test cases were then subsequently independently reviewed by three separate fellowship trained readers (B.A., C.F., D.R.), all with five years or more post-fellowship experience in body MR imaging. Prior to testing, each reader was provided with an instruction manual and the official LI-RADS atlas and glossary published by the ACR to be used at any point as a reference tool. 7 Readers were blinded to the patient history, initial radiology report, consensus LI-RADS score and interpretation of other readers. All 50 cases were independently reviewed, and data were entered into a standardised online data entry web form. For each LI-RADS score, the reader also indicated whether LI-RADS ancillary features for HCC were used to assign that score. The reviewers' consensus for definitively benign or probably benign liver lesions (LI-RADS 1 or 2) was based on: (1) typical or near typical imaging features for a benign aetiology on MRI and (2) interval size stability of at least 12 months. Statistical analysis Categorical variables were expressed as values and percentages. Continuous variables were expressed as the mean ± standard deviation. Statistical tests included: • One-way analysis of variance (ANOVA) to evaluate for significant differences in lesion size between the LI-RADS categories • Diagnostic accuracy measurements including sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated per LI-RADS category for each individual reader • Receiver operating characteristic (ROC) analysis was used to evaluate the area under the receiver operating curve (AUC) for each reader • Fleiss kappa (overall agreement) and weighted quadratic kappa (pairwise agreement) was used to calculate the inter-reader agreement. Ethical considerations This single centre retrospective study was approved by the University of Alberta Health Research Ethics Board (Pro00098131). Patient consent for individual cases was waived as all studies were retrospectively collected from the institutional Picture Archiving and Communication System (PACS) and studies were anonymised prior to review by individual readers. The overall lesion size was 21 mm ± 12 mm with a range from 8 mm to 57 mm. Mean lesion size by LI-RADS category was: 16 mm ± 3 mm for LI-RADS 1, 12 mm ± 3 mm for LI-RADS 2, 21 mm ± 18 mm for LI-RADS 3, 13 mm ± 6 mm for LI-RADS 4 and 29 mm ± 13 mm for LI-RADS 5 (p < 0.001). Post hoc analysis showed a statistically significant difference in lesion size between LI-RADS 2 and LI-RADS 5 lesions. Inter-reader agreement The overall inter-reader agreement for the three readers as a group was 'good' (κ = 0. Discussion This study demonstrates excellent specificities (87% -100%) and NPVs (85% -100%) across the LI-RADS categories, coupled with excellent AUCs (0.93-0.99) indicating that accurate differentiation between benign and malignant liver lesions by individual readers is possible with LI-RADS version 2018. Despite the excellent diagnostic specificities, the sensitivities across the LI-RADS categories were more variable (28% -100%). At least in part, this reflects the complexities in detecting and characterising lesions in the context of the cirrhotic liver. Lesion evaluation may prove to be challenging in circumstances including: (1) detection of small HCCs in a cirrhotic liver with innumerable regenerating nodules; (2) detection of small HCCs in a cirrhotic liver with multiple arterial enhancing perfusion anomalies or with transient hepatic intensity differences (THIDs); (3) regression and fibrosis occurring in benign lesions in the cirrhotic liver resulting in an atypical imaging appearance (e.g. hyalinised haemangiomas); and (4) development of pseudo-lesions that can confound interpretation (THIDs, confluent fibrosis, segmental or lobar dysmorphism). The variable sensitivity is mitigated by the fact that cirrhotic patients typically undergo regular follow up imaging every 3-6 months, thereby increasing the probability of lesion detection at a future examination. Several prior studies have also assessed reader agreement, mostly utilising earlier LI-RADS versions. 12 There was low reproducibility for LI-RADS 2, LI-RADS 3 and LI-RADS 4 categories, much lower than our study (κ = 0.11, 0.26, and 0.28, respectively). 16 A more recent study comparing LI-RADS versions 2017 and 2018 also found that the updated LI-RADS 5 criteria of LI-RADS 2018 yielded significantly better sensitivity (81%) than LI-RADS 2017 (68%) for noninvasive diagnosis of HCC. 15 A recent prospective cohort study by Razek et al. found excellent inter-observer agreement for LI-RADS 1, LI-RADS 2 and LI-RADS 5 using the 2018 LI-RADS lexicon. 12 However, in their study, agreement was poor in LR-3 and LR-4. By contrast, two studies have previously observed good reproducibility for all LI-RADS categories by MRI (κ = 0.609 and κ = 0.926, respectively) using the LI-RADS version 2014 lexicon. 13,14 The reproducibility differences in these previous studies may be related to a combination of reader factors (i.e. modality and readers' liver imaging expertise, prior familiarity with the LI-RADS lexicon, number of years of post residency practice) and study factors (i.e. complexity of lesions, number of readers, whether readers were based at a single institution or multicenter international reader pool, including community practice, academic and mixed practice environments). This study is subject to several limitations. Firstly, our selected reference standard was expert consensus without histological correlation. Validation of the LI-RADS risk stratification and management system has been shown in prior studies. 6,9,10,19 In a recent systematic review and metaanalysis, Van 15,19 However, some lesions labelled as 'benign' in the LI-RADS 2 category may represent malignancy histologically. 6,10 The American College of Radiology guidelines currently recommend serial surveillance for these lesions. 7 Secondly, our study was limited to a testing bank of 50 cases spread across LI-RADS categories. It is possible that small but important differences may exist but were not identified by relatively large CIs in some LI-RADS categories. Thirdly, for practical testing purposes, we excluded some categories of observations including LR-TIV and LR-M, which may limit applicability in a prospective clinical environment. Fourthly, we used a non-consecutive selection of cases over our study period. Although this approach was chosen in an effort to acquire a variable set of LI-RADS observation types and imaging difficulty, it is possible that this approach can predispose to selection bias. Finally, our readers were fellowship trained body imaging radiologists with at least five years of clinical experience, which may limit generalisability. Despite these limitations, the authors believe that the rigorous study design and subsequent results of this study supports the applicability of LI-RADS version 2018 in appropriate cases and with the appropriate use of ACR management guidelines. Conclusion This study demonstrates excellent specificity, NPV and AUC values of the LI-RADS version 2018 for risk stratification of focal liver observations, validating the use of version 2018 for differentiating benign from malignant lesions. Although inter-reader agreement was 'good', there remains ongoing reduced inter-reader agreement amongst intermediate LI-RADS categories, highlighting the need for the risk stratification tool to be used in conjunction with a conservative management recommendation system for high-risk patients.	2022-06-05T15:21:44.434Z	2022-05-19T00:00:00.000	{ "year": 2022, "sha1": "f617bc53aee2d82f95928b4bb01806bf2bf5aa7e", "oa_license": "CCBY", "oa_url": "https://sajr.org.za/index.php/sajr/article/download/2386/3195", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "52e8df50de142c8f13122ea5ece483404dd095c4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
91510770	pes2o/s2orc	v3-fos-license	Population structure of two freshwater amphipods ( Crustacea : Peracarida : Hyalellidae ) from southern Brazil Two recently described amphipods species from southern Brazil, Hyalella georginae Streck and Castiglioni, 2017 and Hyalella gauchensis Streck and Castiglioni, 2017, had their population structures characterized by sex, females’ ovigerous condition, cephalothorax length (mm), size-class frequency distribution, sex-ratio, reproductive period, and recruitment. The specimens were collected with a dip net from a stream source (H. georginae) and from a water reservoir (H. gauchensis) in the Palmeira das Missões municipality, state of Rio Grande do Sul, Brazil, from August 2012 to July 2013 (12 months). Both species showed a bimodal frequency distribution for total and seasonal size classes, with males larger than females. Overall, the sex ratio favored females when analyzed monthly and seasonally. Ovigerous females were recorded throughout the year, with higher frequency in spring (H. georginae) and summer (H. gauchensis), characterizing a seasonal reproduction. Both species showed continuous recruitment, with greater intensity in the spring. The population structure of these two Hyalella species had similar features, orcid.org/0000-0001-7454-5503 orcid.org/0000-0002-3041-5608 original arTiCle This article is part of the special series offered by the Brazilian Crustacean Society in honor to Ludwig Buckup in recognition of his dedication and contributions to the development of Carcinology CORRESPONDING AUTHOR Daniela da Silva Castiglioni danielacastiglioni@yahoo.com.br SUBMITTED 10 October 2017 ACCEPTED 23 August 2018 PUBLISHED 03 December 2018 Guest Editors Alessandra A. de Pádua Bueno and Sandro Santos DOI 10.1590/2358-2936e2018025 inTroDuCTion The study of population structure provides information about the ecological structure of natural populations (Hutchinson, 1981;Santos et al., 1995) such as its stability, productivity, and function within the trophic chain (Cooper, 1965).Understanding the dynamics of a population is useful for providing species conservation measures.Since growth, birth, reproduction, and mortality rates can be inferred, it is possible to predict if a population is expanding or declining to extinction (Cooper, 1965;Muskó, 1992).Population structure of amphipods has been studied through the analysis of density, size and age class distribution, sex-ratio, recruitment, and growth (Guerao, 2003;Appadoo and Myers, 2004;Kevrekidis, 2004Kevrekidis, , 2005;;Subida et al., 2005;Castiglioni and Bond-Buckup, 2008a). The genus Hyalella Smith, 1874 is distributed in the Nearctic and Neotropical biogeographic regions; it is endemic from the Americas, and its species have a restricted distribution (Bousfield, 1996).The genus includes approximately 72 species (Baldinger, 2004;Colla and César, 2015;Streck et al., 2017;Bastos-Pereira et al., 2018.).Hyalella is found in a variety of freshwater environments, attached to aquatic macrophytes, swimming in the water column, or buried in the sediment (Kruschwiyz, 1978;Wellborn, 1995;Grosso and Peralta, 1999).Depending on the species, Hyalella has herbivorous, carnivorous, omnivorous, or detritivore feeding habits (Cooper, 1965, Witt andHebert, 2000;Vainola et al., 2008).Individuals of this genus also act as important links in the transfer of matter and energy in their ecosystems (Moore, 1981;Casset et al., 2011), being the prey of animals such as fishes and birds (Musko, 1992;Casset et al., 2011). The aim of this study was to increase the knowledge on the population ecology of Brazilian Hyalella species.To achieve this aim we made estimates of body size, size-class frequency distribution, sex-ratio, reproductive period, and recruitment of Hyalella georginae and Hyalella gauchensis.Species were collected from Palmeira das Missões municipality, in the northeast region of state of Rio Grande do Sul, Brazil.The species studied in this work were described recently by Streck et al. (2017). MaTerials anD MeThoDs Study sites.To study the population structure, individuals were collected monthly from August 2012 Population of two freshwater amphipods from Brazil Nauplius, 26: e2018025 until July 2013 (spring: September to November; summer: December to February; fall: March to May; winter: June to August), at "Sítio Taqui" in Palmeira das Missões municipality, state of Rio Grande do Sul (27°53′56″ S, 53°18′ 50″ W).Hyalella georginae was sampled from a stream source, while H. gauchensis was sampled from a water reservoir.Palmeira das Missões' county is located in the Brazilian southern region, at the Rio Grande do Sul's North Plateau, and it is of great importance to the state's agriculture and farming.The state of Rio Grande do Sul has a climate classified as temperate Cfa according to the classification of Köppen-Geiger (Peel et al., 2007) and is characterized as mild mesothermic, experiencing periods of drought during spring and summer (IBGE, 2013).The monthly sampling was taken at two sites.Site 1 (S1) consisted of a headwater shaded by small trees and surrounded by Gramineae and Pteridophytae.Although macrophytes of the genus Salvinia were present in the waterbody, H. georginae was only found and collected in the sediment.Site 2 (S2) was a shallow water reservoir (≈30 cm) with a large amount of aquatic Salvinia, which was used as shelter by H. gauchensis.At Site 2 was observed fishes and frogs that may be predators of Hyalella The distance between Site 1 and Site 2 is approximately 20 meters. Sampling of Hyalella individuals.At each site, macrophytes and sediment were collected with a 250 µm mesh dip net during 20 min, placed into plastic bags, and taken to the laboratory.In the field, ovigerous females (with eggs or juveniles in the marsupium) and couples in precopulatory behavior were separated individually into microtubes containing 70% ethanol. In the laboratory, macrophytes and sediment were sieved (0.177 mm mesh) and washed in running water in order to retain the amphipods. All specimens were measured (cephalothorax length, CL in mm) on the ocular micrometer of a stereomicroscope.The CL was taken from the anterior margin of the rostrum to the posterior margin of the cephalothorax.According to Castiglioni and Bond-Buckup (2008a), the CL is correlated with total body length; hence, this measure may represent the actual body size of Hyalella individuals. Data analyses.The minimum, mean, and maximum CL values of males and females were estimated to each species.The mean sizes are given with their standard deviations.The means were compared between sexes and between species using the t test (α = 0.05) (Zar, 1996). To compare the temporal variation of the population age-frequency distribution, we estimated total and seasonal frequency distributions of male and female size classes for each species population.We also analyzed seasonality in the species recruitment processes.The class ranges were determined through ¼ of the average standard deviation of the CL of sampled individuals (Markus, 1971).Normality of frequency distributions were analyzed through the Shapiro-Wilk test (α = 0.05) (Zar, 1996). The sex ratio (males:females) was calculated.The total, monthly, seasonal, and size-class (CL) sex proportions were expressed as the total number of males divided by the total number of females.The Chi-Square test (χ 2 ) with a significance level of 5% (Zar, 1996), was used to verify if the sex-ratio followed the 1:1 ratio. The frequency of ovigerous females in relation to that of adult females of each species was estimated in order to analyze the seasonal and monthly reproductive period.The proportion of ovigerous females was compared between months and seasons using the multinomial proportions test (MANAP; α = 0.05) (Curi and Moraes, 1981).Those H. georginae females with CL > 0.40 mm and H. gauchensis with CL > 0.35 mm were considered adult females. Population of two freshwater amphipods from Brazil Nauplius, 26: e2018025 water reservoir.The two species were not found together at any of the collection sites. Ovigerous females of H. georginae were captured every month except April and May 2013 (Fig. 6).For H. gauchensis, ovigerous females were found across the entire year, and showed a higher frequency fluctuation throughout the year compared with H. georginae (Fig. 6).The higher reproductive intensity of H. georginae occurred in spring (September, 22 th to December, 21 th ; Recruitment peaked in December 2012 for H. georginae, and in October 2012 for H. gauchensis (Figs.8A, B).Recruitment was continuous during the seasons, with higher intensity in spring for both species (Fig. 9), when rainfall and minimum and maximum temperatures in the study area were above the climatological average of the state of Rio Grande do Sul (INMET, 2012). DisCussion In order to understand the differences in body size and abundance found in this study, life-history features and habitat characteristics may be considered, as noted by Castiglioni and Bond-Buckup (2008a) for two other Hyalella species of the "Campos de Cima da Serra" region in state of Rio Grande do Sul, Brazil.Differences between the study sites were observed and they may be related to differences in the species' life-history strategies, such as food availability and microhabitat: H. georginae was captured in the headwater sediment, while H. gauchensis was attached to macrophytes or swimming in the water column. According to Kruschwitz (1978), small-sized individuals reach sexual maturity faster and may reproduce earlier than larger individuals, which may reflect in a population formed by large-sized individuals.Hyalella gauchensis was collected at a water reservoir susceptible to environmental perturbations caused by rainfall oscillations, which were indicated by the large numbers of specimens collected in months with warmer temperatures and lower rainfall, respectively in spring and summer (INMET, 2012).According to Boschi et al. (2011), state of Rio Grande do Sul regularly experiences periods of drought during spring and summer.During those seasons, there were increases in food availability at the study sites, reflected by large amounts of macrophytes and algae at the shelters where H. gauchensis was collected.Some species may be successful in environments subject to periodic disturbance when their populations have a high reproductive output and the progeny are of small size (Townsend et al., 2010).The water regime may influence the macroinvertebrate community structure, mainly those species that have their entire life cycle in the water, such as crustaceans (Wellborn et al., 1996). The characteristic bimodal frequency distribution of H. georginae and H. gauchensis populations was marked by the presence of two distinct groups, juveniles and adults.This feature may be related to the seasonal reproduction and, consequently, to the recruitment peaks of the two populations.Bimodal distributions are apparently advantageous since recruitment occurs in warmer months, when food availability is higher, increasing the survival rates (Appadoo and Myers, 2004).This pattern of population frequency distribution is common to amphipod species such as Corophium multisetosum Stock, 1952 (Cunha et al., 2000), Cymadusa filosa Savigny, 1816, Mallacoota schellenbergi Ledoyer, 1984 (Appadoo andMyers, 2004), and Gammarus chevreuxi Sexton, 1913(Subida et al., 2005). The seasonal frequency distribution of H. georginae and H. gauchensis was bimodal for most seasons, and probably reflects the seasonal reproduction of both species.Castiglioni and Bond-Buckup (2008a) studying the ecological characteristics of two sympatric species of Hyalella also found bimodal size-class frequency distribution for H. pleocuta.The authors concluded that either the reproduction of this species is probably more intense during a few months or there is differential mortality throughout the year. Hyalella georginae males and females had average CL larger than H. gauchensis and H. georginae and H. gauchensis males were significantly larger than females.The body size is considered one of the most significant ecological features and is crucial for the ecological success of the genus Hyalella (Wellborn, 2002).Male and female body sizes may vary according to latitude and environmental conditions (Panov and Macqueen, 1998;Xinqing et al., 2013) or to ecological interactions such as completion and predation (Wellborn, 2002).The larger size of males compared with females has also been reported as a sexual dimorphism in other species of Hyalella, such as H. pleocuta, H. castroi (Castiglioni and Bond-Buckup, 2008a), and H. azteca Saussure, 1858 (Geisler, 1944;Wellborn et al., 2005).Males and females often have similar growth until maturity is reached, then males grow more (Low, 1978).Females grow less due to egg production and incubation (Hartnoll, 1982) while males continue to grow during the mating period, reaching larger body sizes (Wen, 1992).During incubation, females do not molt, which also hampers them from growing at the same rate as males (Cardoso and Veloso, 1996).Besides, during the precopulatory period, males carry the females in their thorax until ovulation and fertilization (Borowsky, 1991).Smaller females are easily carried by males (Adams and Greenwood, 1983;Adams et al., 1985;Castiglioni and Bond-Buckup, 2008b), and larger males outcompete smaller ones during the mating process (Ward, 1983). Total sex-ratio for either H. georginae and H. gauchensis favored females (Tab.1).Amphipods may have populations with a sex-ratio of 1:1, or it can vary depending on the season: males may be more abundant on colder months and females in warmer months (Moore, Population of two freshwater amphipods from Brazil Nauplius, 26: e2018025 1981).As it is known for amphipod populations, the sex-ratio fluctuates seasonally, and females are often more abundant than males (Cardoso and Veloso, 1996;Appadoo and Myers, 2004;Kevrekidis, 2004).The sex-ratio favoring females seen in this study probably reflects the males' behavior of choosing and guarding females, making them more susceptible to predators (Moore, 1981;Kevrekidis, 2005).A higher proportion of females was also found for H. pleocuta, H. castroi (Castiglioni and Bond-Buckup, 2008a), and H. azteca (Strong, 1972).The sex-ratio of H. georginae and H. gauchensis were different depending on the size class considered.It favored females in intermediary classes and males in the upper size classes, which characterizes an anomalous sex ratio pattern.Similar results were found by Wenner (1972) and Castiglioni and Bond-Buckup (2008a).The predominance of males in upper size classes is presumably influenced by the females' prolonged parental behavior, in which they carry the offspring attached to their bodies (Borowski, 1991;Thiel, 2003;Castiglioni and Bond-Buckup, 2007).Due to this behavior, females direct their energetic budget towards offspring care instead of molting; therefore, the molt is delayed, limiting the females' body size (Thiel, 2003). For the seasonal sex-ratio analysis, H. georginae females were more frequent than males only in summer.For H. gauchensis, females were more frequent in the autumn and summer.The seasonal reproduction observed in both species of this study may be related to environmental factors of the study sites such as temperature and rainfall.Temperature is known to be an important factor influencing the life-history of aquatic invertebrates (Panov and Macqueen, 1998).Cooper (1965) and Kruschwitz (1978) reported that for H. azteca, only adult individuals survive winter, since reproduction ceases or is considerably lower then.Differently, in most studies on amphipods, reproduction is considered a continuous event, e.g., Gammarus troglophilus Hubricht and Mackin de 1940 ( Jenio, 1980) and H. azteca (Alcocer et al., 2002).Besides, Castiglioni and Bond-Buckup (2008a) observed a continuous reproduction for H. pleoacuta and H. castroi, but more intense in winter and fall, respectively.Food availability (Xinqing et al., 2013) and quality (Dutra et al., 2011) influenced the reproductive capacity of individuals, causing abundance fluctuations.In those months that the temperature was higher, there was a decrease in water volume and an increase in macrophyte abundance (personal observation), providing a suitable environment for the species reproduction. Food availability for adults and for the development of the complete life cycle may be the most important factor influencing reproduction (Sastry, 1983).In the study by Castiglioni and Bond-Buckup (2009) with H. castroi and H. pleocuta from the "Campos de Cima da Serra", fluctuations in reproductive intensities were related to macrophyte cover.Macrophytes are food and shelter for ovigerous females and juveniles and contribute for the species' reproductive success. The continuous recruitment, with peaks in some months, was also observed in H. pleocuta and H. castroi (Castiglioni and Bond-Buckup, 2008a).The recruitment peaked in December 2012 for H. georginae, and in October 2012 for H. gauchensis.The hypothesis raised by the authors is that reproductive and recruitment peaks can occur in the same season.Considering the embryonic period and parental care duration, females are capable of becoming ovigerous and release the offspring within the same season.The embryonic development, from ovulation to hatching, may last 10-25 days.Hyalella pleocuta and H. castroi have an embryonic period of approximately 12 days (Castiglioni and Bond-Buckup, 2007), and in H. azteca hatching can occur in 9.3-21 days (Geisler, 1944;Cooper, 1965).The parental care in Hyalella is known as the period where juveniles remain in the female's marsupium.The parental care may last roughly six days, as in H. pleocuta and H. castroi (Castiglioni and Bond-Buckup, 2007), or three days as in H. azteca (Geisler, 1944). The population structure of H. georginae and H. gauchensis had similar features.Frequency distributions were similar, males were larger than females, the sexratio favored females, and both species showed seasonal reproduction and continuous recruitment.Those similarities and differences may be related to both species' life-history strategies, which promote adaptations to their habitat variations.The information gathered in this study about H. georginae and H. gauchensis populations may help to understand the ecological stability of these species at the studied site.Also, the results may lead to a better understanding of the species biology in the future. Population of two freshwater amphipods from Brazil Nauplius, 26: e2018025 aCKnowleDgeMenTs We thank to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a fellowship to AVO and financial support to DSC (Proc.nº 405061/2015-3).We are gratefull to colleagues of the Laboratório de Zoologia e Ecologia, Campus de Palmeira das Missões, Universidade Federal de Santa Maria for their assistance during field and laboratory activities.This study was carried out according to state and federal laws concerning wildanimal sampling. Figure 2 . Figure 2. Seasonal size-frequency distribution of males, females and juveniles of Hyalella georginae, Palmeira das Missões, state of Rio Grande do Sul.Brazil.All sizes shown in the graph have at least one measured individual. Figure 1 . Figure 1.Size-frequency distribution of males, females and juveniles of Hyalella georginae (A) e Hyalella gauchensis (B), Palmeira das Missões, state of Rio Grande do Sul.Brazil.All sizes shown in the graph have at least one measured individual. Figure 4 . Figure 4. Seasonal sex-ratio in Hyalella georginae (A) and Hyalella gauchensis (B), Palmeira das Missões, state of Rio Grande do Sul, Brazil.Asterisks above the columns indicate significant diferences between the proportions of males and females (p < 0.05). Figure 3 . Figure 3. Seasonal size-frequency distribution of males, females and juveniles of Hyalella gauchensis, Palmeira das Missões, state of Rio Grande do Sul.Brazil.All sizes shown in the graph have at least one measured individual. Figure 6 . Figure 6.Frequency of ovigerou females of Hyalella georginae and Hyalella gauchensis carrying eggs or juveniles in the brood pouch along year of study, Palmeira das Missões, state of Rio Grande do Sul, Brazil. Figure 7 . Figure 7. Frequency of ovigerous females of Hyalella georginae and Hyalella gauchensis carrying eggs or juveniles in the brood pouch along season of study, Palmeira das Missões, state of Rio Grande do Sul, Brazil.Capital letters correspond to the comparisons of the frequency of ovigerous females of Hyalella georginae among the seasons of the year and the small letters correspond to the comparisons of the frequency of ovigerous females of Hyalella gauchensis among the seasons of the year.Collumns with at least one letter in common did not differ statistically (p < 0.05). Figure 9 . Figure 9. Relative frequency (%) of juveniles of Hyalella georginae and Hyalella gauchensis by season of the year, Palmeira das Missões, state of Rio Grande do Sul, Brazil.Capital letters correspond to the comparisons of the frequency of juveniles of Hyalella georginae among the seasons of the year and the small letters correspond to the comparisons of the frequency of juveniles of Hyalella gauchensis among the seasons of the year.Collumns with at least one letter in common did not differ statistically (p < 0.05). Figure 5 . Figure 5. Sex-ratio by size classes in Hyalella georginae (A) and Hyalella gauchensis (B), Palmeira das Missões, state of Rio Grande do Sul.Brazil.Asterisks above the columns indicate significant diferences between the proportions of males and females (p < 0.05).All sizes shown in the graph have at least one measured individual. Table 1 . Number of specimens sampled monthly for a year, monthly sex ratio and goodness of fit analysis (χ²) of Hyalella georginae, Palmeira das Missões, state of Rio Grande do Sul, Brazil. Table 2 . Number of specimens sampled monthly for a year, monthly sex ratio and goodness of fit analysis (χ²) of Hyalella gauchensis, Palmeira das Missões, state of Rio Grande do Sul Brazil.	2019-04-03T13:08:33.812Z	2018-01-01T00:00:00.000	{ "year": 2018, "sha1": "27fa090551653f4aa6a59c18930d04dfca57c6ac", "oa_license": "CCBY", "oa_url": "http://www.scielo.br/pdf/nau/v26/2358-2936-nau-26-e2018025.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "27fa090551653f4aa6a59c18930d04dfca57c6ac", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology" ] }
118531997	pes2o/s2orc	v3-fos-license	Effect of decoherence on the Berry phase of a spin-half in a rotating magnetic field We investigate the decoherence effect of a bosonic bath on the Berry phase of a spin-1/2 in a time-dependent magnetic field, without making the Markovian approximation. A two-cycle process resulting in a pure Berry phase is considered. The low-frequency quantum noise significantly affects the Berry phase. In the adiabatic limit, the high-frequency quantum noise only has a small effect. The result is also valid in some more general situations. INTRODUCTION Geometric phases [1] have been used as an approach to fault-tolerant quantum computing due to its global geometric feature [2][3][4]. How the geometric phase is really insensitive to classical and quantum noises becomes an interesting topic subject to experimental investigations [5][6][7]. Theoretically, insensitivity to classical control noise in some circumstances has been demonstrated [8]. The effect of quantum noise or decoherence has also been studied, most of which were for Markovian dynamics [9,10], but Non-Markovian dynamics has also been considered to some extent [11]. Also related is the subject of geometric phases of mixed states [12]. In this paper we make a general analysis on the effect of decoherence with non-Markovian dynamics on the Berry phase of spin− 1 2 coupled to a magnetic field, a set-up which is useful for quantum computing [4]. The Markovian limit is also discussed. Using a master equation approach without making Markovian approximation, and avoiding defining the Berry phase for a mixed state, we calculate the degrading of the fidelity due to the coupling with an environment. THE MODEL AND THE MASTER EQUATION Consider a spin- 1 2 coupled to a rotating magnetic field and to an environment, which is a bosonic bath. The total Hamiltonian is with where B(t) ≡ (B x , B y , B z ) = B(sin θ cos Ω 0 t, sin θ sin Ω 0 t, cos θ) is the external field, which rotates around z axis with the angular frequency Ω 0 , θ is the angle between B(t) and z axis, σ ≡ (σ x , σ y , σ z ) are the Pauli matrices, a † k and a k are the creation and annihilation operators of the field mode k, g k is the coupling strength. In an experiment testing the Berry phase of a charge Josephson qubit [5], y is realized by the dipole interaction strength between the qubit and a microwave field with phase Ω 0 t, while B z is realized by the detuning between the qubit transition frequency and the applied microwave frequency. The time-dependent Hamiltonian can be transformed to a time-independent one in the rotating frame by a unitary transformation U 1 (t) ≡ e iσz 2 Ω0t [4]. Subsequently, the magnetic field can be transformed to be along the z axis in a rotated frame by another unitary transformation which equals the energy gap between the ground and excited states. The density matrix of the composite system consisting of both the spin and the environment obeys the Liouville equation, which, in the interaction picture, is withρ I (t) = e i(Hs+He)tρ (t)e −i(Hs+He)t and V I (t) = e i(Hs+He)tṼ e −i(Hs+He)t . As usual, it is assumed that the initial state is a direct product of the states of the spin and the environment, i.e.,ρ I (0) =ρ Is (0) ⊗ρ Ie (0). The coupling of the bath with the system is weak, henceρ Ie (t) ≃ρ Ie (0) ≡ ρ e . By applying the projection operator method [13], one obtains the master equation of the reduced density matrix of the spinρ Is (t), up to second order of the spin-environment coupling, , and T r E means partial trace over the environment, which has been assumed to be initially in thermal equilibrium at temperature T , i.e., ρ e = (1 − e −ω k /T ) k e −ω k a † k a k /T . Our calculation is based on the above master equation, without making the Markovian approximation, i.e., replacing the upper limit of the time integral as infinity, as in some previous studies. The effect of the environment can be captured by the force autocorrelation function where N (ω k ) ≡ (e ω k /T − 1) −1 denotes the average number of bosons in a mode with frequency ω k , J(ω) is the environment spectrum. We solve the master equation by using a secular approximation [13] to remove high-frequency terms such as e inEt , n being an integer, on the right-hand side. Then we obtain the solution of the reduced density matrix , whose matrix elements arẽ , where the energy E has been approximated as B, κ(s) ≡ ǫ(s)ǫ(0) andκ(s) ≡ e −iBs κ(s). It indicates that the interaction with the environment induces dephasing, energy dissipation and Lamb-like shift, all of which affect the Berry phase to different degrees. Discussions below will be made in the original frame by using To characterize the environmentally induced decoherence, we compare the density matrix ρ s (t) of the system coupled with the environment, i.e. evolving under H(t), with the density matrix ρ s 0 (t) of the isolated system, i.e., evolving under H s (t). This is done by using the fidelity defined as by setting ρ s (0) = ρ s 0 (0). Without decoherence, we would have F (t) = 1. With decoherence, we have F (t) < 1. We study how the fidelity F (t), as a function of time, is affected by different environmental spectrum. The initial state ρ s (0) is set to be an equal superposition of ground and excited states, as in recent experiments [5], thus ρ s The fidelity is calculated to be exactly We focus on Ohmic spectrum J(ω) = λ 2 ωe − ω Ω , where λ is a coupling constant and Ω is the cut-off frequency. If the correlation time scale of the noise τ c = 1/Ω is comparable with the adiabatic time scale of the system τ 0 = 1/Ω 0 , which is also the time scale of the dynamics described by the master equation, then the environmental memory affects the system dynamics, which is then non-Markovian. Hence the Markovian case is defined by Ω ≫ Ω 0 , which implies that the short-term dynamical fluctuation of the system caused by the feedback of the environment is averaged out [14]. The dependence of the fidelity on the azimuthal angle of the rotational magnetic field θ is clearly shown. The inset in Fig. 1 shows the oscillation of the fidelity in the beginning if θ is large enough, which indicates feedback from the environment. After a certain period of time, the non-Markovian effect becomes remarkable. The smaller the value of θ, the smaller the fidelity. Now we consider the following evolution from the initial state ρ s (0), which is designed to cancel the dynamical phase and result in purely the Berry phase [4], where R Ω0 represents an adiabatical rotation with constant angular frequency Ω 0 for a time period T 0 = 2π/Ω 0 , which is followed by an a instantaneous π pulse represented as Πρ ≡ AρA † , where A ≡ \|e(T 0 ) g(T 0 )\| + \|g(T 0 ) e(T 0 )\|) exchanges \|e(T 0 ) and \|g(T 0 ) . It is then followed by a reversed rotation represented as R −Ω0 . Consequently the Berry phase doubles while the dynamical phase cancels. The relative phase between \|e(2T 0 ) and \|g(2T 0 ) can be measured by state tomography. Note that the process consists of two parts, each of which is subject to a constant rotation, while the two parts are connected by an instantaneous reversal of rotation is direction, with the final state of the first part being the initial state of the second part. For an isolated system, under the adiabatic approximation, the final state is where Φ = π(1 − cos θ) is the Berry phase of \|g . PURE BERRY PHASE UNDER ADIABATIC APPROXIMATION In the adiabatic-limit i.e., ζ 1 ≈ ζ 2 ≈ 0, one obtains In F (2T 0 ), the absence of energy dissipation terms n 1,2 (T 0 ) and m 1,2 (T 0 ) in the adiabatic limit is due to the equal superposition of \|g and \|e in the initial state. Thus the dephasing factors and spectrum-induced phase shifts have strong effects on the Berry phase. The phase shifts k 1 and k 2 partially cancel each other, but the dephasing factors l 1 and l 2 add. In the adiabatic limit, E 1,2 ≈ B ∓ Ω 0 cos θ, we have 4Φ − T 0 (E 1 − E 2 ) = 4π, which goes away. The phase correction is thus δΦ = k 1 (T 0 ) − k 2 (T 0 ) ≃ 4Ω 0 sin 2 θ cos θ T0 0 t 0 s cos(Bs)Re[κ(s)]dsdt, from which we note that δΦ is proportional to sin 2 θ cos θ, no matter what the environment spectrum is. The dephasing factor is l 1 (T 0 ) + l 2 (T 0 ) ≃ = πδ(B − ω). Taking the limit t → ∞ is equivalent to the Markovian approximation. The result is consistent with the result in Ref. [10], suggesting that the geometric nature of the phase correction and dephasing is model-insensitive. We would like to emphasize that in the adiabatic limit, there are two major factors degrading the Berry phase, that is, the dephasing effect and the environment-induced phase shift, which are intertwined together. The dependence of the fidelity F (2T 0 ) of the final state on the time period T 0 is shown in Fig. 2, where the behavior of an isolated system is also shown for comparison. Note that the oscillation at small values of T 0 , which exists in all cases including the isolated system, is a manifestation of non-adiabatic error, which has been analyzed previously [15]. In Fig. 2, it can be seen that in Markovian-limit and a low temperature environment, there is a wide range of T 0 where Berry phase can be observed as the fidelity is close to 1. The non-Markovian spectral density is significant for low frequencies, where the effect of Berry phase is strongly degraded. This is similar to an experimental result [5]. We plot n 1 (T 0 ), l 1 (T 0 ), k 1 (T 0 ), and k 1 (T 0 ) − k 2 (T 0 ) together in Fig. 3. m 1,2 (T 0 ) is too close to zero to be plotted. In the adiabatic limit, the energy dissipation does =20 =2 not appear in the fidelity expression, while the Lamb shift is eliminated by the spin-echo technique, the dephasing effect plays a crucial role in degrading the coherence. Furthermore, an interesting characteristic exhibited in Fig. 4 is that the dependence of the fidelity F (2T 0 ) on the azimuthal angle θ varies with the noise spectrum. We note that in the expression of l(t), the second term is negligible in the non-Markovian limit as it oscillates with frequency B, and thus l(T 0 ) ∝ cos 2 θ. But in the Markovian limit, l(T 0 ) ∝ sin 2 θ. Therefore we can see that the fidelity of the final state at a certain temperature is a monotonically increasing function of the azimuthal angle θ in the non-Markovian case, but is a monotonically decreasing function of θ in the Markovian limit. MORE GENERAL SITUATIONS The above analysis can be extended to the situation that the magnetic field adiabatically travels along an arbitrary closed path: = B(sin θ(t) cos ϕ(t), sin θ(t) sin ϕ(t), cos θ(t)), leading to a Berry phase Φ = 1 2 (1 − cos θ)dϕ for \|g(t) , or −Φ for \|e(t) . The transformed Hamiltonian To proceed, we treatH s under first-order adiabatic approximation, while treatṼ ′ to zeroth-order i.e.,H ′ . Then it can be obtained that n(t), m(t), l(t) and k(t) replaced as n ′ (t), m ′ (t), l ′ (t) and k ′ (t) respectively, given by n ′ (t) = 4 As an example, consider the closed path traveled by the magnetic field is given by as shown in Fig.5. Noting θ 2 (t) = θ 1 (T 0 − t), we can investigate the response of the Berry phase for the noise direction defined by γ. The dependence on γ indicates that for a certain noise environment we can always choose an optimal loop to minimize the decoherence effect. Fig. 6 also shows that in addition to the noise spectrum, the azimuthal angle θ ′ itself, as an intrinsic geometry parameter for the Berry phase, strongly affects the fidelity. some features of the fidelity F (2T 0 ) for a smoothly rotating field persist in the present more general case. We can also consider the multi-noise case based on Eq. (1) by assuming that the frequency Ω 0 is precisely controlled, this seems achievable in realistic devices, but an additional bosonic bath couples to the rotating component B in the xy plane denoting the dipole interaction in the solid-qubit device [5]. In the rotating frame the Hamiltonian can be written asH = E 2 (cos ασ z + sin ασ x ) + σ z k g zk (a † zk + a zk ) + σ x k g xk (a † xk + a xk ) + k ω zk a † zk a zk + k ω xk a † xk a xk . Assuming the two independent baths possess the same autocorrelation function, i.e., ǫ z (t)ǫ z (s) = ǫ x (t)ǫ x (s) , we found that the decoherence no longer depends on the azimuthal angle θ, and that the spectrum-induced phase shift δΦ = 0. The gen- eral feature of F (2T 0 ) observed above is still valid now. However, the dephasing effect caused by low-frequency noise has been enhanced. SUMMARY To summarize, we have analyzed the effect of decoherence on the Berry phase by calculating the fidelity between the reduced density matrix of the system coupled with an environment and the exact state of a closed system, both starting from a same initial state. This approach does not rely on any definition of the geometric phase in a mixed state. We use the master equation without any constraint on the correlation time of the bath, hence our discussions cover both the non-Markovian dynamics and Markovian limit. It is found that in the adiabatic limit, with a high frequency quantum noise, the deviation of the fidelity from 1 is quite small, implying that the Berry phase is robust. With a low frequency quantum noise, the fidelity is significantly lowered, implying that the Berry phase is significantly degraded. Our finding is beyond what can be obtained by making Markovian approximation, and is in accordance with the experimental result [5]. We also note that the result is valid in the more general cases of an arbitrary path of the cycle and of the multi-noise. It is also noted that for the initial state considered, dephasing clearly dominates over the energy dissipation and the Lamb shift. As the dephasing is path dependent, an optimal evolution loop can be chosen to protect the coherence. We hope that our analysis is useful for designing quantum gates based on geometric phases. Finally, we note that it is interesting to combine the geometric phase approach with the dynamical decoupling approach [16], where it is also found that non-Markovian environmental noise causes phase randomization [17].	2013-08-13T11:00:26.000Z	2013-08-13T00:00:00.000	{ "year": 2013, "sha1": "9eddd1dbc9f0f7e9e50a0ad03891a9c47932b60d", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1308.2821", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "9eddd1dbc9f0f7e9e50a0ad03891a9c47932b60d", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
257069214	pes2o/s2orc	v3-fos-license	Are we ready for telemonitoring inflammatory bowel disease? A review of advances, enablers, and barriers This review summarizes the evidence about telemonitoring in patients with inflammatory bowel disease (IBD). To give an overview of the advances performed, as well as the enablers and barriers which favoured/hindered telemonitoring implementation. We performed a literature search in PubMed, EMBASE, MEDLINE, Cochrane Database, Web of Science and Conference Proceedings. Titles and abstracts published up to September 2022 were screened for a set of inclusion criteria: telemonitoring intervention, IBD as the main disease, and a primary study performed. Ninety-seven reports were selected for full review. Finally, 20 were included for data extraction and critical appraisal. Most studies used telemonitoring combined with tele-education, and programs evolved from home telemanagement systems towards web portals through mHealth applications. Web systems demonstrated patients’ acceptance, improvement in quality of life, disease activity and knowledge, with a good cost-effectiveness profile in the short-term. Initially, telemonitoring was almost restricted to ulcerative colitis, but new patient reported outcome measures, home-based tests and mobile devices favoured its expansion to different patients´ categories. However, technological and knowledge advances led to legal, ethical, economical and logistic issues. Standardization of remote healthcare is necessary, to improve the interoperability of systems as well as to address liability concerns and users´ preferences. Telemonitoring IBD is well accepted and improves clinical outcomes at a lower cost in the short-term. Funders, policymakers, providers, and patients need to align their interests to overcome the emerging barriers for its full implementation. INTRODUCTION Inflammatory bowel disease (IBD) is a group of disorders characterized by the chronic and recurrent inflammation of different segments of the gastrointestinal tract, which usually associates extraintestinal manifestations and complications due to sustained activity. Unlike other chronic pathologies, IBD mainly affects young individuals in their optimal period of personal and professional development. As such, IBD is related to high levels of school absenteeism and work disability[1], interference with social activities, and impaired health-related quality of life (QoL) [2]. Therefore, IBD has a significant medical, social, and financial impact, further increased by the global increase in its incidence and prevalence in recent years [3]. It is suggested that the "treat-to-target" strategy leads to better outcomes [4]. However, in the conventional management of IBD, scheduled outpatient visits show difficulties to address the disease evolution in each patient, with frequent discrepancies between medical practice and guideline recommendations. Furthermore, patients have little involvement in decision-making, and nearly 50% of adults[5] and an even higher percentage of adolescents with IBD[6,7] are nonadherent to treatment. All these factors prevent the effectiveness of traditional interventions in disease control and increase health expenses [8], especially considering that patients with IBD use health care resources more often than patients with other conditions [9]. Nowadays, health systems are facing financial problems, and telemedicine has been proposed as an alternative to provide an efficient and equitable use of health resources. Information and communication technologies (ICTs) have the potential advantages of providing better communication between healthcare providers and patients, as well as educational resources adapted to patients´ needs. On the one hand, communication improvements could overcome limitations of health access in remote areas, also developing telementoring systems and contact between different specialists in centres where multidisciplinary teams are not available. On the other hand, educational elements could favour patients' empowerment and treatment optimization throughout the disease course [10,11], also addressing behavioural and psychological factors related to nonadherence [5]. Telemedicine has been successfully used in other chronic diseases such as congestive heart failure[12, 13], diabetes mellitus [14,15] or chronic obstructive pulmonary disease [16,17] and showed excellent acceptance by patients, improvement in health related QoL and a reduction in hospitalizations [12,16,18]. Owing to these positive results, telemedicine systems have been evaluated in patients with IBD, especially in mild to moderate ulcerative colitis (UC) [19,20]. Telemedicine in IBD started with the adaptation of telemonitoring programs previously used in other chronic pathologies [21], but these were subsequently replaced by web and m-health systems, which represented more attractive options to maintain patients adherence to remote follow-up. Telemonitoring is the main form of telemedicine in IBD. It is based on the provision of health services at a distance, related to diagnosis, treatment, follow-up or education. It is characterized by the structured and continuous monitoring of clinical data that is self-reported by patients in their usual environment, and then sent to health providers. The objective is the early detection and intervention on complications related to the disease itself or its treatment. It usually includes tele-education interventions and shares many features with other domains of use of ICTs in the health-care setting (Figure 1). Web telemonitoring in IBD is safe and reduces the duration of disease flares [22]. Moreover, patients´ empowerment has been related to a reduction in outpatient visits and hospitalizations [23][24][25][26], which represent potential cost savings [22,24,27]. The development of more sophisticated telemonitoring programs and point of care (PoC) testing during recent years provided additional value to remote follow-up in the IBD context, improving the ability to cover different patients' profiles more objectively. These advances gained special interest after the advent of coronavirus disease 2019 (COVID-19) outbreak, as distance management offered new elements to overcome healthcare challenges posed during the pandemic[28]. However, telemedicine was represented mainly by telephone and e-mail, previously available in many centres [29,30], but the development of mature telemedicine programs integrated with electronic health records were still the exception, due to a series of remaining barriers. In this review we focus on the advances performed in telemonitoring of patients with IBD, taking into consideration the elements which enabled its use and how technological achievements led to other barriers for its full implementation. The search strategy is detailed in the Supplementary material. TELEMEDICINE IN CHRONIC DISEASES There is a wide heterogeneity of telemedicine programs considering the different types of technological resources used, the different diseases and populations in which they are applied, and the objectives pursued with their use. The variability in the quality and design of the published studies (randomized clinical trials, before-and-after studies, qualitative studies, etc.) can partially explain the variable results obtained. Furthermore, in some studies these systems are part of wider interventions, rendering the comparability between programs even more difficult. These factors limit the quality of evidence regarding the efficacy of telemedicine to improve outcomes in chronic diseases. Despite this, ICTs have been used in a wide range of pathologies, with improvement of patients´ empowerment and with good acceptance [31]. With the aim of giving response to the rise in chronic diseases and multimorbidity worldwide, different projects in Europe have studied the use of telehealth, mainly in diabetes mellitus, cardiovascular diseases, depression, and chronic obstructive pulmonary disease (COPD). There is moderate evidence about the efficacy of telehealth systems in the improvement of glycaemic control, mainly in terms of HbA1c in patients with type 2 diabetes mellitus. Active telemonitoring including providers´ feedback, as well as tele-education have shown positive results compared with usual practice [14]. In fact, the highest impact was seen in the combination of telemonitoring and teleeducation for both patients and providers, allowing for shared decision-making [15]. In patients with heart failure, telemonitoring has been shown to reduce global mortality and hospitalizations compared to usual care [12]. Many telemonitoring systems were part of multidisciplinary programs managed by specialized nurses and incorporated tele-education and action plans before hospital discharge [13]. Interactive monitoring with healthcare providers has also shown to improve blood pressure in hypertensive patients, weight control and lipidic profile [32]. Video consultation and biosensors are especially useful in cardiovascular diseases, with reduced costs compared with other pathologies [33]. Most digital resources used in the mental health context refer to the application of cognitive behavioural therapy (CBT) in patients with depression. Both traditional and e-Health CBT are effective [34], but its use with telemedicine programs could offer additional advantages, such as better accessibility. However, many studies with psychotherapies show a high rate of nonadherence to follow-up [35]. Similarly, in patients with IBD one clinical trial showed a significant improvement in QoL after 12 wk of self-administered computerized CBT, but this outcome was not maintained at 6 mo, with a high rate of dropouts [36]. In patients with asthma, the use of multiplatform programs combining tele-education, telemonitoring and individualized action plans reduced hospitalizations compared with traditional care, mainly in more severe patients [18]. In patients with COPD, the use of telemedicine also reduces hospitalizations, but without an improvement of global mortality [16]. With the development of mHealth, the use of SMS combined with telephone support is associated with an improvement in respiratory function and QoL in patients with asthma[32], but telemonitoring in COPD has not demonstrated any improvement in these outcomes [17,37]. Telemonitoring of patients with COPD is more expensive due to associated multimorbidity [33]. The use of telemedicine in digestive diseases is more limited, and most studies focused on IBD and irritable bowel syndrome. Unlike other chronic diseases such as diabetes mellitus, IBD implies the consideration of many clinical, biological, endoscopic, and even histologic variables to reach disease control. However, early detection of complications usually requires invasive tests in IBD. The absence of validated tools with adequate cost and accuracy to measure disease activity at a distance have represented an important limitation. Most studies about telemedicine in IBD have emerged in the last decade. The development of mHealth, the validation of patient reported outcome measures (PROMs), PoC and home-based tests to measure fecal calprotectin (FC) near the patient improved the ability to evaluate more types of patients with IBD at a distance, even in more complex cases. PROGRAMS FOR TELEMONITORING IBD The increase in the capacity of data transmission and storage, as well as the evolution of wireless communications provided many resources that are easy to use and adaptable to IBD telemonitoring. Initially, telemonitoring systems for IBD allowed communication between health centres and patient´s home using computers. Afterwards, the development of web-based systems permitted easy-touse and cheaper telemonitoring programs. In the last years, mobile devices (Smartphone, Tablet, etc.) made it possible to establish the communication process with the patient during his/her daily activities. Furthermore, in other settings (such as cardiovascular diseases) the transmission of continuous physiological data through biotelemetry has evolved with the incorporation of wearables. In the IBD setting, telemonitoring is a safe, acceptable, and effective option to improve clinical outcomes[38,39], but the results of studies are still variable. In this context, telemonitoring has mainly used programs requiring home installation or web-systems, although e-mail and telephone have supported some of these programs. The main telemonitoring platforms used in IBD are summarized in Table 1. Home telemanagement systems The Cross group was the first to apply ICTs in adult patients with IBD, mainly with UC. This research team developed a remote-control system (home automated telemanagement system: HAT system) made up of 3 stations, adapted from a program previously used in self-management of patients with asthma [40]. The Home Unit was made up of a portable computer that collected patients' information (symptoms, adverse effects, medication, etc.), and these data were then sent to a decision-support server connected to a provider's PC [21]. The computer created alerts if the values collected in a web portal surpassed pre-established thresholds. Moreover, the HAT system incorporated educative elements. Different exploratory studies showed good acceptance with the use of this system. In 2 studies with 10 and 23 patients with IBD, all of them considered that the HAT system was simple and increased patients´ knowledge [21,41]. To confirm the acceptability and adherence to follow-up with this program, the authors performed a subsequent study with 25 patients followed-up over 6 mo. Adherence to the weekly questionnaire was 91% and 86% had a prescribed medication adherence over 80%. This good adherence corresponded to a trend towards improvement in disease activity and QoL levels, together with a statistically significant improvement in understanding the disease (P = 0.0015). These good results led to the hypothesis that the HAT system could be feasible for telemonitoring patients with IBD. Subsequently, the same group designed a randomized clinical trial including 47 patients with mild to moderate UC. Twenty-five patients were controlled with the HAT system and 22 followed usual inperson visits together with educational support and individualized action plans to make groups more comparable [42]. The groups had similar baseline characteristics, except for the use of immunosuppressants in 56% of the study group and 27% of the control group (P = 0.05), which would indicate a higher level of disease activity in the experimental group. There were no statistically significant differences for improvement of disease activity, treatment adherence, and quality-of-life values between both groups at 12 mo. These results could be related to the small sample size as well as a higher dropout rate in the intervention group, possibly due to the platform design, which required installation and eventual repairs at home. To avoid these problems, Cross and cols developed web telemonitoring using mobile devices[25]. Web-based systems In the last decade, telemonitoring systems have progressively evolved with web programs and mHealth solutions. Web applications are less expensive, safe, and feasible in the management of IBD not only in adults but also in adolescents [43][44][45], and they are associated with a reduction in outpatient visits and hospitalizations [22,24,27,43,45]. A Danish group developed telemonitoring through the web under the concept of "Constant-care". The system was developed through the web http://www.constant-care.dk, which also incorporated an educational centre. These investigators designed a randomized controlled trial with 333 UC patients treated with 5-aminosalicylates (5-ASA) from different hospitals in Denmark and Ireland [22]. The intervention group introduced clinical data and analytic results in the web to guide changes in the follow-up schedule and treatment. This intervention was compared to usual care. After 12 mo of follow-up, in both the Danish and Irish population 88% of patients showed good acceptance with the web telemonitoring. There was a statistically significant improvement in adherence to treatment after 4 wk and a lower duration of disease flares. This was related to the use of high doses of 5-ASA in 100% of patients from the intervention group in Denmark, who also had improved QoL and disease knowledge. However, these outcomes were not reproduced in the Irish population. Moreover, in the Danish population telemonitoring reduced outpatient and emergency department visits, which led to direct cost-savings of 189 euros per patient-year, but also to an increase of e-mails and telephone contacts. The use of this web-system in paediatric patients also reduced outpatient visits and school absenteeism, without differences in disease activity, QoL and adherence to treatment compared to the control group [43]. In another study developed in the University of California with a telemonitoring program combined with tele-education, patients followed remotely used less corticosteroids and suffered less hospitalizations and emergency department visits, with cost reductions of 16%[23]. In short, these studies show that web-telemonitoring is feasible, safe and could reduce health costs, although there are reproducibility differences depending on the population in which telemonitoring is applied [19]. Moreover, web-systems have been used to individualize the treatment according to the disease course. In a prospective study with 95 patients with mild to moderate UC, web control allowed the adjustment of 5-ASA doses and improved adherence. This was related with a significant improvement in clinical activity and FC values after 3 mo of follow-up [11]. Telemonitoring has even been used to individualize the treatment schedule with infliximab. After 1 year of follow-up, there were no significant changes in disease activity and QoL, although there was an estimated cost-saving of 699 euros/patient, compared with a historic control group[10]. In the same line and to avoid problems generated with the HAT system in the pioneering studies, the Cross group developed a web system for the management of patients with IBD (TELE-IBD) through text messages. In a randomized clinical trial with 3 parallel groups (TELE-IBD weekly, TELE-IBD every other week and control group) in 3 reference centres for IBD, they included 348 patients who had at least one disease flare in the last 2 years. Seventy-five percent completed the study, with an improvement in disease activity and QoL in the 3 groups, but without a higher improvement in these outcomes, depressive symptoms, or self-efficacy in the web control group, although in another study self-efficacy improved when tailored counselling was associated [46]. Moreover, telemonitoring was associated with a change in the profile of health expenses. Less hospitalizations were seen in the telemonitoring group but with higher use of non-invasive tests and telephone or e-mail [25,47,48]. The largest clinical trial with a telemonitoring program to date was performed with the Dutch web myIBDcoach (http://www.mijnibdcoach.nl). This web allows distance monitoring of disease activity, treatment adherence and side effects, as well as nutritional status, smoking habits, QoL, fatigue, stress, February 21, 2023 Volume 29 Issue 7 anxiety and depression. As other platforms, it provides educational elements to improve empowerment. Patients showed good acceptance with its use in a pilot study [49]. In a clinical trial including 909 patients with different disease characteristics, the use of this system reduced outpatient visits and hospitalizations compared to standard care after 12 mo of follow-up [24]. Similarly, a reduction in outpatient visits was also obtained in adolescents [43,45] and in adults who used home-based tests to measure FC [50]. In a pilot study performed in France with the EasyMICI-MaMICI ® platform, a reduction in outpatient visits was also associated with a significant improvement in QoL and satisfaction[51]. Our study group evaluated the impact on health outcomes of the telemonitoring web platform TECCU (Telemonitorización de la Enfermedad de Crohn y Colitis Ulcerosa or Telemonitoring of Crohn's Disease and Ulcerative Colitis), as compared to standard care and telephone care. In a 3-arm randomized clinical trial, 63 patients (21 per arm) with complex IBD were managed with each follow-up method over 24 wk. At the end of the study, the percentage of patients in remission was higher in the TECCU group (17/21, 81%) compared to nurse assisted telephone care (14/21, 66.7%) and standard care (15/21, 71.4%). The telemonitoring group had more improvement in disease activity, and this was associated with a larger reduction in FC values. All completers adhered to treatment in the TECCU group, while QoL, social activities, and satisfaction improved in all 3 groups[52]. Telephone and e-mail support in web-systems Telephone and e-mail are resources attended by both medical doctors and specialized nurses in some IBD units, with high capacity to solve problems at less cost [53][54][55]. These tools have also been used to coordinate action plans in telemonitoring systems. In Spain, the Crohn´s and Colitis Care Unit model has been used since 1999 as a multidisciplinary model of continuous care for patients with IBD. This model manages health demands with distance management mainly through telephone or e-mail with the support of a web page, which includes educational elements. The number of users has risen over the years, with a reduction of in-person care [56]. In Illinois, the Sonar Project is based on monthly web monitoring of symptoms in patients with IBD. Nurses exert a central role and use telephone contact for those patients who send results out of normal ranges, and together with medical health providers management adjustments are performed. This system also reduced hospitalizations, emergency visits and costs [57]. Therefore, beyond the use of telephone and e-mail in units which work as centres for resource coordination, telemedicine in IBD is expanding through the use of web and mHealth systems. These include telemonitoring, tele-education and videocalls in some cases. Its application allows the development of projects to provide health resources in remote areas [58,59], mainly with the use of mobile apps and the integration of some of these platforms into the electronic medical records, as is the case of the app HealthPROMISE and mynexuzhealth [60,61]. These models promote collaboration and mentoring between specialists, which could reduce variability in medical practice and modify the structure of future health systems if they demonstrate to be cost-effective. Cost-effectiveness of telemonitoring IBD Although many data about cost-savings have been published, they refer almost exclusively to direct costs [22,27], without considering costs of installation and maintenance of platforms or indirect costs. In the IBD setting, our research group published the first cost-effectiveness and cost-utility analysis of a telemonitoring program compared to telephone and standard care [62,63]. The differences between groups and statistical uncertainty in disease activity, quality-adjusted life-years, and costs were calculated using nonparametric bootstrap estimations. Even though our trial only included 63 patients, we imputed the original dataset 5 times, and the bootstrapping estimations allowed us to extract 1000 random samples (of 21 patients per arm) from each of the 5 imputations, thus generating 5000 bootstrap replications. We concluded that there is a high probability (79.96%) that the use of the TECCU web platform for telemonitoring complex IBD patients produces a greater improvement in disease activity at a lower societal cost, compared with standard care. Telemonitoring through the TECCU platform saved €2250 per additional patient in remission (95%CI: €-15363 to 11086) vs telephone care, and telephone care saved €538 compared with standard care (95%CI: €-6475 to 5303). Moreover, the use of the TECCU platform and telephone care showed an 84% and 67% probability, respectively, of producing a cost saving per additional quality-adjusted life-year (QALY) compared with usual care, even considering the simulations that involved negative incremental QALYs. With a similar methodology, our results were reproduced by de Jong et al [64] who concluded that telemedicine with myIBDcoach is cost-saving and has a high probability of being cost-effective, without a decline in QoL. In this study, telemedicine resulted in lower mean annual costs of €547/patient (95%CI: €1029-2143) without changing quality adjusted life years. At the Dutch threshold of €80000 per quality adjusted life year, the intervention had an increased incremental cost-effectiveness over standard care in 83% of replications. The authors included all subtypes of IBD, whereas our study with the TECCU platform recruited complex patients with IBD who needed to start immunosuppressants and/or biologic agents. According to our conclusions, the big sample size recruited in this article is useful to confirm our prior results, and February 21, 2023 Volume 29 Issue 7 the reproducibility of the favourable cost-effectiveness profile of telemedicine applied in IBD across countries and patients' characteristics. In fact, during the COVID-19 pandemic, similar cost-savings with a higher gain of QALYs have been observed with the use of telemonitoring for IBD in Hong Kong [65]. ENABLERS AND BARRIERS FOR THE IMPLEMENTATION OF TELEMONITORING IN IBD Unlike the use of ICTs in other fields (streaming entertainment services, grocery delivery, e-banking, etc.), telemonitoring interventions deal with a series of barriers which hinder their definitive implementation to reorganize health systems, despite other associated advantages (Table 2)[66-71]. The factors which favoured and limited these changes can be classified in 5 groups: technological, organizational, legal, acceptability and costs [72]. MODELS OF TELEMONITORING IN IBD Telemonitoring theoretically include three different diagnostic models: patient self-diagnosis, remote providers´ diagnosis and computer-assisted diagnosis. They usually work as triage systems but, beyond diagnostic capabilities, telemonitoring platforms allow for remote management of other aspects such as disease treatment or education. In the IBD setting, they usually combine self-management, remote providers´ management and computer-assisted telemanagement [ Patient self-management Self-management refers to a dynamic, interactive, and daily process in which individuals engage to manage a chronic illness [73]. This process includes the ability to deal with their own symptoms, treatment, physical and social consequences, and lifestyle changes to maintain a satisfactory QoL [74]. In this sense, telemonitoring platforms for IBD have incorporated PROMS and home-based tests that allowed patients to self-report their health status. This information has been even used to guide treatment adjustments by themselves [22]. Resources for patients´ self-management: PROMs, home-based tests and wearable devices Considering the "treat-to-target" strategy, the Selecting Therapeutic Targets in Inflammatory Bowel Disease (STRIDE) project recognized different evidence and consensus-based recommendations to optimize outcomes in patients with IBD. Among the different targets proposed, clinical remission and endoscopic healing were confirmed in the STRIDE-II actualization, while normalization of serum and fecal markers of inflammation have been determined as short-term targets [4]. There was agreement to evaluate disease remission with clinical indexes, PROMs and endoscopic criteria [or also radiologic criteria in Crohn's disease (CD)]. Usually, endoscopic disease activity has been considered the gold standard to measure inflammation and to consider mucosal healing, but endoscopy is invasive and expensive. In this sense, with the aim of measuring inflammation non-invasively, different PROMs and PoC tests have been developed over last years. Moreover, some of these tools have been specifically validated for their use in telemedicine programs. PROMs: A PROM is a measurement of any aspect of a patient's health status that comes directly from the patient, without the interpretation of the patient's responses by healthcare providers and without the need of laboratory tests [75]. PROMs are designed for screening of disease activity, and then they need to be sensitive enough, especially if it implies more false positive results. The Simple Clinical Colitis Activity Index (SCCAI) has shown a high correlation and good agreement between patient and clinician reported versions [76]. Compared to UC, PROMs used in the context of CD have shown worse correlation with other markers of clinical or endoscopic activity. The Harvey-Bradshaw index (HBI) had high correlation but only moderate agreement between versions registered by the patient or the clinician [77], although a recent version of the HBI self-administered by the patient through a mobile app showed a high percentage of agreement with in-clinic physician assessment, with a remarkably high PPV for remission [78]. Both SCCAI and HBI show good agreement between paper and online versions [78][79][80], and represent attractive tools for telemonitoring IBD. Few PROMs have had their correlation with endoscopic activity evaluated. The global assessment of the patient, based on an analogic visual scale about how they felt regarding their UC during the previous 2 d, only showed moderate correlation with endoscopic activity [81]. The subscore of the global medical assessment and the 6-point Mayo index (which includes stool frequency and rectal bleeding) have a high correlation with the whole Mayo index [82]. Moreover, the 6-point Mayo index has an AUC of 0.80 compared to the endoscopic subscore [83]. Recently, the mobile Health Index was validated to monitor IBD activity through mHealth systems. In patients with CD, it showed high correlation and agreement with the Crohn´s Disease Activity Index and the HBI, as well as in patients with UC when it was compared to the partial Mayo index [84]. The intraclass correlation coefficient for test-retest reliability was high for CD and for UC. Nevertheless, its agreement with endoscopic scores was poor in CD and moderate in UC. QoL and absence of disability are other targets of the STRIDE-II initiative. The Inflammatory Bowel Disease Questionnaire (IBDQ) was specifically validated in patients with IBD and has a moderate to high correlation with treatment response and the endoscopic Mayo index. However, their 32 and 36 items versions require a lot of time for their interpretation, so the reduced versions of 9 and 10 questions were subsequently validated [85,86]. On the other hand, the IBD disability index predicts active disease, nonadherence, and treatment with corticosteroids when high disability values are obtained [87]. Finally, health-related fatigue was incorporated in the Monitor IBD At Home index, but it is still not considered a specific target. Probably the accuracy of PROMs increases when used in combination with FC. Thus, the Monitor IBD At Home index was developed to predict the endoscopic activity in patients with IBD. The association of FC to both the CD and UC versions showed high sensibility and NPV to rule out endoscopic activity [88]. The development of home-based FC tests that can be measured by the patient represent a potential option to measure disease activity in telemonitoring programs. PoC tests and home-based tests: The use of PoC tests refers to patient specimens assayed at or near the patient with the assumption that test results will be available instantly or in a very short timeframe to assist caregivers with immediate diagnosis and/or clinical intervention [89]. In the IBD setting, the interest has centered on FC, and even though lactoferrin tests have been developed with adequate accuracy[90], FC offers better sensibility at certain cutoffs [90,91]. FC has good correlation with endoscopic activity in both UC [92,93] and CD [94][95][96]. FC helps to differentiate between functional and inflammatory diseases in patients with digestive symptoms. Moreover, in patients already diagnosed of IBD it allows the evaluation of disease activity, response to treatment, post-surgical recurrence and it predicts relapses after the withdrawal of anti-TNF agents [97,98]. These features, its non-invasiveness and a relative low cost makes FC tests in a useful tool in the diagnosis, monitoring and treatment adjustment in IBD. Furthermore, the diagnostic accuracy of FC in different clinical scenarios has increased the interest in its use in telemonitoring IBD programs. In line with a patient-centered care and to favour empowerment, during the last years and the COVID-19 pandemic, different home-based FC tests have February 21, 2023 Volume 29 Issue 7 been developed as an additional tool for a home-based follow-up [99]. These tests are based on kits that analysed faecal samples through immunochromatography. Then, the results are read with a smartphone camera, and they are sent through a specific app to a server accessible by providers (Figure 2). Comparison between different home-based FC tests: Three main FC tests have been developed for its use by patients at home: CalproSmart, IBDoc and QuantonCal. In a recent study, these 3 tests were compared with the ELISA method of the same manufacturer [100]. Considering the importance of obtaining good agreement in the low range of FC values (rulingout disease activity), IBDoc, QuantonCal and CalproSmart have an 87%, 82% and 76% agreement, respectively, compared with their corresponding ELISA readings. In any case, the error range between FC measured with home-based tests and their corresponding ELISA method was high. This happens especially when FC values are > 500 µg/g. With values ≤ 500 µg/g, differences were also over the acceptable range of 200 µg/g (+/-100 µg/g), but they were not wide enough to induce errors in the interpretation of inflammatory activity. Therefore, home-based tests are considered useful to rule-out inflammation at a distance when FC values are < 500 µg/g, but when values are > 500 µg/g disease activity should be evaluated with other methods. Home-drug monitoring: Home therapeutic drug monitoring of monoclonal antibodies appears to be an innovative possibility to improve and simplify IBD management. To date, these tests are performed only in some hospital laboratories and results are not immediate. This delay, of months in some cases, impairs drug monitoring and dose adjustment, compromising its utility. To solve this issue, home drug monitoring using dried blood samples is being evaluated in different inflammatory diseases. The first data were published by Kneepkens et al[102] in patients with rheumatic inflammatory diseases. Adalimumab and anti-adalimumab antibodies concentration measurements in finger prick dried blood spots were compared with simultaneous serum measurements. They found that both drug levels and antibody concentrations from the finger prick method correlated well with serum measurements (correlation coefficient > 0.87). However, some disadvantages should be considered, such as loss in precision, workload and elevated costs. Berends and colleagues did a similar study with 40 IBD patients, comparing infliximab concentrations in dried blood samples and serum. Home-based test infliximab concentrations showed a good correlation (correlation coefficient: 0.671) with serum measurements [103]. This author also published data with adalimumab treatment one year later. A high correlation was found (Pearson's correlation: ≥ 0.96) between dried blood test and venipuncture results when performed at the same time during the outpatient clinic. Moderate correlation (Pearson coefficient = 0.51) was reported between home selfperformed finger test and estimated adalimumab concentrations [104]. Larger studies are needed to confirm the reliability, accuracy, and cost effectiveness of home-based testing as a new telemonitoring tool in IBD. Wearable devices: Wearable devices are electronic gadgets that consumers wear to track health-relevant physiological data to monitor and improve health [105]. IBD patient preferences and interest in wearable technology were evaluated by Hirten and colleagues using a 28-question survey. Four hundred patients completed the survey. Of these, 42.7% reported prior or current use of wearable devices, mainly smart watches (34.5%) and wrist band devices (29.1%). Almost 90% of subjects believed these gadgets could provide important information about their health and 93.8% reported that they would use them if it could help doctors manage their disease [106]. In the last few years, several studies have tried to demonstrate the utility of wearable devices in IBD telemonitoring [107]. The first available data was published by Yvellez et al [108] in 2018. They prospectively assessed daily health-related QoL, pain and sleep data using validated indexes through a mobile application and a Fitbit ® device. Fitbit ® compliance was almost 80%, suggesting this technology is feasible [108]. The Fitbit ® device has also been used to predict disease activity. In one study a significant reduction in daily steps has been shown over the week before CRP or FC elevation, but without differences in daily resting heart rate [109]. Two years later, however, Hirten et al [110] demonstrated that significant changes in heart rate variability measured using VitalPatch ® were observed before the development of symptomatic or inflammatory flare in ulcerative colitis patients. Step count and sleep monitoring have also been used, not to predict flares but the intent to determine post-operative length of stay. Overall, step count and sleep duration/efficiency did not predict length of stay. However, in a multivariable linear regression model, significant interaction was found between postoperative complications and step count, suggesting that increased physical activity was associated with a reduction in duration of hospital stay [111]. February 21, 2023 Volume 29 Issue 7 Remote providers´ management All the telemonitoring interventions in IBD reported in this review comprise healthcare providers´ advice. Most systems employ store and forward programs, where nurses acquire a central role in tracking the information received and making contact between patients and specialists to set up healthcare plans. Communication is established through websites, usually with the support of telephone and e-mail, as reported above. Computer-assisted telemanagement Computerized systems have been used for telemonitoring IBD and they usually work as a triage system to identify which patients might require further evaluation. Many of them can generate automatic action plans through the integration of different monitorable indicators in decision-making algorithms. In most telemonitoring programs tested thus far, these tools are combined with self-management and the remote providers´ management models seen above. Telemonitoring systems in IBD are integrated by personal computers or mobile devices used by patients, a decision support server and a website for staff and providers. The website provides an interface to collect data from testing sessions, and these platforms usually generate automated reminders to favour adherence to follow-up. The structure of most eHealth tools in IBD are based on a traffic light system [10,11,22,25,27,43,45,52,112]. Patients usually enter their symptoms in scheduled online controls, mainly in a structured manner through PROMS, but many apps also include a comment box to freely express anything outside the questionnaires. Then, the patient´s status appears as red, yellow or green when disease is highly, moderately active, or quiescent, respectively. Some systems combine self-reported symptoms with the level of FC in a total inflammation burden score. In fact, recent clinical trials have incorporated FC tests performed by patients at home [45,50,112]. This status is sometimes supplemented with disease activity and QoL graphs [10,11,22]. Depending on the level of alert, simultaneous action plans and email alerts are sent to the participant and providers, who review the information to decide if further management changes are necessary. Altogether, these new models and resources for patients´ self-management represent advances to reach the implementation of telemonitoring in daily practice, but still some legal, ethical, economical and logistic barriers need to be solved (Figure 3). DISCUSSION Telemonitoring is about communication, and the development of faster and wireless systems at a lower cost has supported the use of proactive remote monitoring. As well, the increase in data storage also favoured the incorporation of tele-education in most telemonitoring programs in the IBD setting. However, during the pandemic, e-mail and telephone still represented the main resources used [29,30], while the application of mature telemonitoring programs was the exception. As different enablers encouraged advances of telemonitoring in IBD, many other barriers emerged and hindered its full implementation in daily practice. Pioneering studies evaluated telemonitoring programs previously used in other chronic diseases [21,42,113,114]. Feasibility and patients´ acceptance of these applications was excellent. Yet, they were not able to clearly demonstrate an improvement in QoL, disease activity, and treatment adherence. These systems were based on remote monitoring through computers, they needed to be adapted to the IBD context and required eventual repairs at home, which were time expensive. Telemonitoring subsequently evolved towards the use of web-based systems, which were cheaper and easy to use. Remote monitoring through the web demonstrated its feasibility and excellent patients' acceptance, with an improvement in QoL, disease activity, and disease knowledge [22,115]. In the last few years, telemonitoring prioritized the use of mHealth resources [24,25,116]. Beyond the improvement in clinical outcomes, mHealth telemonitoring associated cost-savings in outpatient visits and hospitalizations [23][24][25][26]. Almost parallel to the mHealth evolution, many PROMs have been validated to self-report disease activity. In line with patient-centered care, empowered patients can use new home-based calprotectin tests, which are accurate enough and useful to rule-out disease activity at low FC values [100,101]. In fact, new home drug monitoring is being developed to measure levels of monoclonal antibodies near the patient [103,104]. Furthermore, the development of mobile devices even enabled the increasing use of wearables to monitor physiological predictors of disease activity and postoperative length of stay [110,111]. These new tools could represent one of the first steps towards ubiquitous Health in IBD, and in a near future machine learning may allow the integration of large data sets in personalized algorithms. Despite the technological and knowledge advances reached, the effect of telemonitoring on health outcomes is not consistent in different populations and health systems [22,24,25,42,44,45,52]. Initially, remote monitoring was mostly restricted to UC patients, while the design of new apps, PROMs and home-based tests allowed to progressively expand its use to a broad range of patients´ profiles. However, telemonitoring has not been demonstrated to improve QoL or clinical/endoscopic remission in the long-term. In addition, biomarkers in IBD are less accurate compared to other chronic diseases such as diabetes mellitus, and the early recognition of complications in IBD still require invasive tests in many cases. On the other hand, although telemedicine has been traditionally considered cost-effective, cost-saving data previously published referred almost exclusively to direct costs [22,27]. The implementation of telemonitoring services represents a short-term high initial cost, not only from a technological point of view, but also by changes in the organization of the IBD units. Thus, decision-makers have had difficulties to support the implementation and investment in telemedicine due to a lack of solid evidence so far. In addition, these decisions become even more complicated in areas where reimbursement is an important factor in the setup of clinical activity. In this regard, recent studies suggested a good cost-effectiveness profile of telemonitoring [62][63][64][65], even after considering the costs of installation and maintenance of platforms, as well as indirect costs. The availability of more powerful and cheaper communication tools turned technical challenges into legal, ethical, economical, and logistic issues [28]. To standardize remote medical practice, in the US the Interstate Medical Licensure Compact was created to increase efficiency in multistate licensing of physicians[66] but such a proposal is lacking in Europe. Besides, only a few examples of full integration of telemonitoring programs into electronic medical records are available to date [60,61]. In this sense, interoperability of systems while maintaining the confidentiality of data cannot be guaranteed in many centres. Moreover, the provision of remote health safely also requires a specific European regulation to protect remote medical practice and to lift some existing legal barriers. Finally, to keep adherence to follow-up, it is essential to adapt telemedicine programs according to patients' and providers´ character-	2023-02-22T16:17:01.962Z	2023-02-21T00:00:00.000	{ "year": 2023, "sha1": "4cf6bcefbf2fe6b77e4ad4cadd58b6fb1b0c170a", "oa_license": "CCBYNC", "oa_url": "https://doi.org/10.3748/wjg.v29.i7.1139", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "acddef21fb12bde26b64092093626db60c92be16", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
41515634	pes2o/s2orc	v3-fos-license	Feasibility Study of Ethanol Production from Food Wastes by Consolidated Continuous Solid-State Fermentation To save the cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation (CCSSF) system composed of a rotating drum reactor, a humidifier and a condenser has been developed. In this research, the feasibility of using this system for production of ethanol from food wastes was carried out. The ethanol conversion of bread crust and rice grain (uncooked rice) as substrates reached up to 100.9% ± 5.1% and 108.0% ± 7.9% (against theoretical yield), respectively. Even for bread crust, a processed starchy material which contained lower carbohydrate content than rice grain, the amount of ethanol obtained in a unit of CCSSF per year was higher due to easy saccharification and fermentation. The salt contained in potato chips directly affected yeast activity resulting to low ethanol conversion (80.7% ± 4.7% against theoretical yield). Introduction Ethanol production from biomass is the focus of much interest worldwide because bioethanol is a renewable fuel contributing to the reduction of the global warming effect and negative environmental impact generated by the worldwide utilization of fossil fuels.Waste biomass such as corn stover, waste wood, and waste foods is very attractive since it is cheap raw material for ethanol production [1].Food waste is a kind of organics solid waste discharged from households, restaurants and food processing factory [2].In Japan, the estimated amount of food waste generated annually is approximately 45.1 million tons (2010).The major conventional recycling method has been to employ food wastes as animal feed and fertilizer yielding approximately 8.6 million tons [3].However, large amounts of wastewater are generated when desalting the food waste for fertilizer production, and animal feeds produced from this material often create hygiene problems for feeding animals [4].The rest of food wastes, approximately 35.7 million tons, are sent to incineration or landfill.Nevertheless, a landfill is not a suitable choice for handling these wastes, since space is limited in Japan and uncontrolled fermentation of organic wastes in landfill causes emission of greenhouse gases, such as methane and carbon dioxide.The incineration of food waste is also impractical because of high water content and the possibility of dioxin generation [5].Therefore, it is imperative to overcome the technological and systematic dilemma of the conventional recycling method for food waste and simultaneously develop an environment-friendly recycling method that can convert food wastes to high value product as ethanol fuel. With an aim to develop a recycling system for food waste, Yan et al. [6] conducted a study to evaluate the critical variables that affect saccharification rate which is the rate limiting step in ethanol production from food wastes.Cekmecelioglu and Uncu [7] also tried to reduce the production cost by improving the pretreatment method of food wastes. Generally, ethanol production utilizes the separate hydrolysis and fermentation (SHF) process, which includes a series of steps such as liquefaction, saccharification and fermentation [8][9][10].A challenging perspective of our previous study was to apply a one-step process called the "consolidated continuous solid-state fermentation (CC-SSF)" system which was composed of a rotating drum reactor, a humidifier and a condenser.The process combines enzymatic hydrolysis, ethanol fermentation and re-covery of ethanol into a single operation.It could save overall fermentation time and the capital investment for reactors.Another advantage of CCSSF system is that the continuous repetitive fermentation allows for the yeast and enzyme to be reused.Furthermore, the CCSSF system uses minimum amount of water which reduces the volume and cost for waste treatment [11]. Typically, food waste is characterized by a high organic content since it contains soluble sugar, starch, lipids, proteins, cellulose and other compounds that make it a source of potential fermentative substrates [12].In this experiment, three kinds of starchy food wastes were studied as model cases 1) bread crust; a processed starchy material that has low carbohydrate content and high amounts of moisture, 2) potato chips; a processed food material with high salt content and, 3) rice grain; nonprocessed starchy material (as a model of off-spec rice grain).Since the CCSSF system uses minimum amounts of water, the concentration of organic compounds in food wastes would be concentrated and might affect the activity of yeast and saccharifying enzyme.In this study, the feasibility of ethanol production using food wastes was investigated in a lab scale reactor.The ethanol production from each food waste per unit of CCSSF was compared.Furthermore, the effect of salt (as NaCl) on yeast and saccharifying enzyme was evaluated. Yeast A commercial dry yeast, Super Camellia (Nisshin, Tokyo) was used. Samples of Food Waste Bread crust, potato chips and rice grain (Japanese rice) used in this study were obtained from the market in Osaka prefecture, Japan.Bread crust was chopped into small pieces (about 5 × 5 mm), while potato chips were ground using ceramic mortar and pestle (particle size  < 2 mm).Rice grain was ground into small particles (particle size  < 355 m) using a blender (wonder blender Model WB-1, Osaka chemical Co. Ltd., Osaka). CCSSF System The system consisted of a rotating drum reactor, a Liebig condenser and a humidifier [11].The temperature of the reactor, condenser and humidifier were controlled at 32˚C, −10˚C and 37˚C, respectively. Ethanol Fermentation The fermentation mixture was composed of 3 g of the commercial dry yeast, 64.5 ml of water, 2645 GAU of Stargen TM 002 (contains Aspergillus kawachi alpha-amylase expressed in Trichoderma reesei and a glucoamylase from Trichoderma reesei, Genencor; one glucoamylase unit (GAU) produces 1 g of reducing sugar calculated as glucose per hour from soluble starch substrate at pH 4.5, 48˚C).During fermentation, the pH of the fermentation mixture was maintained at 4.5 -5.0 by adding 28% ammonium water (Wako Pure Chemical Industries, Ltd., Osaka), and the reactor was rotated at 5 rpm to prevent the sedimentation of fermentation mixture.When the ethanol content in the fermentation mixture reached a set value (40 g•kg-mixture −1 ), the circulation of the headspace gas to the condenser and the humidifier was initiated and ethanol content was maintained within a range of 30 -50 g•kg-mixture −1 by changing the flow rate of the pump manually in accordance with the control protocol reported by Moukamnerd et al. [11].Appropriate amount of the materials was added every 6 or 8 without withdrawing the residue.Since ethanol produced by fermentation acts as an antimicrobial agent, the fermentations were carried out under non-sterilized condition [13]. Analyses For determination of carbohydrate content, 0.5 g of food waste was hydrolyzed with 72% sulfuric acid (Wako Pure Chemical Industries, Ltd., Osaka) and the carbohydrate content was measured by phenol-sulfuric acid method using glucose as standard [14].Dry weight was analyzed by oven-drying the materials at 80˚C for 24 h.The ethanol and glucose contents were determined using a biosensor (Biosensor BF5, Oji Scientific Instruments Co., Ltd., Hyogo).The salt (NaCl) content was measured using a salt meter (Pocket PAL-ES1, Atago Co., Ltd., Tokyo).The specific rate of ethanol production from glucose was determined as follows.The fermentation mixture (about 0.2 g) harvested from the reactor was resuspended in 5 ml of YPD medium (10 g•L −1 yeast extract, 20 g•L −1 polypeptone and 30 g•L −1 glucose).The suspension was transferred to a 15-ml plastic tube equipped with a check valve followed by degassing by an aspirator.The tube was incubated for 80 min at 32˚C and the ethanol concentration in the suspension was measured every 20 min.Fermentative activity was expressed as the specific rate of ethanol production from glucose (g•g-drycell −1 •h −1 ). Raw Materials In this study, three kinds of food waste materials namely bread crust, potato chips and rice grain were used.Table 1 summarizes the result of CCSSF for the three substrates used.Rice grain was mainly consisting of carbohydrate (0.76 ± 0.05 g•g-raw-material −1 ) whereas bread a Average values and standard deviation of three independent fermentations are shown (n = 3); b Against theoretical yield (0.57 g-ethanol g-carbohydrate −1 ). crust and potato chips contain protein, fat and salt. Repetitive Fermentation Since one of the advantages of the CCSSF system is that the running cost for saccharifying enzymes and yeast can be reduced by repetitive addition of raw material, repetitive fermentation was conducted for each food waste. Ethanol content was maintained in the range of 30 -50 g•kg-mixture −1 because the fermentative activity of yeast decreased markedly at an ethanol content above 50 g•kg-mixture −1 whereas the concentration of recovered ethanol increased.Figure 1 shows representative time courses of two or more CCSSF for bread crust, potato chips and rice grain.The arrows indicate the timing of addition of raw material which was different depending on saccharification rate of each raw material. For the CCSSF of bread crust (Figure 1(a)), 40 gram of bread crust was added to the reactor every 6 h for 5 times, and the fermentation needed to stop after the fifth batch since the reactor was filled with residue.A total of 200 g of bread crust produced 54 g of ethanol within 30 h.Total ethanol production corresponded to 100.9% ± 5.1% of ethanol conversion (against theoretical yield).The ethanol was continuously recovered and condenses to a clear solution with 37% (w/v) of ethanol concentration.grain was added into the reactor separately every 6 h.Within 51 h, 60 g of ethanol was produced and the ethanol conversion was 108.0% ± 7.9%.The concentration of recovered ethanol reached up to 40% (w/v). In the CCSSF of potato chips, glucose started to accumulate after three batches, whereas no glucose accumulation was observed in both case of bread crust and rice grain.The residues from fermentation of bread crust, potato chips and rice grain were 167, 148 and 67 g with moisture of 76%, 68% and 64%, respectively (Table 1). The time-course changes in the fermentative activity of yeast during fermentation were investigated and shown in Figure 2(a).The fermentative activity in the CCSSF of potato chips was drastically reduced after three batches, while the reduction was more gradual using rice grain and increased in the case of bread crust.During fermentation, the amount of salt (as NaCl) increased in case of bread crust and potato chips as shown in Figure 2(b).When NaCl concentration in the fermentation mixture exceeded 20 g•L −1 , the fermentative activeity of yeast decreased significantly, resulting in accumulation glucose (Figure 1(b), closed triangles). Effect of Salt Content The effects of salt on yeast and saccharification enzyme was evaluated since salt is always contained in food wastes.The effect of salt on ethanol fermentation was assessed by comparing the glucose and ethanol production within 24 h of incubation (Figure 3).No significant effect of salt on saccharifying enzyme was observed (Stargen 002), however, it directly affected the yeast activity.The ethanol production rate decreased when salt content was increased. CCSSF Conditions Yeast is more tolerant to low-moisture conditions compared with other microorganisms.The commercial yeast used in the present study maintains a sufficient fermentative activity for CCSSF even at a moisture content of 40% (Data not shown).Although it is possible to perform CCSSF at moisture content less than 40%, it would become difficult to mix the fermentation mixture homogeneously, resulting in a non-homogeneous ethanol content that leads to further decrease in fermentative activity.Therefore, CCSSF should be performed at a moisture content of approximately 50% for homogeneous mixing, whereas the present CCSSF was conducted at 60% moisture content to ensure reproducible sampling. Influences of the Composition of Raw Material on Fermentation There was no glucose accumulation observed even after the third addition of bread crust and rice grain (Figure 1), indicating that the rate of glucose consumption by yeast cells could be maintained higher than the rate of glucose production by the saccharifying enzymes.However, the glucose level was found to increase in the case of potato chips after three batches.This increase would be due to a reduction in the activity of yeast.To clarify the reason for the accumulation of glucose, the effects of salt (NaCl) concentration on the activity of the yeast and the enzyme were investigated.As shown in Figure 3, the fermentative activity decreased with an increase in the NaCl concentration, whereas the enzyme activity was not affected.Previous studies reported that gradual increase of NaCl in the growth medium for yeasts can cause a cell growth arrest depending on the NaCl concentration [4, [15][16][17][18].In addition, the reduction of water activity, even with salt concentrations as low as 5 g•L −1 can still affect cell growth [19].When CCSSF was carried out with minimum moisture content, the water activity was low, and therefore, the effect of salt was more pronounced than in liquid fermentation.These imply that although concentration of NaCl in CCSSF system is low, it could inhibit sugar uptake causing the period of ethanol fermentation to be prolonged.Therefore, the results suggest that when the fermentation mixture accumulated more than a certain level of NaCl that would affect yeast activity, and the reaction need to be stopped.On the other hand, a salt tolerant yeast strain should be utilized in order for the system to achieve higher efficiency of ethanol fermentation from food wastes. The Amount of Ethanol Produced by a Unit of CCSSF System For estimation of the profit for each food waste, it is necessary to know the amount of ethanol that can be produced by one unit of CCSSF system in a year.In a CCSSF system, the amount of ethanol [20], E (kL•year −1 ), that can be produced by one unit of CCSSF system in a year is given as: Where Mw g and Mw e are the molecular weights of glucose unit in carbohydrate and ethanol corresponding to 162 g-carbohydrate•mol −1 and 46 g-ethanol•mol −1 , respectively;  e is the specific gravity of ethanol equivalent to 0.79 kg•L −1 ; M is capacity of CCSSF system (kg•batch −1 ); D is operation period (day•year −1 ); d is fermentation time (day•batch −1 ); X is the carbohydrate content of the material (g-carbohydrate•g-raw-material −1 ); and Y is ethanol yield (against theoretical yield). To calculate the amount of ethanol produced by a unit of CCSSF system, the actual size of CCSSF system is assumed to be 3 m in diameter and 6 m in height.This diameter is the maximum width that can be transported by land in Japan.Based on the given dimensions, the inner volume of the reactor will be about 40 m 3 .The capacity of the CCSSF system, M, was temporarily set at 5 × 10 3 kg•batch −1 .The operation period per year, D, was assumed to be 300 days in a year considering the maintenance period.The time required for a batch of fermentation, d, was calculated based on the experimental data.Similarly, the carbohydrate content, X and the ethanol yield, Y, were estimated from experimental data (Table 2). The amount of ethanol produced by a unit of CCSSF in a year, E, from bread crust, potato chips and rice grain were calculated to be 527, 345 and 341 kL•year −1 , respectively.Even bread crust and potato chips were contained lower carbohydrate levels than rice grain, higher ethanol production by a unit of CCSSF in a year could be obtained.Jain et al. [21] reported that cooking rice af- accharification rate.To increase the ethanol production Conclusion ction from food wastes was success- Acknowledgements y Low Carbon Technology s and reduce the operation time using rice grain, a pretreatment process may be necessary. The ethanol produ fully carried out using the CCSSF system.In this system, ethanol was recovered continuously from the reactor and the ethanol conversions of bread crust, potato chips and rice grain were 100.9% ± 5.1%, 80.7% ± 4.7% and 108% ± 7.9%, respectively.It was found that salt contained in food wastes had a direct negative effect on yeast activity.In order to improve the system, salt tolerant yeast should be applied to the system.The results of this study indicate that the CCSSF system can be used to recycle food wastes. Figure 2 . Figure 2. Fermentative activity of yeast and salt concentration during fermentation of bread crust (closed circle), potato chips (open circle) and rice grain (closed triangles). Figure 3 . Figure 3.Effect of NaCl on yeast and saccharifying enzyme.Closed circle, glucose production rate; open circle, ethanol production rate. Table 2 . Estimated values for the parameters. fects the amylose content in starch by influencing the	2017-08-15T16:23:33.660Z	2013-06-25T00:00:00.000	{ "year": 2013, "sha1": "cb0cbb3a7b46ebe85e8d20e331aef5f9f26cae9e", "oa_license": "CCBY", "oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=33628", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "cb0cbb3a7b46ebe85e8d20e331aef5f9f26cae9e", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Engineering", "Environmental Science" ], "extfieldsofstudy": [ "Chemistry" ] }
264993514	pes2o/s2orc	v3-fos-license	Evaluation of Community Restaurants Linked to Government Food and Nutrition Safety Programs: A Scope Review Community restaurants linked to government food and nutritional security programs are establishments created to offer meals to the population in socially vulnerable situations. The objective was to identify the methods, approaches, criteria, and indicators used to evaluate restaurants linked to government food and nutrition security programs. A scoping review based on the Joanna Briggs Institute’s methodology and the international guide’s recommendations of preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews was conducted. Medline databases via PubMed, Lilacs, Scopus, Cochrane, Web of Science, and ScienceDirect were used. Primary observational studies, systematic reviews and meta-analyses, ethnographies, documentary studies, and case studies were included, with a quantitative, qualitative, and/or mixed approach. A total of 2498 studies were identified. After taking out 180 duplicated articles, another 2202 articles were excluded by the title. Among the 71 studies selected for complete reading, 10 did not correlate with the research objective, and 12 were included after analyzing the references, totaling 73 included studies. In this review, evaluative approaches were mapped and systematized on the menu, food consumption, food health, food security and/or insecurity, nutritional education, and human right to adequate food; users’ profile and health, implantation, history, perceptions, senses, and meanings; handlers/workers; hygienic–sanitary quality; evaluation and monitoring; physical–functional planning, and rest–intake. The presented data provide elements that can be adapted in future evaluations and describe the panorama of academic production in this area. Introduction Community restaurants (CRs) linked to government food and nutrition security programs are world-renowned food and nutrition security establishments whose objective is the production and distribution of free or low-price meals for people in situations of social vulnerability in order to increase access to food and guarantee human rights to adequate food and fight hunger [1][2][3][4][5][6][7][8]. Several CR experiences can be found in countries such as Peru (Comedores Populares), Chile (Servicios de restaurantes populares), Spain (Comedores Sociales), Argentina (Comedores comunitarios), Canada (Community/collective kitchens), and Australia (Community Kitchens).Like community kitchens in Brazil, soup kitchens are located in countries such as Mexico, Peru, Uruguay, the United States of America, and Colombia [9]. The evaluation and monitoring of food and nutrition security are essential axes for programs and public policies to promote access to food.Through assessments, tools can be offered for improvement, management, and strengthening, especially regarding political and economic instability and threats to social rights.The evaluation must be a continuous and permanent object, aiming to reorient actions and subsidize public agents in decision making, interventions, and the implementation of public policies [10,11]. In this sense, there is an increase in investments by public agents in social program evaluations.However, evaluating food promotion programs is challenging due to the multiplicity of actions, multidisciplinary nature, heterogeneity of local and regional problems, and cultural and socioeconomic diversity [12].Therefore, evaluations must be developed with knowledge and practices that may be influenced by different approaches, scientific disciplines, and theoretical-methodological traditions, which can reveal relevant evidence for the program [13,14]. In addition, considering that health service managers need to monitor programs to obtain information on the daily decision-making process and that population surveys are carried out but not with the desired frequency, it is necessary to develop and improve approaches, techniques, and methods for evaluation based on data produced by health information systems [15]. Thus, systematizing findings from CR assessments aims to improve practices and policies so that researchers can identify possible gaps and understand how such researchers have conducted studies in this area.In this way, the scoping review can help produce new evidence when the existing scientific production is recent and/or incipient and examine how research is being developed in consolidated areas, which can generate knowledge with the potential to guide decisions and actions in public policies. The present study aims to identify the methods, approaches, criteria, and indicators used to evaluate community restaurants linked to government food and nutrition security programs. Protocol and Registration This scoping review study aims to synthesize research evidence to map the literature on a previously determined subject (nature, characteristics, and volume), identifying knowledge gaps [16].This review had its study protocol registered in the Open Science Framework on 23 December 2022 (https://osf.io/eptfv,accessed on December 23,2022).The protocol was developed based on the recommendations of the international guide's preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews (PRISMA-ScR) [17] and the Joanna Briggs Institute (JBI) method [18]. The structure consists of six main consecutive steps: (I) identification of the question and research objective; (II) identification of relevant studies that would enable the breadth and scope of the review's purposes; (III) study selection, according to predefined criteria; (IV) data mapping; (V) summarization of results, through a qualitative thematic analysis about the objective and question; (VI) presentation of results, identifying implications for policy, practice, or research.The acronym PCC-Population, Concept, and Context-was adopted with the following question: "What is the scientific evidence produced about the evaluation approaches carried out in community restaurants linked to governmental programs of Food and Nutrition Security?".Therefore, they were defined based on the guiding question: population-users (target audience), professionals, and managers involved in program evaluations; concept-methods, approaches, criteria, and indicators used and evaluation results; and context-community restaurants linked to government food and nutritional security programs. Inclusion Criteria This study included observational studies (cross-sectional, case-control, cohort, and ecological studies), documentary studies, and case studies, with a quantitative, qualitative, and/or mixed approach.There was no restriction regarding publication date, language, geographic region, or country.Also included were studies that addressed the methods, criteria, and indicators used in the evaluations of CRs, carried out with users of such restaurants, professionals, and/or managers who work in these food services where eligible. CRs linked to government food and nutritional security programs were considered to be food services financed by the municipal, state, and federal government or with public funding such as community restaurants, comedores populares, comedores sociais, comedores comunitários, budget restaurants, economy restaurants, popular restaurant, community restaurants, government-subsidized kitchens, social kitchens, community kitchens, community food programs, soup kitchens, and self-service.These are programs that offer food to the population with social vulnerability. Exclusion Criteria Studies and publications whose research context was not exclusively the evaluation of CRs, reviews, editorials, comments, perspectives, conference abstracts, reports, opinion polls, master's theses, doctoral theses, or systematic or systematized reviews were excluded.Studies conducted in restaurants located at universities or private companies were also excluded. Information Sources and Search Strategy The last searches were conducted in June 2023 in the databases Medical Literature and Retrieval System online (Medline/via PubMed), Latin American and Caribbean Health Sciences Literature (Lilacs/via Virtual Health Library), Scopus, Cochrane, Web of Science, and ScienceDirect. The descriptors and their synonyms were identified in the Medical Subject Headings (MeSH) and Health Sciences Descriptors (DeCS): "community restaurants", "comedores populares", "comedores sociais", "comedores comunitários", "budget restaurants", "economy restaurants", "popular restaurant", "community restaurants", "government-subsidized kitchens", "social kitchens", "community kitchens", "community food programs", and "soup kitchens".These were associated with the terms referring to the evaluation, that is, "evaluate", "assess", and "assessment".Along with the descriptors, the Boolean operators AND/OR were used to compose the search strategies in the databases.The strategy was specifically adapted to each database.The results were exported to the online reference manager EndNote ® , where duplicate references were excluded.After this step, the other documents were exported to Rayyan ® , where another evaluation of the duplicates was carried out, and the selection steps of phases I and II were carried out. Data Selection and Extraction Phase I was performed by two reviewers independently.Eligibility criteria were applied for selection by titles and abstracts.Then, in phase II, also carried out by two independent reviewers, the full texts were analyzed, again applying the adopted eligibility criteria.Also, the authors evaluated the list of references from the included studies.In phase II, the exclusions were justified.At all stages, disagreements were resolved in a consensus meeting.Contacts were made with experts to identify whether any study was left out of the search, as described in Figure 1. The review results are presented in a descriptive format, using tables to summarize data from the studies, following the JBI recommendations [18]. Summary of Results The results were synthesized by qualitative analysis, with information presented in narrative, tabular, and/or graphic form.The studies were evaluated by identifying the used methodologies and the prevalence of the methods, approaches, criteria, and indicators reported from the proportions based on the number of included studies. In addition, the information was synthesized and subdivided into ten groups: user profile; users' health; handlers/workers; menu, food consumption and food health; hygienicsanitary quality; assessment of (in) food and nutrition security, nutrition education, and the human right to adequate food; implementation, history, perceptions, senses, and meanings; physical-functional planning; rest intake; evaluation and monitoring.The review results are presented in a descriptive format, using tables to summarize data from the studies, following the JBI recommendations [18]. Summary of Results The results were synthesized by qualitative analysis, with information presented in narrative, tabular, and/or graphic form.The studies were evaluated by identifying the used methodologies and the prevalence of the methods, approaches, criteria, and indicators reported from the proportions based on the number of included studies. In addition, the information was synthesized and subdivided into ten groups: user profile; users' health; handlers/workers; menu, food consumption and food health; hy- Results We found 2498 studies, of which 180 duplicates were excluded.After reading the titles, another 2202 articles were excluded.Of the 116 studies selected for abstract reading, 45 were excluded for not meeting the eligibility criteria.Among the 71 full-text reading studies, 10 did not correlate with the research objective, and 12 were included after reference analysis, totaling 73 studies in the scoping review.Figure 1 illustrates the selection process. Of the included studies, 57 had a quantitative approach, 14 had a qualitative approach, and 2 were mixed (quantitative/qualitative).Of the 73 studies, the main study design was cross-sectional (n = 60; 82.2%), followed by case studies (n = 10; 13.7%) ( Most of the studies were conducted in Brazil (52), 9 in the United States, 5 in Peru, 2 in Mexico, 2 in Canada, 2 in Australia, and 1 in Argentina (Table 2).Adapted from the JBI Model source of evidence details, characteristics, and result extraction instrument [18]. Regarding the type of evaluation, 20 studies evaluated the menu, food consumption, and food health; 17 Food and nutritional security, nutrition education, and the human right to adequate food; 13 the user's profile; 12 the health of users; 12 the implantation, history, perceptions, senses, and meanings; 8 handlers/workers; 4 evaluation and monitoring; 3 hygienic-sanitary quality; 3 physical-functional planning; and 2 intake/rest/consumption (Table 3).A study was allocated to more than one category when it performed more than one type of evaluation.The description of the methods, criteria, and indicators used in evaluating CRs are presented in Table 4. Brazilian Food Insecurity Scale (EBIA) 5.9 Questionnaire about new knowledge learned under the Program (Likert scales) 5.9 Questionnaire about behavior since they use a community kitchen (Likert scales) 5.9 Questionnaire of current practices, barriers, and ideas to improve the nutrition of homeless people 5.9 Nutritional Knowledge Scale, Central Food Safety Module (CFSM) 5.9 Normative evaluation 5.9 Assessment matrix of two community restaurants in Brazil 5.9 Questionnaire on the understanding of the Human Right to Adequate Food and the Community Restaurant Program Discussion Community restaurants have been evaluated since the 1990s, initially seeking to describe the population that accessed these facilities, focusing on sociodemographic profiles and the reasons that led these users to frequent such spaces.After this initial period, other research aimed to evaluate the quality of the food served and its impact on the health of users and the situation of food insecurity.Studies have focused on evaluating community restaurant programs, shifting from looking at users to focusing on the program concerning achieving its objectives for society. The main approach in the evaluations of the CR was the quantitative one, which is defined by the work carried out with variables expressed in the form of numerical data, which rigid resources and statistical techniques are used to classify and analyze, such as the percentage, mean, standard deviation, correlation coefficient, and regressions, among others.As they express greater precision and reliability, quantitative studies are more suitable for planning group actions, as their results are likely to be generalized, primarily when the surveyed samples faithfully represent the population from which they were taken.On the other hand, the qualitative approach seeks to understand specific complex phenomena of a social and cultural nature through descriptions, interpretations, and comparisons without considering their numerical aspects (mathematical and statistical rules) [85,86]. For this dichotomy to be overcome, some researchers seek mixed methods.The choice of quantitative, qualitative, or mixed studies is mainly based on the research questions and chosen variables.However, quantitative studies have been the most chosen option, not only to answer such research questions but mainly because such studies are used for the direct or indirect evaluation of food and nutritional security programs.Subsidies are offered when considering the maintenance of programs with indicators such as cost/benefits or compliance with the user profile to be covered by policies to overcome the population's social vulnerability. As for the study design, the cross-sectional design is the most predominant.Compared to other designs, this type of study is easily carried out, fast, economical, and very useful in public health.In addition, it offers a better cost/benefit ratio for planning and evaluating public programs, as already mentioned.This study design analyzes well-defined populations, with its fundamental characteristic being the measurement at a single moment [85,86].However, such designs have limitations.In general, it is impossible to determine causality to ensure that confounding factors will be equally distributed between the groups, and there is no way to guarantee that they are not compromised by prevalence bias (cured and deceased people are excluded).Nevertheless, the sample's internal groups may have very different sizes, resulting in a loss of statistical efficiency.Still, its strengths outweigh its limitations [87]. Another type of study that stood out was the case studies, characterized as studies of a well-defined entity such as a program, an institution, an educational system, a person, some people, or a social unit.This study design seeks to understand in depth how and why a particular situation, which is supposed to be unique in many aspects, leads to such behavior.Therefore, it seeks to discover what is more important and characteristic about this object from the participants' point of view.It always intends to analyze the case objectively and pragmatically or present a global perspective of the event (case) [86,88]. Regarding the origin of the studies, Brazil, followed by the United States, gained greater prominence.This arises from the fact that Brazil currently has a well-structured program that offers meals, preferably to the low-income population, as part of the food assistance strategies integrated into the Brazilian federal government s network of social inclusion and hunger-fighting policies [7,26,65,67].The United States, due to its federalism model and regarding the preparation of the meals and the financing of these initiatives, presents state or municipal initiatives with the participation of the local government and volunteers.These initiatives are vital to the food security of the North American low-income community [5,89]. Observing the subdivisions adopted by this study, the evaluation of the served menu, food consumption, and dietary health were the most studied (20 studies).This fact comes from the researchers' concern for evaluating whether these restaurants, even offering the meal for free or at subsidized prices (USD 0.20 to 1.02), produce a quality meal that promotes users' health.To this end, they used dietary surveys through food history, which met the inclusion criterion of a frequency of meals no less than three times a week during the last six months.Obtaining data related to the users' dietary information included the technique of retrospective dietary assessment (35%), application of food frequency (25%), the 24 h recall (30%), and/or direct weighing of food [90][91][92][93][94]. Retrospective methods included the 24 h recall, food frequency questionnaires, and food history.When applying the recording method, the food and drinks consumed in the last 24 h were quantified, describing the type of food and drink, size and/or volume, preparation method, and fractionation.The food frequency consisted of applying a list of foods in which the interviewee indicated how often each food was consumed in a given period.The direct weighing of food consisted of a prospective method carried out by weighing the food discounting the inedible parts and leftovers.The various methods for the quantitative assessment of food consumption provide information not only on the meals consumed in the CR but also on how they contribute to the user's day and the coverage of nutritional needs.According to the Council of the Institute of Medicine, Food, and Nutrition, three consecutive days of assessment of food consumption is representative of an individual's diet.It constitutes the gold standard for obtaining data on the food consumption of individuals and/or populations [93]. For the food consumption investigation, part of the questionnaire for surveillance risk and protective factors for chronic diseases by telephone survey (VIGITEL) was used.VIGITEL is a Brazilian survey carried out by the Ministry of Health, created to monitor risk and protective factors for chronic non-communicable diseases in all capitals of the Brazilian states and in the federal district.Data collection is carried out through telephone calls to interviewees, and the questionnaire is composed of sociodemographic and health variables, providing information on the population's habits in relation to food, physical activity, smoking and the consumption of alcoholic beverages, and the existence of diseases such as diabetes, hypertension, and depression, among others [95], in addition to the offer of the total caloric value, the caloric density of the meal [40,96], regional food offer, the number of calories provided by dietary liquid protein [97], and macro and micronutrients [98,99], to verify if the daily supply of calories, protein, and nutrients is adequate.Although the methods used to determine consumption are highly different, the choice for a certain method was undoubtedly due to the cost/benefit ratio, not the precision.VIGITEL, for example, is an investigation carried out through telephone calls and, therefore, subject to biases inherent to the desirability and memory of individuals. The nutritional composition of the meal (45% of the studies) was evaluated using technical preparation files [100], direct food weight, and food composition tables [101].Technical preparation files consist of an operational management support instrument, which aims to survey costs, order preparation, and calculate the nutritional value of the preparation to be carried out.They help support menu planning.Meal suitability values were compared to requirements specified by the Food and Drug Administration standards [101], Centers for Disease Control and Prevention [96], Food and Agriculture Organization of the United Nations [102], and National Health Surveillance Agency [97].The choice of these strategies that determine and evaluate consumption and adequacy were adopted, possibly because they can be replicated by subsequent studies and compared with national or international standards. These studies aimed to assess the adequate supply of nutrients by restaurants.The studies indicated that meals served at the CR presented an energy density above the recommendations.However, these values can be justified because these restaurants have consumers that usually just have one meal a day, and this meal is consumed at the CR.Regarding the nutritional composition of the meals, the majority attended the nutritional needs for the lunch period, the most served meal at the CR.These studies aimed to assess the adequate supply of nutrients by restaurants.It is important to ensure the adequacy of the nutritional composition of menus, thus creating conditions for users to have a nutritionally healthy meal. For the assessment of food safety/insecurity, in Brazil, there is the Brazilian Food Insecurity Scale [103].In the United States, the Nutritional Knowledge Scale, Central Food Safety Module was used [104], and in Mexico, the Latin American Food Security Scale [105].They all directly assess one of the dimensions of food and nutritional security in a population through the perception and experience of hunger.Hunger perception scales are direct indicators for assessing food insecurity but do not measure the nutritional dimension.Using the score obtained, the scales classify the assessed households into food security, mild food insecurity, moderate food insecurity, or severe food insecurity.They have become an assessment standard, as they can express food access and provide high reliability, reflecting the experience of life with food insecurity and hunger. Nutrition education was developed through workshops, recreational activities, and focus groups to promote education for citizenship and create conditions for empowering the population regarding food and nutritional security issues and the right to food.This technique can be improved and transcribed to the local reality and culture.This allows for some instruments to be adjusted and validated in different countries and cultures, conducting studies resulting from applying such instruments, subject to comparison.In addition, a legal and institutional approach was used to evaluate the human right to adequate food, analyzing the limits and possibilities of the advances and preservation of the guarantee of the human right to adequate food [106].These studies aimed at developing active citizenship among restaurant users who participate in social programs, in which society mobilizes and can demand from public authorities the fulfillment of the right to adequate food from a legal point of view. Profile studies of restaurant users (13 studies) aimed to characterize frequent users to verify whether they serve the population with higher rates of social vulnerabilities.The main sociodemographic indicators used in these assessments were gender, age group, marital status, skin color, education, head of household, family composition, place of residence, housing condition, type of housing material, profession, per capita income, social class, formal contract, place of work, possession of goods, means of transportation used, motivation for having the meal, days on which meals are served, place where meals are served on weekends, beneficiaries of social programs, drug or alcoholic beverage users, practice of physical activity, and the presence of chronic diseases.In Brazil, most studies used the Brazilian Institute of Public Opinion instrument as their basis, which is a pioneering study on the evaluation of user profiles [107].Carrying out this type of analysis enables a more detailed diagnosis of each region's socioeconomic inequalities and specificities, thus facilitating the planning and execution of corrective actions consistent with the local reality.Majorly, CR users are low-income, non-white race, and have little educational background.Therefore, CRs follow their goals to offer meals to the most vulnerable population.Food access to this population is essential since low-income people eat less or no food, and frequently, do not have resources to buy it. In the evaluations of the users' health (12 studies), the studies aimed at evaluating whether the food served by the CR presented a connection with users' diseases such as metabolic syndrome, cardiovascular diseases, chronic non-transmissible diseases, anemia, total cholesterol, triglycerides, high-density lipoproteins (HDL), low-density lipoprotein (LDL), and very low high-density lipoprotein (VLDL) [96,[108][109][110] using methods already scientifically established by the WHO.Dyslipidemia, anemia, and blood glucose were measured in a fasting blood sample, and to determine glucose, total cholesterol, and triglycerides, enzymatic colorimetric methods were used.HDL was determined using the low-and very-low-density lipoprotein precipitation method using the cholesterol oxidase/peroxidase enzyme system with colorimetry.Blood pressure was measured twice with a properly calibrated aneroid sphygmomanometer.The concern in evaluating users' health arises from the low cost of the food, making it possible to have concerns about the quality of the final product served to the consumer.The recommendations of the World Health Organization and the International Diabetes Federation were used to assess nutritional status (41.6% of the studies).The studies evaluated the user's health to verify their current state of health and whether the food served by the restaurants had promoted the improvement of their health or the onset of diseases [98,108,110,111].The CR's primary objective is to serve low-cost food that is nutritionally healthy and does not harm the health of its guests. Body mass index (BMI) and waist and abdominal circumference were also evaluated and correlated with the onset of health problems.To obtain the body mass index, the ratio of weight in kilograms to the square of height in meters (kg/m 2 ) was calculated, while circumference measurements were obtained using a measuring tape.This type of assessment seeks to recognize the users' dietary needs so that it is possible to intervene appropriately for health maintenance or recovery.The assessment of nutritional status through BMI is a good indicator of the accumulation of adipose tissue due to excess energy, and it is equally reliable for both genders and different ages.Other methods that could be used in this type of evaluation are densitometry and bioimpedance, which are quick, as they are performed in up to 12 min and do not require preparation.However, among the disadvantages are the high cost, the use of radiation during the evaluation, and the difficulty in transporting the equipment to different locations [112]. To evaluate the implementation, history, perceptions, senses, and meanings, the authors resorted to a sociohistorical analysis (41.6% of the 12 studies) [113,114], bibliographic research, documentary, and field research, and direct observation [115,116].They created focus groups with managers, handlers, and users, seeking to understand each person s life story and their perceptions about these food and nutrition establishments, which aim to provide access to food and fight hunger.However, this investigation method may not be as efficient in terms of the coverage of a specific topic compared to individual interviews because there is little depth on the subject.There is a possibility that members may not honestly express their personal opinions, especially if their ideas differ from those of other members [117].According to Sordini [118], for the users of these spaces, the main perception is that the practices, from preparing food to eating the meals served by the CR, are based on love, trust, and hope as a possibility of meeting with the others, and it implies a standard view of possible, desirable, and shared horizons of action. Occupational psychosocial characteristics were studied using the Job Content Questionnaire (JCQ), a validated and self-administered instrument designed to measure workers' social and psychological characteristics.It is often used for the analysis of micro-level job characteristics, such as assessing the relative risks of individual exposures to different work settings, to predict the development of work-related illnesses, psychological distress, coronary heart disease, musculoskeletal diseases, and reproductive disorders [121].The food and nutritional security level of workers/handlers was also evaluated using the food insecurity scales [103][104][105].A questionnaire based on current Brazilian legislation on good handling practices (National Health Surveillance Agency) was applied to assess Brazilian food handlers' knowledge and self-reported practice in two studies [123,125].These studies served as a basis for revealing the characteristics of workers whose well-being is reflected in their health and their daily work practices. Within the scope of the evaluation and monitoring studies, the construction of the proposal of the theoretical-logical model of Brazilian community restaurants was carried out [55] as a representation of the program and its movements and relationships, translating into theoretical and practical propositions for the evaluated object.A proposal for an assessment matrix was also developed [56] containing the restaurants' dimensions, sub-dimensions, and evaluation indicators with their respective justifications.To build the theoretical-logical model and matrix, an in-depth literature review and consensus workshops were carried out, totaling 12 h, using the traditional committee technique.The matrix was evaluated by experts external to the research group with experience in CR implementation and management.In addition, the criteria of efficiency and effectiveness were used to create a value judgment of CRs [14,126].To evaluate the effectiveness of the CR program, the proportion of coverage of the "target audience," defined within the scope of the CR program, was estimated.Access to food was considered adequate effectiveness when the CR, within their possibilities, served meals to 50% to 70% of users considered as the program's "target audience".Data envelopment analysis (DEA) was used as the most appropriate methodology for the efficiency of public spending.For this purpose, the software MaxDEA version 12.0 for data envelopment analysis was used.For the evaluation and monitoring of public policies and/or instruments, the authors resorted to evaluation techniques according to the criteria of effectiveness and efficiency, which are guidelines for the planning and improvement of programs in public management [113,127,128]. The assessment of the hygienic-sanitary quality was verified using the sanitary inspection script based on the Resolution RDC nº 216/2004 of the National Health Surveillance Agency [123], from the checklist of Manual de buenas practicas de manipulación de alimentos para restaurantes y servicios afines [129] and the Official Mexican Standard NOM-093-SSA1-1994 de Practicas de hijiene y sanidad en la preparación de alimentos que se ofrecen en establecimientos Àjos [130].They all aim to help traders and handlers prepare, store, and sell food appropriately, hygienically, and safely [123].The evaluations were aimed at verifying the quality of the hygienic-sanitary conditions and identifying non-conformities that could interfere with the quality of the served meals, calculating the adequacy percentage of the hygienic-sanitary conditions [131,132].These studies used standards and norms from the national legislation of each country.The main evaluated items were buildings, installations, furniture, and fixtures; the hygiene of facilities, equipment, furniture, and accessories; the integrated control of vectors and urban pests; water supply; waste management; handlers; raw materials, ingredients, and packaging; food preparation; the storage and transport of prepared foods; exposure to the consumption of prepared foods; documentation and records; standard operating procedures; and, finally, responsibility.For application in other countries, it is necessary to follow the guidelines and checklists approved by the national health surveillance agencies.In this sense, such instruments offer the possibility of determining which conditions are considered ideal for producing meals and which points require correction to obtain the ideal conditions.Furthermore, it is possible to analyze the conditions of restaurants in different countries after the required adaptations. The physical-functional planning studies aimed to assist in the implementation of CRs.To this end, a list describing the equipment, utensils, and consumables by sector and their respective quantities was drawn up.It also describes the average cost of meals and base menu planning for five days (Monday to Friday).The analysis of the quantity of human resources necessary for the establishment's operation was used as a basis for the calculations, using several parameters described by Teixeira et al. (2007) [83].The organization chart of the staff was prepared, describing the positions and functions.Furthermore, when evaluating the installation of CRs, aspects related to location, zoning, sectors, and environment were observed based on the roadmap for the implementation of community restaurants published by the Ministry of Social Development and Fight against Hunger [133].The studies in this subdivision are Brazilian and followed the federal government's rules.The other studies did not assess physical-functional planning. To determine the intake (consumption) of meals, the studies used the proportion between the food returned by users and the quantity of food distributed.By performing the calculation, the formula proposed by Vaz (2006) [84] was used to obtain the food waste index and was adopted in the evaluation of CRs.Thus, it was considered as a synonym of poor quality when the indexes were above the recommended percentages, which can be avoided through planning.This type of evaluation's main objective is to promote diners' awareness and minimize food waste.It should be noted that only two studies assessed this issue, one in Brazil and one in the United States.In both cases, the assessment of food losses was carried out using the same formula.It is crucial to raise awareness of the need to reduce waste as one of the strategies for the sustainability of restaurants.Food waste is one of the sustainability assessment pillars in restaurants [134].In this view, sustainable actions must be developed within the most diverse stages involving the meal production process, thus contributing to increasing the quality of the service provided and sustainability [135]. Systematizing and disseminating findings regarding CRs assessments can contribute to identifying the most prevalent state practices and policies, research gaps that may indicate which methods and tools should be created and consolidated, and how research in this area is being conducted.In this way, the scoping review can help the reviewer examine emerging evidence when the existing scientific production is recent and/or incipient and examine how research is being conducted in already consolidated areas that can generate knowledge with the potential to guide decisions and actions in public policy. This study presents limitations inherent to systematic reviews, or not, such as, in some cases, not all studies are included in the main databases, and it does not propose, in the case of the scoping review, to evaluate the quality of the included studies.At the same time, materials and research that are not published in scientific journals and databases, such as government documents, are not included and could have provided more information about CRs. The search used eight databases, allowing a greater number of studies in the evaluated area to be found.Accordingly, the review includes a variety of studies published since the 1990s on evaluations carried out on CRs, which allowed the identification of different evaluative approaches using quantitative, qualitative, and mixed methods, in addition to presenting remarkable experiences in several countries. Conclusions Seventy-three studies were published since the 1990s in different countries, mainly in Brazil.The evaluative approaches dealt with the menu, food consumption, food health, food security and/or insecurity, nutrition education, the human right to adequate food; user profile and health; implantation, history, perceptions, senses, and meanings; handlers/workers; hygienic-sanitary quality; evaluation and monitoring; physical-functional planning; and rest intake.The results increase the comprehension of evaluation methods performed at the CR.They provide details on methods, approaches, criteria, and indicators that can be used and/or adapted in future evaluations.The results also describe the area's academic production panorama. In this scenario, progress on the methods, criteria, and indicators used in CRs is necessary to better investigate the nutritional and food security framework.The evaluations performed in these establishments must be strategies inherent to the programs, being fundamental for their qualification and goal achievement. Furthermore, the scoping review is appropriate to examine studies for decision making in the theoretical-methodological field, from mapping theories to methodologies that should inform researchers.Systematizing and disseminating findings fulfill the objective of contributing to practices and policies. Table 1 . Epidemiological approach and types of studies included in the scoping review; Brazil, 2023. Table 2 . Studies included in the scoping review according to authorship/year of publication, title, country of origin, and publication type; Brazil, 2023.consumer preferences for healthy eating from Community Kitchens in low-income urban areas: a discrete choice experiment of Comedores Populares in Peru. 6 Araújo; Almeida; Bastos [21] (2007).Food and Nutritional Aspects of Users of "Restaurante Popular Mesa do Povo".Brazil 7 Assunção et al., [22] (2017).Socioeconomic, demographic, and food profile of users of the community restaurant in Juiz de Fora, MG.Brazil 8 Balam-Gómez et al. [3] (2013).Evaluation of community kitchens in Tizimín, Yucatán, Mexico: perceptions and proposals of staff and beneficiaries.Mexico 9 Bento et al. [23] (2016).Factors associated with the eating behavior phases of users of community restaurants in Belo Horizonte/MG-Brazil. Brazil Boas et al. [24] (2021).Access to regional food in Brazilian community restaurants to strengthen the sustainability of local food systems.Brazil Branquinho et al. [26] (2015).Health and sociodemographic profile of the clientele of restaurants linked to the Brazilian social program.Brazil Braun; Costa [73] (2019).Impact of community restaurants on health and social development of users: the case of Toledo (PR).Brazil Buttorff et al. [74] (2015). Table 2 . Cont.Association between the nutritional status and the presence of non-communicable chronic diseases. [37]ilFideles et al.[37](2021).Food Insecurity among Low-Income Food Handlers: A Nationwide Study in Brazilian Community Restaurants.Brazil Fideles et al. [38] (2022).Brazilian Food Handlers' Years of Work in the Foodservice and Excess Weight: A Nationwide Cross-Sectional Study.Brazil Freedman; Bartoli [39] (2013).Food intake patterns and plate waste among community meal center guests show room for improvement. Food insecurity among the elderly: Cross-sectional study with soup kitchen users.Brazil 73 Zanette et al. [71] (2021).Systemic arterial hypertension and associated factors in users of the popular restaurant in Caxias do Sul-RS.Brazil Table 3 . Study division according to the evaluation area; Brazil, 2023. Table 4 . The methods, criteria, and indicators used to evaluate community restaurants linked to government food and nutritional safety programs; Brazil, 2023. Table 4 . Cont.Semi-structured questionnaire on the type of service provided and contract, number of meals produced, type and composition of the menu, meal distribution system, opening hours of the restaurant, and composition of preparations.	2023-11-04T15:06:52.137Z	2023-11-01T00:00:00.000	{ "year": 2023, "sha1": "3bbbe40b5d0a6eb14899cd3bbf36c92d552ed4a6", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2304-8158/12/21/4009/pdf?version=1698926654", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1fcf90235f4cb987772209fd5288d010437b5904", "s2fieldsofstudy": [ "Business", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
234707447	pes2o/s2orc	v3-fos-license	Smart Bran Dispenser for Animal Husbandry Purpose The creation of technology in this century changes people’s life. Technology plays an important role that benefits young people and has increased agriculture production’s efficiency and profitability. Innovative technology mainly involved in animal feeding automation is currently one of Smart Bran Dispensers’ new inventions. This project approaches an innovative animal husbandry management system to improve the agricultural system’s efficiency, particularly livestock nutrition and feed resources. The benefit of this project is to facilitate animal feeding for breeders, which can be remotely controlled and detected by a tracking module that transmits a signal to the user and informs them of the status of the bran dispenser through the Blynk server. NodeMCU ESP8266 and Arduino UNO were implemented as the main controller. Keywords— animal feeding, bran dispenser, Arduino, NodeMCU ESP8266 I. INTRODUCTION Animal husbandry is about applying to breed and taking care of the animals, such as cows, goats, horses, sheep and dogs, for some advantage, especially in business. Speaking of animal husbandry, it is all about raising and maintaining livestock. Nowadays, we even have no time to care for ourselves, so how could we look after our animals or pets by consistently filling up their stock and monitoring their healthiness too. From the other point of view, breeders, who ran a large business scale, are fully occupied with never-ending schedules. They sometimes forget which part needs to be filled and which one is already done. Thus, the establishment of technology, particularly the Internet of Things (IoT), somehow helps in advancing the livestock's control, the animal husbandry management system, and its safety as well [1][2] [3]. There are several projects in this field employing this kind of technology, such as fish feeder [4] [5], dog feeder [6], psychological and environmental monitoring system [7], and general pet feeding [8] [9]. Some system has been developed to detect the presence of animal [10]. Despite that, an autonomous dispenser has successfully been expanded for multi-purpose use for pill mechanizing [11] [12]. With all of these kinds of technology developed, this project aims to foster an animal husbandry management system involving large-scale businesses. This improves the agricultural system's efficiency, particularly in livestock nutrition and feed resources purpose, by helping the breeders maintain all the animal's needs. The breeder always encounters time constraint problems that might contribute to a lack of nutrition, disturbing the animal's growth process due to insufficient nutrients. A bran dispenser system should be constructed to support breeders in animal husbandry to maintain a regular feeding schedule for their animals. Therefore, in this project, a WiFi system moduled by ESP8266 will diversify breeders to control the bran dispenser to feed at the right schedule's time. It provides a GPS locator device so that the Blynk server could solve the problem regarding the food feeder's stock location and identify the food's availability in the storage box. Once the microprocessor collects these values, it performs the actions required to run the solenoid valve (food valve) [4]. The GPS facilitates the breeder by locating which stock has run out of food. This technology helps the breeder minimize the cost to hire staff due to the full system being autonomously controlled by the system of the Internet of Things (IoT). Furthermore, livestock owners would also worry less because this smart bran dispenser provides sufficient quantities of food and drink to their animals. In this project, a smart bran dispenser prototype is developed using NodeMCU ESP8266 to integrate the Blynk server to provide the food dispenser system's location. NodeMCU ESP8266 plays a major role as it is a brain for the whole system. It is a programmed microcontroller circuit board with a circuit connection of analog and digital input and output pins. Blynk is a server used in most countries for mobile communication and an alert system. This project aims to develop a system that manages to facilitate humans like breeders to preserve their animals. These are two specific objectives to be achieved: developing an efficient device for managing feeding's schedule for animal husbandry purpose and installing Smart Bran Dispenser by embedding WiFi module (ESP8266) and Blynk server to locate and control the product from elsewhere. II. METHODOLOGY In essence, as discussed previously, the main purpose of this project is to help the breeder to look after those animals easily and properly. In other words, it benefits the breeder to control the feeding system. The main electronic board of this project employs the NodeMCU ESP8266 microcontroller that has been successfully programmed. In addition, Arduino UNO acts as a second microcontroller for the water feeding system. Fig. 1 shows the whole picture of the methodology in this project. B. Hardware Implementation for NodeMCU ESP8266 NodeMCU ESP8266 has been selected as a WiFi module to allow the microcontroller access to a WiFi network. Having the network will make the systems function well in receiving the data about its location by Geolocation API. When the bran's stock in the storage box runs out, the breeder will receive notification through the Blynk application, where the HC-SR04 Ultrasonic Sensor is used to measure the bran's existence. Furthermore, if feeding occurs at the time set, the breeder will receive notification by Blynk that feeding is done. If the dispenser failed to drop the bran, the breeder would notify that the attempted process failed. Fig. 3 shows the schematic diagram connection of NodeMCU. C. Hardware Implementation for Arduino UNO Arduino UNO has been used in this project as a medium to control the water feeding system. Once the feeding schedule was set, the components are connected to the NodeMCU. Here, the water pump and HC-SR04 will operate by pumping the water from the container's source. This process only occurs when the smart bran dispenser has completed water feeding the animal, which means this tool is used for the feeding system. If the feeding process occurs over the time set coded in the IDE, the water pump will stop working. Fig. 4 shows the schematic diagram connection of Arduino UNO with a water pump and ultrasonic sensor HC-SR04. Table 1 displays the components that have been used in this project to construct the smart bran dispenser for animal husbandry purpose. E. Software Implementation Blynk server and Geolocation API is employed for software implementation in this project. Before that, the NodeMCU ESP8266 must be programmed using the C language to start the application. This process is to instruct the NodeMCU ESP8266 of the WiFi module required. NodeMCU plays the main role in this project as it controls the whole system by interfacing with some hardware devices. The remaining other hardware devices are then connected to the Arduino UNO. NodeMCU controls this project's main system: the bran feeding system and bran storage system, having each inputs signal. Meanwhile, the Arduino is for controlling the water feeding system. This system is then set up to send the notification through the Blynk server to the mobile phone by the NodeMCU ESP8266 programmed. This project helps the breeder locate the bran dispenser's location from somewhere that runs out of bran in the storage and details the longitude and latitude through the Geolocation API. Fig. 5 interprets the flowchart for the Geolocation API. F. Measuring Implementation Three units of HC-SR04 ultrasonic sensors are used in this project to execute input signals to the NodeMCU and Arduino as information for the outputs. HC-SR04 ultrasonic sensor works when this component detects a high level of food sufficient, which sends data to NodeMCU to stop the process of Servo MG90S that occurs for the feeding system. The HC-SR04 Ultrasonic sensor works to detect the presence of bran in the food storage box and measure the high water level in the container used for the water feeding system. Fig. 6 shows how the NodeMCU functioning in the bran feeding system while Fig. 7 shows the water feeding system's flowchart. Fig. 6 The flowchart on how the NodeMCU functioning in the bran feeding system Fig. 7 The flowchart of the water feeding system III. EXPERIMENTAL RESULTS Firstly, the systems should be checked to ensure the process runs well after implementing its hardware and software. Some tests are performed showing that it can function as expected for this Smart Bran Dispenser. Every system of this tool, such as the food feeding system, water feeding system, and food storage system, functions very well. The result of each test is shown and discussed in this section. A. Hardware and Software Implementation Test Smart bran dispenser starts to operate with the feeding system. This process is set-up to occur twice daily and programmed trough the Blynk server at 8 am and 5 pm a day. When the process happens, the HC-SR04 starts operating to detect the presence of bran in the container. Moreover, when the bran reaches the maximum high, 3 cm from where the ultrasonic is placed, the servo will stop working. Then, NodeMCU starts to send data and notifications to the mobile phone through the Blynk application, such as "feeding done". Figure 8 shows the notification image via the Blynk application. Once the bran level in the storage box reaches 20 cm or more than the programmed distance from the HC-SR04 placed at the storage box, the breeder receives notification that the bran is exhausted and the bran dispenser location. The tool's notifications and location are sent through the use of the Blynk application with Internet access by ESP8266. The purpose of submitting location is to facilitate the breeder to track which location has lots of tools. Fig. 9 (a and b) below indicates the food storage system at three different food level conditions, while Fig. 10 (a to c) shows the appearance of notification by the Blynk application. Finally, the water feeding system would also be tested. This system is programmed to be always working as long as this Smart Bran Dispenser functions. However, this system is programmed to start operating when the high water level, the distance of water from HC-SR04 placed at the water container reaches 3 cm and above. When the water distance and HC-SR04 reach 3 cm, the water pump will stop working for the water feeding system. Fig. 11 shows the combination circuit of the food feeding and food storage system of the Smart Bran Dispenser; meanwhile, Fig. 12 shows the circuit of the water feeding system. Table II indicates the summary for each part of the devices that have been implemented in the food feeding system for the Smart Bran Dispenser. Table IV indicates the summary for each part of the device implemented in the Smart Bran Dispenser water feeding system. Table V indicates the summary for each part of the device implemented in the Smart Bran Dispenser as a whole system. IV. CONCLUSION The advancement of the Internet of Things (IoT) had a significant impact on humans' lives. One of the biggest effects of IoT in technology is making things easier, such as controlling something remotely without interrupting daily life like the Smart Bran Dispenser. Therefore, it was successfully designed and developed to assists breeders. In the past, breeders had to work hard for the feeding schedule to maintain their livestock. In addition, the innovation of this dispenser by embedding IoT benefits both breeders and livestock as the breeders could save energy and time in managing their livestock. In contrast, the livestock would benefit from having the right food at the right time with the right quantity. Furthermore, the Smart Bran Dispenser system is convenient for users during emergencies or on vacation because this dispenser can be controlled through mobile phones.	2021-05-18T00:04:14.482Z	2021-03-30T00:00:00.000	{ "year": 2021, "sha1": "e6a8654d1e8a77a572c7dfcdcaa14d082ac93e55", "oa_license": "CCBY", "oa_url": "https://mjosht.usim.edu.my/index.php/mjosht/article/download/159/91", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "2c3971f888652fa8409d8e95540682294d064745", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Business" ] }
218651758	pes2o/s2orc	v3-fos-license	Dual-Function Enhancer for Near-Infrared Photopolymerization: Kinetic Modeling for Improved Efficacy by Suppressed Oxygen Inhibition There are many strategies for improved conversion efficacy such as the reduction of oxygen inhibition effects (OIH). Three-component system using the phosphine to reduce the OIH effects during the free radical polymerization of (meth)acrylate monomers has been reported. Addition, near-infrared (NIR) photopolymerization offers advantages of safer, less light diffusion and scattering, and deeper penetration into the materials. This study presents the detailed kinetics, and modeling the conversion efficacy associated with the experimental results of Bonardi et al. (Macromolecules, 2018, 51, 1314–1324). The dual function of the enhancer additive includes: (i) regenerating the photoinitiator, and (ii) producing extra reactive radical. The temporal profiles of the concentration of each of the 3-component system and the associate conversion efficacy are numerically produced. In this study, several new findings showing unique features of various factors influencing the conversion will be demonstrated. For examples, reverse trends (roles) are found in: (i) the light intensity and enhancer concentration, and (ii) the coupling rate constants of radical-oxygen and radical-monomer. The monomer conversion is an increasing function of enhancer, oxygen concentration, and the light intensity. However, they have significantly different steady state features. The steady-state conversion increases from 10% without the enhancer (with enhancer concentration [B]0 = 0) to (30%, 50%, 80%) for [B]0 = (0.5, 1.0, 2.0)%. High conversion also requires a long lifetime of the free radical. Finally, the measured conversion profiles at various conditions reported by Bonardi et al. (Macromolecules, 2018, 51, 1314–1324) are compared and analyzed by our modeling. I. INTRODUCTION Free radical photopolymerization consists of two types of photoinitiation (PI), in which type-I is related to the direct coupling of the UV-light initiated radical and the monomers; whereas type-II is related to oxygen-mediated crosslink, or visible-light initiated radicals which coupled to the co-initiator [1], [2]. UV lights (360-400 nm) have been commonly used in most type-I photopolymerization of (meth)acrylate monomers [1]- [3]. However, the UV The associate editor coordinating the review of this manuscript and approving it for publication was Navanietha Krishnaraj Krishnaraj Rathinam. wavelength suffers the disadvantages of being unsafe to skin and eyes, small penetration depth and larger light scattering in tissues [1]. Camphorquinone (CQ), due to its good visible absorption properties, is the most common type-II PI of (meth)acrylates under visible light [4]- [8]. The first threecomponent system of (CQ)/amine/ (aryliodonium ylides) was reported by Kirschner et al. [8]. In comparison, near-infrared (NIR) light offers advantages of safer, less light diffusion and scattering, and deeper penetration into the materials [1], [2]. Thus, the curing of a thick and filled material can be potentially enhanced compared to curing with UV or visible light. However, the use of NIR VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ photoinitiation systems such as cyanine is often associated with a low reactivity and requires a high light intensity [1]. Phthalocyanines and conjugated macrocycles have been used as commercial pigments and dyes having a high molar absorptivity coefficient in the red and NIR wavelength of 650-810 nm [9], [10]. Efficient polymerization conversions using NIR photoinitiation by cyanine/iodonium salt couples are reported by Strehmel et al [9]. Three-component system with a dye as a photosensitizer absorbing in the NIR range, an iodonium salt (as an initiator), and a phosphine (as a co-initiator) was reported, in which the phosphine is used to reduce oxygen inhibition (OIH) during the free radical polymerization of (meth)acrylate monomers [10], [11]. There are many other strategies to reduce (OIH) including: working in an inert or closed environment, increasing the photoinitiator concentration, increasing the light dose, or light intensity, use of multiple photoinitiators with different rate of initiation, or addition of oxygen scavengers [12], [13]. Furthermore, functionalized monomers which are insensitive to oxygen, such as the thiol-ene and thiol-acrylate-Michael additive systems were also reported [14], [15]. Additive enhancer-monomer were also proposed to improve the curing (crosslink) efficacy by either reducing the oxygen inhibition effect by stable-monomer, or increase the lifetime of radicals in clinical applications [16], [17]. In addition, dual-wavelength (red and UV) photopolymerization was reported, in which pre-irradiation of red-light was used to prereduce the OIH [18]. Recently, Bonardi et al [19] reported the first threecomponent system for high performance NIR (785 nm) photopolymerization of thick methacrylates, which used (i) a borate dye used as a NIR photosensitizer (PS), (ii) an iodonium salt as a photoinitiator (PI) for the free radical polymerization of the (meth)acrylates, and (iii) a dual-function enhancer (phosphine) to prevent oxygen inhibition, and to regenerate the PS upon irradiation, in which a stable radical is coupled with the enhancer. This study presents the detailed kinetics, and modeling the conversion efficacy associated with the experimental results of Bonardi et al [19]. The dual function of the enhancer additive includes (i) prevention of OIH, and (ii) regeneration of the PS, which will be explored numerically and analytically in a 3-initiator system. The temporal profiles of the concentration of each of the 3-component and the associate conversion efficacy are numerically produced. In this study, several new findings showing unique features of various factors influencing the conversion will be demonstrated. For examples, the reverse trends (roles) are found in: (i) the light intensity and enhancer concentration on the PS concentration; and (ii) the coupling rate constants of radical-oxygen and radical-monomer coupling. Finally, the measured conversion profiles at various conditions reported by Bonardi et al [19] are compared and analyzed by our modeling. We note that due to the complexity of the kinetics, this article is highly theoretical. Therefore, for those readers without a strong theoretical background may skip the In addition, readers may read the measured data reported by Bonardi et al [19] which also explained the important features as predicted by our analytic formulas. The kinetic equations for our previous single-initiator systems are revised for the 3-initiator system as follows [9]- [12] ∂ II. MATERIALS AND METHODS where b=83.6a'wq, with w being the NIR light wavelength (in cm) and q is the excited state C * quantum yield, a' is the mole absorption constant, in (1/mM/%) and light intensity, I (z, t) in mW/cm2. We will use the so-called quasi-steady state assumption [5] described as follows. The life time of radical C * and [RO] are very short (ns to µs time scale) since they either decay or react with the substrate monomer after they are created. However, [ROO] is a rather stable radical and could not be assumed at a steady-state. The steady-state solutions of Eq. (4) to (6) The dynamic light intensity is given by [9], [10] ∂I(z,t) ∂z where a' and b' are the extinction coefficients of Initiator-C and the photolysis product, respectively; Q' is the absorption coefficient of the monomer at NIR wavelength. Greater detail may be found in Ref. [9], [10]. We note that the dynamic feature of Eq. (17) due to the depletion of C(z,t) and the spatial dependence of both I(z,t) and C(z,t) are critical in optically-thick polymers [10]. The steady-state radical of Eq. (13) is given by where For the radical [R] dominant case with 8k T bIC G 2 , we obtain an approximate radical given by which is a decreasing function of the oxygen inhibition term G. We note that the conversion is a decreasing function of the oxygen initial concentration, whereas conversion is improved by enhancer Solutions of Eq. (10) to (18) are available by the approximated analytic formulas for I j (z,t) as follow [2], [4] where A 1 = 2.3(a'-b')C0I0bz. We note that the -A 1 t term represents the decrease of A', or increase of light intensity due to PS depletion, which is important for optically-thick polymers. Using Eq. For analytic solution of Eq. (23), we could solve for the approximated solution of Eq. (11) and (12) Therefore, by the definition of efficacy C EFF = 1-M/M 0 , we obtain where radical [R] is proportional to bICg, but is a decreasing function of oxygen, as shown by Eq. (20). We note that g= k 7 /(k 5 . We note the coupling constant bI, a product of b and light intensity (I), will be considered as one parameter which also represents the light intensity for a given absorption constant (b). Figure 3 could be further interpreted as follows. As shown by Figure 1 and Eq. (5) and (6) ] are, respectively, an increasing and decreasing function of time. Therefore, the combined effect leads to the optimal (or peak) of the temporal profile of [RO]. Furthermore, a larger co-initiator concentration ([B] 0 leads to a larger radical [RO] as shown in Figure 3 (A). In contrast, a higher light intensity leads to a faster oxygen depletion and thus a faster drop of [RO] profile as shown in Figure 3 (B). Figure 4 and 5 show the concentration profiles of initiator C(t), and oxygen [O 2 ] associate to Figure 2(A) and 2(B), respectively. We note that C(t) has a higher value in the presence of enhancer ([B]) due to the regeneration of C, shown by Figure 4 having various [B] 0 . In contrast, a reversed trend is shown by Figure 5 having various coupling constant of b'= bI0, in which a larger b' leads to a stronger depletion and lower value of C(t). , which is an increasing function of oxygen. In the absence of oxygen, radical [RO]=0, the efficacy is given by gbIC and k'R, as shown by curve-1 of Figure 6(B). In contrast, for the k'R dominant case, conversion is reduced by OIH, as shown by Eq. (20), in which bimolecular termination leads to a reverse-trend of light intensity, i.e., a lower intensity leads to a higher steady state conversion. [19] could be compared to our modeled results as follows. Figure 8 shows that higher coupling constant b'= bI0 leads to higher efficacy, which is also an increasing function of light intensity, as shown by Figures 4, 6 and 8 of Bonardi et al [19]. Furthermore, the photolysis (% decomposition of the peak at 800 nm) upon laser diode at 785 nm, shown by Figure 10 of Bonardi et al [19] could be compared with our Figure 4(A) for the initiator concentration C(t), in which a higher enhancer concentration [B] leads to a higher C(t), or less depletion due to the regeneration of C(t) by the enhancer [B]. We note that the photolysis or the relative peak spectrum height at 800 nm, D(t) reported by Bonardi et al [19], is related to C(t) by D We suggest that readers should read the measured data reported by Bonardi et al [19], which also explained the important features as predicted by our analytic formulas and our numerical results. For a more comprehensive modeling, readers may refer to Ref. [20]. This article explores the kinetics of a single-wavelength system. We also suggest readers to read our other articles which further explored 2-wavelength and 3-wavelength polymerizations [23], [24] for the applications in 3D bioprinting. IV. CONCLUSION We have demonstrated that efficacy of NIR photopoly- CONFLICTS OF INTEREST Jui-Teng Lin is the CEO of Photon Vision Corp., Taipei, Taiwan. 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.	2020-04-30T09:04:12.390Z	2020-01-01T00:00:00.000	{ "year": 2020, "sha1": "4ac10be97724c719b31aadcb234eb4889d0a3f44", "oa_license": "CCBY", "oa_url": "https://ieeexplore.ieee.org/ielx7/6287639/8948470/09076690.pdf", "oa_status": "GOLD", "pdf_src": "IEEE", "pdf_hash": "3b7c9f18646cf5c4f10725fb11bdb45f019640e1", "s2fieldsofstudy": [ "Materials Science" ], "extfieldsofstudy": [ "Computer Science", "Chemistry" ] }
257913281	pes2o/s2orc	v3-fos-license	Spontaneous rotational symmetry breaking in KTaO3 heterointerface superconductors Broken symmetries play a fundamental role in superconductivity and influence many of its properties in a profound way. Understanding these symmetry breaking states is essential to elucidate the various exotic quantum behaviors in non-trivial superconductors. Here, we report an experimental observation of spontaneous rotational symmetry breaking of superconductivity at the heterointerface of amorphous (a)-YAlO3/KTaO3(111) with a superconducting transition temperature of 1.86 K. Both the magnetoresistance and superconducting critical field in an in-plane field manifest striking twofold symmetric oscillations deep inside the superconducting state, whereas the anisotropy vanishes in the normal state, demonstrating that it is an intrinsic property of the superconducting phase. We attribute this behavior to the mixed-parity superconducting state, which is an admixture of s-wave and p-wave pairing components induced by strong spin-orbit coupling inherent to inversion symmetry breaking at the heterointerface of a-YAlO3/KTaO3. Our work suggests an unconventional nature of the underlying pairing interaction in the KTaO3 heterointerface superconductors, and brings a new broad of perspective on understanding non-trivial superconducting properties at the artificial heterointerfaces. Unfortunately, the extremely low T c of SrTiO 3 heterointerface superconductors is a critical challenge, preventing extensive attentions to further unveil the origin of these emergent quantum phases. Very recently, unexpected superconductivity is experimentally observed at the heterointerface between polycrystalline (p)-EuO [or amorphous (a)- LaAlO 3 ] and KTaO 3 single-crystal substrates which shows a T c~2 K 15,16 , approximately one order of magnitude higher than that of c-LaAlO 3 /SrTiO 3 5 , evoking an exciting opportunity to study the physical properties of heterointerface superconductivity. Although KTaO 3 shares many common features with SrTiO 3 [15][16][17][18] , the superconductivity of KTaO 3 heterointerfaces behaves in a quite different manner. Remarkably, the superconductivity of these heterointerfaces exhibits a strong dependence on the KTaO 3 crystalline orientations by compared to the SrTiO 3 crystalline orientation independence of superconductivity [19][20][21][22][23][24] . Furthermore, considering the fact that the strong spin-orbit coupling associated with the heavy Ta in 5d orbitals of KTaO 3 heterointerfaces is comparable to the bandwidth and the accompanying strong on-site Coulomb repulsion [25][26][27] , the combination of strong spin-orbit coupling and the electron-electron interaction is theoretically expected to result in an unconventional superconductivity, including a mix of spin-singlet and spin-triplet components 28 as a manifestation of rotational symmetry breaking. Experimentally, an indication of strong in-plane anisotropic electrical resistance in the normal state has been reported at the ferromagnetic heterointerface of p-EuO/KTaO 3 , implying a possible existence of "stripe"-like superconducting phase 15 . This anisotropy, however, is alternatively attributed to be an extrinsic property of the ferromagnetic p-EuO in theory 29 , leading to that a consensus on the rotational symmetry breaking of superconductivity in KTaO 3 heterointerface superconductors remains elusive. Here, we carry out an experimental study on nonmagnetic a-YAlO 3 thin films with a wide energy gap of 7.9 eV grown on the polar KTaO 3 (111) single-crystal substrates. This energy gap is significantly larger than that of a-LaAlO 3 (5.6 eV) 30 , enabling strong confinement potential to restrict the interfacial conducting electrons to a thinner interfacial layer, thus prompting an intriguing quantum behaviors at their interface 31 . Electrical transport measurements on the as-grown films reveal two-dimensional superconductivity with a T c of 1.86 K, and a superconducting layer thickness of 4.5 nm. By tuning the in-plane azimuthal angle φ-dependent magnetic field, both the magnetoresistance and superconducting critical field display pronounced twofold symmetric oscillations deep inside the superconducting state, whereas they vanish in the normal state. These results unambiguously demonstrate that the anisotropy with in-plane rotational symmetry breaking is an intrinsic property of the superconducting phase in a-YAlO 3 /KTaO 3 . Through group theory study, we thus classify the inversion symmetry breaking KTaO 3 heterointerface superconductors as a mixed-parity unconventional superconductivity with an admixture of s-wave and p-wave pairing components, a candidate platform for realizing Majorana modes 32 . Results The a-YAlO 3 /KTaO 3 heterostructures are prepared by depositing a-YAlO 3 films on (111)-oriented KTaO 3 single-crystal substrates using pulsed laser deposition. Atomic force microscopy characterizations show that the surface of KTaO 3 substrates and a-YAlO 3 films are atomically flat (see Supplementary Fig. 1). X-ray diffraction confirms the absence of epitaxial peaks of YAlO 3 (see Supplementary Fig. 2 and Supplementary Fig. 3), thus suggesting that the YAlO 3 film is not in a well-defined crystalline phase. The microstructure of the interface is further examined by aberration-corrected scanning transmission electron microscopy (STEM). From the high angle annular dark field (HAADF)-STEM image shown in Fig. 1a, it can be seen that the homogeneous and amorphous phase YAlO 3 thin film is grown on the KTaO 3 (111) substrate (also see Supplementary Fig. 4). Looking at the sample from a larger field of view, the thickness of the a-YAlO 3 film is found to be about 60 nm. High-resolution (HR)-STEM imaging shown in Fig. 1a and Supplementary Fig. 4, and energy dispersive X-ray spectroscopy (EDX) elemental mapping shown in Fig. 1b indicate that the abrupt and smooth interface between KTaO 3 single-crystal substrate and a-YAlO 3 film is resolved structurally and chemically. These results are consistent with previous studies on a-LaAlO 3 /KTaO 3 (111) 15,16 , a-LaAlO 3 /KTaO 3 (110) 23 , a-LaAlO 3 /KTaO 3 (001) 33 , and a-AlO x /KTaO 3 (111) 34 . Figure 2a shows the temperature-dependent sheet resistance R s on two representative as-grown a-YAlO 3 thin films (Samples #1 and #2 with growth temperatures of 780 and 650°C, respectively) with the Hall bar configuration, schematically illustrated in Fig. 1d. A typical metallic behavior is visible in a wide temperature range, indicating that a two-dimensional electron gas is formed at their interface induced by a candidate mechanism of the formation of oxygen vacancies at the surface of KTaO 3 23,35 , similar to that in the sister a-LaAlO 3 /SrTiO 3 36 . The transverse Hall resistance R xy is obtained from Hall measurements at 5 K, and reveals that the charge carriers in the a-YAlO 3 /KTaO 3 are electrons. The estimated carrier density is about 1.45 × 10 14 and 6.62 × 10 13 cm −2 for Samples #1 and #2, respectively. The electron mobility for Samples #1 and #2 is thus 193.6 and 159.7 cm 2 V −1 s −1 . These results are highly universal and reproducible (see Supplementary Table 1) and reasonably consistent with previous electrical transport studies on the KTaO 3 heterointerfaces 15,23 . Remarkably, as the temperature is further decreased, the resistance R s undergoes a narrow and sharp transition with a transition width of less than 0.5 K to a zero-resistance state, signaling the appearance of superconductivity at the heterointerface of a-YAlO 3 /KTaO 3 . The critical temperature is determined to be T c = 1.86 and 0.92 K for Samples #1 and #2, respectively, as defined by where the resistance is at the midpoint of the normal electrical resistance at 5 K, i.e. R s (T c ) = 0.5 × R s (5 K). To further characterize the superconducting behaviors in a-YAlO 3 /KTaO 3 , we measure the magnetoresistance R s (μ 0 H) (here, μ 0 is the vacuum permeability) at various temperatures between 0.5 and 5 K with fields parallel (μ 0 H ∥ ) and perpendicular (μ 0 H ⊥ ) to the plane surface of Sample #1, as shown in Fig. 2b, c, respectively. The fundamental superconducting behavior is clearly perceived. Indeed, the magnetoresistance R s (μ 0 H) varies differently with μ 0 H ∥ and μ 0 H ⊥ , and both the upper critical fields μ 0 H k c2 and μ 0 H ? c2 parallelly shift to a lower value with the increase of the temperature, where μ 0 H c2 are evaluated at the midpoints of the normal state resistance at 5 K. These results provide an indication of a two-dimensional superconducting feature in a-YAlO 3 /KTaO 3 . The temperature-dependent upper critical fields μ 0 H c2 are summarized in Fig. 2d and are well fitted by the phenomenological two-dimensional Ginzburg-Landau where Φ 0 , ξ GL , and d SC denote a flux quantum, the in-plane superconducting coherence length at T = 0 K, and the effective thickness of superconductivity, respectively. Using the extrapolated μ 0 H ? c2 ð0Þ = 0.98 T and μ 0 H k c2 ð0Þ = 13.81 T, we find ξ GL = 18.4 nm and d SC = 4.5 nm, where ξ GL is significantly larger than d SC , suggesting a twodimensional nature of superconductivity. Additionally, the in-plane μ 0 H k c2 ð0Þ is substantially larger than the Pauli-paramagnetic pairbreaking field B P ≈ 3.46 T based on the BCS theory in the weakcoupling limit 38,39 . High values of μ 0 H k c2 ð0Þ excessing B P could be realized in the presence of strong spin-orbit coupling owing to the elastic scattering, which results in the suppression of spin paramagnetism effects. The violation of this paramagnetic limit is a common phenomenon in heterointerface superconductors 15,40 , especially when the superconducting layer thickness is in the range d SC < 20 nm. However, the underlying mechanism for realizing 37 . This result could be intuitively expected, since the strong confinement potential induced by YAlO 3 significantly restricts the superconducting electrons to a thinner superconducting layer 31 . On the other hand, the out-of-plane polar angle θ-dependent upper critical field H θ c2 at 1.5 K quantitatively verifies the behavior expected from a two-dimensional structure in a-YAlO 3 /KTaO 3 , as shown in Supplementary Fig. 6. The θ-dependent μ 0 H θ c2 are quantitatively fitted by the two-dimensional Tinkham formula and the threedimensional anisotropic G-L model, given by 41,42 . A cusp-like peak is clearly observed at θ = 90°(see Supplementary Fig. 6), which is well described by the two-dimensional Tinkham model, as frequently observed in heterointerface superconductivity 42,43 and layered transition metal dichalcogenides 44,45 . Since the superconductivity in a-YAlO 3 /KTaO 3 is two-dimensional, the Berezinskii-Kosterlitz-Thouless (BKT) transition describes superconducting phase coherence 46,47 . Here, the BKT transition temperature defines the vortex unbinding transition, and can be determined using current-voltage (I-V) measurements as a function of temperature T, as shown in Fig. 3a. Below T c , we find a critical current I c , whose value decreases with increase in temperature. The maximal value of I c is~330 μA at 0.5 K, which is substantially larger than that previously observed in the KTaO 3 heterointerfaces 15,23 . Such a high critical current value probably originates from the high charge carrier concentration (about 1.45 × 10 14 cm −2 , Sample #1 in Fig. 2a) confined in a thinner superconducting layer of a-YAlO 3 /KTaO 3 , promising for large-scale applications in superconductor-based devices. In Fig. 3b, we also plot the characteristics I-V on a log-log scale, and observe that the slope of the I-V curve smoothly evolves from the normal ohmic state, V ∝ I, to a steeper power law resulting from the current exciting free-moving vortices, V ∝ I α(T) , with α(T BKT ) = 3. In Fig. 3c, a value T BKT = 1.7 K is interpolated, which is consistent with T c as defined in Fig. 2a. In addition, close to T BKT , an R s = R 0 exp½ÀbðT=T BKT À 1Þ À1=2 dependence, where R 0 and b are material parameters, is expected 48 . As shown in Fig. 3d, the measured R s (T) is also consistent with this behavior and yields T BKT = 1.85 K, in good agreement with the analysis of the α exponent shown in Fig. 3c. Next, we turn to discuss the in-plane anisotropy of superconductivity in a-YAlO 3 /KTaO 3 using an in-plane azimuthal angle φdependent magnetoresistance, where φ is defined as the azimuthal angle between the magnetic field and the ½1 10-axis of the lattice, as indicated in Fig. 1d. Care has been taken to rule out the inevitable misalignment effects of an accidental out-of-plane component of the field, when the vector magnet is utilized. In the normal state (T = 1.8 K in Fig. 4a) of Sample #4 (Supplementary Fig. 7), the magnetoresistance R s is found to be essentially independent of φ, displaying an isotropic behavior. Whereas in the superconducting state (T = 1.5 K in Fig. 4a), we observe a pronounced twofold symmetric oscillations of the R s (see Fig. 4b), which is consistent across multiple samples including the heterointerfaces of a-YAlO 3 /KTaO 3 (111) and sister a-LaAlO 3 /KTaO 3 (111) (see Supplementary Fig. 8 − Supplementary Fig. 10 and Supplementary Note 2). In this case, the anisotropic R s attains the maximum value when the magnetic field is directed along the special ½1 21-axis (φ = − 30°or 150°) that is in the direction of one of the principal axes of KTaO 3 (111) shown in Fig. 1c, and becomes minimum when the position with respect to that of maximum is shifted by 90°(φ = 60°or 240°). This finding implies that an extrinsic error from the experimental setup is unlikely to the source of the observed twofold anisotropy of magnetoresistance in the superconducting state at the heterointerface of a-YAlO 3 /KTaO 3 . In particular, the significantly large anisotropic ratio of R s (φ = 60°)/R s (φ = 150°) = 0.03 at 1.5 K corresponds to a putative misalignment angle estimated up to 88.03°(cos 88.03°= 0.03) between the field and the basal plane 49 , which is impossible for such a large angle misalignment in the vector magnet, we could exclude the possible contribution from an accidental misalignment of the field with the film plane. Since the magnetoresistance minima approach zero in R s (φ) curve measured at 1.5 K (see Fig. 4a), and considering the fact that the existence of conspicuous twofold symmetry in magnetoresistance manifests deep inside the superconducting region, which vanishes in the normal state (see Fig. 4a and Supplementary Fig. 11), we could further rule out the possibilities of extrinsic contributions, such as the magnetic field induced Lorentz force effect 50 and the Fermi surface inherent to the KTaO 3 with respect to the underlying threefold lattice symmetry 26 ( Fig. 1c and Supplementary Fig. 3), and thus demonstrate that this anisotropy with rotational symmetry breaking is an intrinsic property of the superconducting phase in a-YAlO 3 /KTaO 3 . To further reveal the twofold symmetric superconductivity in a-YAlO 3 /KTaO 3 in terms of the superconducting gap structure, we extract the upper critical field μ 0 H c2 from the φ-dependent magnetoresistance R s in the superconducting region determined by the criterion of 90% sheet resistance dropped from normal state, as shown in Fig. 4c. Here, it should be pointed out that although the values of H c2 are changed by different criteria, the symmetry of H c2 itself remains qualitatively (see Supplementary Fig. 12). In addition, it should be noted that the data shown here have been taken by averaging the raw data with positive and negative magnetic fields to avoid the possible asymmetric problem. Remarkably, the in-plane φ-dependent μ 0 H φ c2 also displays twofold symmetric oscillations (see Fig. 4d), providing additional strong evidence for the twofold rotational symmetry of the superconductivity in a-YAlO 3 /KTaO 3 . Furthermore, the oscillation of μ 0 H φ c2 has a π phase shift compared with that of the R s (see Fig. 4b) such that for the φ value where superconductivity is hardest to suppress, μ 0 H φ c2 is the largest and R s is the lowest (Fig. 4b, d), as expected from our intuitions 50-52 . Since μ 0 H φ c2 achieves its maximum for the field applied perpendicular to the main crystallographic axis, and minimum for the direction along the main crystallographic axis (see Figs. 1d and 4d), the superconducting gap leads to a maximum (or minimum) direction perpendicular (or parallel) to the main crystallographic axis, manifesting a rotational symmetry breaking state of superconducting a-YAlO 3 /KTaO 3 with the direction of the minimum gap spontaneously pinned to the main crystallographic axis. Discussion Having experimentally established the intrinsic twofold anisotropy of the superconducting state of a-YAlO 3 /KTaO 3 , we now proceed to elaborate about its origin using the underlying symmetries of the crystal structure without requiring the details of the pairing mechanisms based on the group theoretical formulation of the Ginzburg-Landau theory 53 (also see Supplementary Note 2 and Supplementary Note 3 in details). This allows us to deduce fundamental information about the superconducting ground state in the a-YAlO 3 /KTaO 3 heterointerface superconductors. From the viewpoint of group symmetry, if a superconductor possesses an inversion symmetry, the Pauli principle requires a totally antisymmetric Cooper pair wavefunction, which imposes the condition that the superconducting states should be either spin-singlet or spin-triplet, whereas mixed-parity states are forbidden 53 . In the a-YAlO 3 /KTaO 3 the lack of inversion symmetry, however, tends to mix spin-singlet and spin-triplet driven by strong spin-orbit coupling 54 . Indeed, the conducting electrons with strong spin-orbit coupling originating from the heavy Ta 5d orbitals has been elucidated at the KTaO 3 heterointerfaces 26,33,55-60 (also see Supplementary Fig. 13). Due to the absence of a mirror plane parallel to the interface of a-YAlO 3 /KTaO 3 , the point group of a-YAlO 3 /KTaO 3 is C 3v , which does not contain the symmetry element of an inversion. This situation is analogue to non-centrosymmetric superconductors 54 superconducting state only belongs to the A 1 +E-representation with the possible basis function of s+p. Notably, the two-dimensional irreducible representation of E could spontaneously break the threefold rotational symmetry of the crystal (see Fig. 1c and Supplementary Fig. 3), leading to a subsidiary uniaxial anisotropy or nematic superconductivity 62,63 , such as a uniaxial p x -wave or p y -wave pairing. Since the upper critical field is proportional to the square of the superconducting gap amplitude based on the Ginzburg-Landau theory and the Pippard definition of the coherence length 49 , μ 0 H φ c2 / \|Δ gap ðφÞ\| 2 , only the s+p x -wave pairing with the gap function of Δ gap ðkÞ = i½Δ sσ0 + Δ p sinðk x Þσ 3 σ 2 (here, Δ s and Δ p are the pairing amplitudes of s-wave and p-wave, respectively,σ is the vector of Pauli matrices, and k is the momentum) 61,64 , could give rise to an overall twofold anisotropic gap and well reproduce the exact topology of the anisotropic H φ c2 shown in Fig. 4d. Therefore, the mix of s-wave and p-wave pairings driven by strong spin-orbit coupling is suggested to the source of the experimentally observed twofold anisotropic superconductivity at the KTaO 3 heterointerfaces, which has long been a topic of interest sought in condensed matter physics. Further experiments, including probes of the superconducting gap by tunneling spectroscopy and/or Josephson junction experiments, will also be helpful for clarifying the underlying mixed-parity pairing nature of the twofold symmetric superconductivity that we observe. In summary, we have experimentally observed spontaneous rotational symmetry breaking from threefold to twofold in the superconducting state of KTaO 3 (111) heterointerfaces with respect to an application of in-plane magnetic field. This in-plane anisotropic superconductivity is theoretically attributed to the intrinsic nature of mixed-parity unconventional superconductivity with an admixture of s-wave and p-wave pairing components, bringing with it fresh new insights into the study of emergent fascinating and non-trivial superconducting properties at the heterointerfaces with inversion symmetry breaking. Methods Thin film growth and structural characterizations a-YAlO 3 thin films are grown on KTaO 3 (111) single-crystal substrates (5 × 5 × 0.5 mm 3 ) by pulsed laser deposition in an ultrahigh vacuum chamber (base pressure of 10 −9 Torr). Prior to growth, the KTaO 3 substrates are annealed at 600°C for 30 mins in ultrahigh-vacuum to obtain a smooth surface ( Supplementary Fig. 1). During deposition, a single crystal YAlO 3 target (Kurt J. Lesker Company) is used to grow the a-YAlO 3 films with a KrF excimer laser (Coherent 102, wavelength: λ = 248 nm). A pulse energy density of 1.5 J/cm 2 and a repetition rate of 2 Hz are used. The a-YAlO 3 films are deposited at temperatures ranging from 600 to 900°C in a vacuum chamber to promote growth of the superconducting phase. All the samples are cooled to room temperature at a constant rate of 20°C/min in vacuum after deposition. The quality of a-YAlO 3 films under ambient conditions is examined by atomic force microscopy (AFM, Asylum Research MFP-3D Classic) and by four-circle X-ray diffraction (XRD, Bruker D8 Discover, Cu Kα radiation, λ = 1.5406 Å) operated in HR mode using a three-bounce symmetric Ge (022) crystal monochromator. STEM measurements Cross-sectional specimens for electron microscopy are prepared with Focused Ion Beam (FIB) (Helios-G4-CX, Thermo Fisher Scientific) using lift-out method. The HR-STEM images are performed on a double aberration corrected field-emission STEM (Themis Z, Thermo Fisher Scientific) operated at 300 kV. For HAADF-STEM imaging, the semiconvergent angle of the probe forming lens is set to 21.4 mrad. The geometric aberrations within the probe forming lens aperture have been effectively tuned to zero using probe corrector (SCORR, CEOS GmbH). The semi-collection angle of the HAADF detector is 76-200 mrad. Furthermore, the chemical composition of the interface is qualitatively analyzed using EDX in STEM spectrum imaging mode. The EDX are collected using 4 silicon drift detector (SDD) system (Super X detector, Thermo Fisher Scientific). The beam current for STEM-EDX analysis is about 200 pA. Electrical transport measurements The electrical transport measurements are performed using a commercial cryostat with temperature ranging from 1.5 to 300 K (Oxford Instruments TeslatronPT cryostat system), physical properties measurement system with temperature ranging from 0.5 to 300 K (PPMS, Quantum Design), and 10 mK dilution refrigerator with vector magnet (Oxford Instruments Triton 200). The Hall bar structure (Fig. 1d) is fabricated by ion-beam etching to systemically measure the electrical transport properties. The vector magnet is utilized to reveal the inplane anisotropy of magnetoresistance in the superconducting state shown in Fig. 4a, and the samples are mounted on a mechanical rotator in a 4 He cryostat to clarify the anisotropy of H c2 shown in Fig. 4c. The misalignment of the field with the film plane is estimated to be less than 2°and 7°for vector magnet and mechanical rotator, respectively, as our experimental errors. Data availability The relevant data supporting our key findings are available within the article and the Supplementary Information file. All raw data generated during our current study are available from the corresponding authors upon reasonable request.	2021-11-11T02:31:54.989Z	2021-11-10T00:00:00.000	{ "year": 2023, "sha1": "538c1c08a21739b8a0d64a4e1a997edc308f9a21", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "8c2f05b2b0568fb6d9aaa81c7012c0b188f3b19a", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
139355502	pes2o/s2orc	v3-fos-license	Nanocatalytic Conversion of Waste Palm Oil Grade III and Poplar Plant’s Wood Sawdust into Fuel The aim of this research was to study the pyrolysis oil production from Palm Oil Grade III (POG-III) and popular wood sawdust crude oil. Today, worldwide studies have been undertaken on the biomass usage and co-conversion of biomass and coal to seek out alternative fuels for supplying energy in an environmental friendly way. Substitute fuels have become more and more vital due to descending petroleum reserves, increasing economic circumstances and awareness of the increased environmental penalties of emissions from petroleumfuelled engines. In this study, biodiesel fuel was manufactured by the nanocatalytic to elucidate their thermal behaviour under pyrolysis conditions and to assess major decomposition products in terms of their yields. Transesterification of Palm Oil Grade III (POG-III) at 80°C temperature by using nano Co as nanocatalyst. The prepared biodiesel was characterized by FT-IR and GC-MS. Results revealed the presence of esters, alkanes, alkenes, saturated and unsaturated hydrocarbons with carbon chain in the range C9-C27. Prepared biodiesel is cost effective and highly efficient. Besides Palm oil popular wood sawdust crude oil was also used. All products were obtained at low temperature and at low atmospheric pressure. Introduction In recent years, diminishing fossil fuel sources, growing demand, cost of petroleum-based fuels, and environmental hazards as a result of their combustion have encouraged researchers to investigate possibility of using alternative fuels as new energy resource. It is necessary to reduce consumption of the petro fuels due to the negative effects on human life. As known fossil energy sources have been exhausted rapidly nowadays, it is predicted that fossil fuel sources will be depleted in the near future [1]. Waste energy sources were focused in different research works presented in the literature as renewable energy source [2]. Recycling is the applied process for advancement of petroleumbased wastes by converting them into recyclable products such as diesel fuel, gasoline and heavy oil. Production of diesel-like fuel from waste oils such as industrial and engine waste oils, wood pyrolytic oils, fresh and waste fats and vegetable oils is an excellent way for producing alternative fuel sources. Industrial and engine waste oils, wood pyrolysis oils, fresh and waste fats and vegetable oils have been proposed as pyrolytic raw material to produce gasoline and diesel-like fuels. There is plenty of the waste engine oil in the world. Average amounts of used engine lubricating oils are produced worldwide every year [3]. Municipal and industrial wastes like Waste Cooking Oil (WCO), Waste Plastics Oil (WPO) and Waste Lubricating Oil (WLO) which have a high calorific value are considered as efficient source of energy production [4]. Among the important substitute fuel sources waste lubricant oils and biofuel are the best replacements for existing petro fuels, since waste generated oils represent more than 60% of used lubricant oils. It should be collected and re-used in order to decrease detrimental effects on environment, and underground and surface water, since it pollutes the atmospheric air as a result of burning, and has negative effect on living organisms, underground and surface water when it is discharged into soil or water. Conversion of the waste engine oils into diesel like fuels by using pyrolytic distillation, and by utilization of the product as a diesel fuel has positive effects on environment and atmospheric air, and also has economic value [5]. A very large amount of used oils are disposed through dumping on the ground or in water and land fillings [6]. Fuels or lubricating oil base stock can be produced by refining and treating the used oils. Used oils on the other hand are a severe threat to the environment as they contain metal content and other contaminants [7]. Due to the hazardous properties waste oils that are found in rivers, lakes are damaging to the aquatic life. A single liter of that waste oil can be damaging to a million liters of clean water. Soil pollution is one of the outcomes of waste oil spill [8]. Several classes of heterogeneous catalysts, such as supported enzymes oxides and Oxo salts impregnated oxides inorganic complexes, layered double hydroxides metal carboxylates zeolites and ion-exchange resins [9][10][11][12][13][14][15][16] have been reported in the literature as being active for the transesterification (alcoholysis) of vegetable oils. Currently, most of the biodiesel production processes are based on the Transesterification of vegetable oils using heterogeneous nano catalysts, which are known for their high activity and low cost, making them suitable for large-scale industrial operation [17]. In fact, this class of catalysts offers economical, ecological, and technical advantages, which are critical parameters for improvement of the efficiency and sustainability of biodiesel, manufacture [18]. Transition metal oxides and oxosalts have also been investigated as transesterification catalysts due to their elevated Lewis acidity, depending on the metal oxidation state and ion radius size [19]. In this sense, molybdenum-and tungsten-containing solids have been extensively studied in a number of reactions in which the acidity profile plays an important role, such as isomerization. Cyclic alkene oxidation alcohol dehydration and vegetable oils transesterification [20]. Transesterification is the chemical process between triglycerides and short-chain alcohol in the presence of basic or acidic catalyst to produce monoalkylester [21]. The long-and branched-chain triglyceride molecules are transformed to monoalkylesters and glycerol. Commonly used short-chain alcohols are methanol, ethanol, propanol, and butanol [22]. Methanol is commercially used due to its availability and cost effectiveness [23]. The subsequent useful product is Fatty Acid Methyl Ester (FAME) commonly known as biodiesel. Palm oil grade III is the world's cheapest waste vegetable oil, therefore crude palm oil itself presents a promising alternative as a feedstock for Fatty Acid Methyl Ester (FAME). Nevertheless, the study on converting grade III palm oil to Fatty Acid Methyl Ester (FAME) using heterogeneous reaction is still very limited and only being explored in Asian countries, most probably due to its availability [24]. Experimental (A) Chemicals The cobalt nitrate hexahydrate was purchased from Sigma Aldrich USA and Sodium hydroxide was purchased from Fluka. All solvents were of analytical reagent grade and were used without further purification. Synthesis of CoO nano particles through co-precipitation method For the synthesis of CoO nanoparticles, 0.1 molar solution of cobalt nitrate hexahydrate and 0.1 molar solution of sodium hydroxide were separately prepared in distilled water. The sodium hydroxide solution was taken in dropping funnel and very slowly dropped to the cobalt nitrate solution with constant stirring at a temperature of 40-50°C. The dark brown precipitates of cobalt Co(OH) 2 appeared after almost one third (1/3) of sodium hydroxide solution was added to cobalt nitrate solution respectively. The addition of the sodium hydroxide solution to the salt solution continued till precipitates formation was complete in the reaction mixture. The PH was plus 10. The precipitates were then filtered and washed two times with distilled water to remove the un-reacted sodium hydroxide and the salts. The precipitates were dried in oven at 250 o C. The precipitates were taken in china dish and placed in furnace. The temperature of the furnace was raised to 500°C at a heating rate of 0.5°C min -1 and the contents were kept at 500°C for 24 h and then allowed to cool to room temperature. The calcined product was ground into powder and formed CoO nano particles. The nanoparticles were studied at XRD, SEM and BET. Sample collection Palm oil samples were collected from Ferozsons Tank Terminal (Pvt.) Ltd., Karachi Plot # 50, and Molasses and Edible Oil Area Port Muhammad bin Qasim, Karachi. The following initial physiochemical parameters were carried out; conductivity, organic matter, Free Fatty Acids (FFA), Dirt content and oil content [25]. Determination of moisture and volatile content A mass of 10 g of thoroughly mixed palm oil sample was weighed into a known weight of clean petri dish which had been previously dried and cooled in desiccators. It was placed in the oven for 4 h, allowed to cool to room temperature in a desiccator for 45 min and weighed till a constant weight was obtained. The moisture and volatile matter content was expressed in percentage by mass using the formula. % Moisture and Volatile content=((Mb-Md)/(Mb-M)) × 100 (1) Where M=Mass (g) of petri dish; Mb=Mass (g) of petri dish+sample; Md=Mass (g) of petri dish+test sample after dying. Determination of oil content Dry palm oil sample (10 g) was weighed into thimble and extraction was done for 6 h using 150 ml of hexane as an extracting solvent. The oil extract was dried at atmospheric pressure at 103°C for 2 h, cooled for 30 min and weighed. The oil content was expressed as percentage by using the formula. Determination of dirt content The dirt content of the sample was analyzed as 8.50 g of the sludge was weighed into a conical flask, 100 ml of hexane was added, and then mixture was filtered through glass fiber filter paper in a crucible and dried in the oven at 103°C for an hour. Percentage dirt content was expressed as: (Mass of crucible+filter paper+dirt)-(Mass of crucible+filter paper/ Mass of sample) × 100 (3) Determination of free fatty acids (FFAs) Sample (2.5 g) was weighed into conical flask, 50 ml of neutralizing solvent was carefully added to the weighed sample in the conical flask, placed on hot plate at 40°C mixed gently and titrated with standard potassium hydroxide (0.5M). Values were calculated using the formula, Free Fatty Acids=25.6× Molarity of KOH × Volume of KOH/mass of sample (4) Experimental set up for catalytic conversion of palm oil into biodiesel Experiment was carried out in around bottom flask, equipped with L-shaped tube, connected with a condenser. About 50 ml of pretreated palm oil grade III was mixed with 10 ml of methanol was taken in round bottom flask and 1 g of Co nano catalyst was added. The flask was placed in the furnace at 80°C. The temperature was maintained at 80°C for 1 h. The liquid was condensed in L-shaped tube and collected in a beaker. Conversion of popular wood sawdust into biodiesel by using nanoparticles during the gasification process Cobalt nanoparticles were used during the gasification process with the sawdust of popular wood. Sawdust used=30 g; Oil extracted after the completion of gasification=17.5 ml; Muffle furnace temperature was maintained at 300°C. Nanoparticles help the gasification to complete at a very low temperature as compared to the gasification done without nano particles. The oil obtained was much pure as compared to the oil extracted without using nano particles. Experimental set up for catalytic gasification of popular wood sawdust crude oil. As shown in Figure 1 the catalytic gasification of popular wood sawdust was carried out in around bottom flask, equipped with L-shaped tube, connected with a condenser. About 50 g of pre-treated popular ash was taken in round bottom flask and was mixed with 0.5 g Co nano catalysts. The flask was placed in the furnace and started the reaction. The temperature of the furnace was increased from room temperature to 300°C with a rate of 10°C/ min. The temperature was maintained at 400°C for one hour. Dark brown oil starts vaporized in L-shaped tube which was condensed by condenser and collected in a beaker. This oil product was named as popular wood sawdust oil. Crude oil after extraction was then treated with Methyl alcohol in the ratio 1:6 respectively. 10 ml oil was taken in the round bottom flask and mixed with 60 ml methanol. First methanol and nano catalyst was mixed and allowed them to stir for 1 h at 45 °C till the methoxide was formed. Placed round bottom flask in Muffle furnace. Condenser was used to condense the gases formed during the process. 10 ml of the crude oil was pour into the round bottom flask already containing the mixture of nanocatalyst. After 2 h heating was stopped and the 2 layers are formed in the round bottom flask. Upper layer is of biodiesel and the lower layer is of soap and glycerin. Solution was then filtered with the help of Whatman filter paper. Biodiesel was separated and was collected for the analysis. Fourier transforms infrared spectroscopy FTIR analysis was performed on a BRUKER VECTOR 22 spectrophotometer device. Table 1 shows the functional group obtained from FTIR spectra. The biodiesel produced from popular wood sawdust crude oil were analyzed using FTIR spectrophotometry. The spectra as shown in Figure 2. The broad band located at approximately 3383 cm -1 are related to O-H vibrations of the hydroxyl groups The band located at approximately 2967 cm -1 are related to C-H vibrations of the methylene groups and to stretching and contraction vibrations of the methyl group. The intense peak located at 1708 cm -1 corresponds to carbonyl radical and is characteristic of esters. The band located at 1371 cm -1 corresponds to the asymmetric stretching of the C-H bond and the asymmetric bending of the functional group. There was a set of bands between 1371 and 1288 cm -1 that were associated with asymmetric vibrations of the C-C (=O)-O and O-C-C bonds. The high intensity peak located at 1009 cm -1 was attributed to the symmetric angular deformation of the C-H bond of olefins. Results and Discussions X-ray diffraction analysis: Generally, XRD can be used to characterize the crystallinity of nanoparticles, and it gives the average diameters of the nanoparticles. The precipitated fine particles were characterized by XRD for structural determination and estimation of crystallite size. Further, the formation of oxide could be confirmed from the X-ray diffraction analysis of the samples. Figure 3 indicates the X-ray diffractograms of the cobalt oxide nanoparticles obtained by co-precipitation. Each diffractogram show the peaks characteristic for the cobalt oxide of ~ 31.4°, 36.1°, and 65.7°. The crystallite size of cobalt oxide nanoparticles were 46 nm. The crystallite size determined by Scherer formula. Figure 4 Support the microcrystalline nature of the particle after calcinations with least degree of agglomeration. Particles seem to have an irregular shape with chemical homogeneity with uniform morphology and with good porosity due to an inter particle surface connectivity. It was observed as the annealing temperature increases the crystalline nature of the particle changes. Brunauer-emmett-teller (BET) The surface area and pore volume was found to be 33.0715 m 2 /g supporting the presence of limited porosity with a pore size distribution. These values support their industrial applications as a catalyst in organic reactions [26]. Fourier transforms infrared spectroscopy The functional group analysis of pyrolysis oil was carried out by using Fourier Transform Infrared Spectroscopy (FTIR). The FTIR spectra obtained for pyrolysed oil was given in Figure 5. FTIR analysis was performed on a BRUKER VECTOR 22 spectrophotometer device. Table 2 shows the functional group obtained from FTIR spectra. The biodiesel produced from palm oil were analyzed using FTIR spectrophotometry. The spectra as shown in Figure 5. The band located at approximately 2924 and 2852 cm -1 are related to C-H vibrations of the methylene groups and to stretching and contraction vibrations of the methyl group. The intense peaks located at 1713 cm-1 correspond to carbonyl radical and is characteristic of esters. The band located at 1457 cm -1 corresponds to the asymmetric stretching of the C-H bond and the asymmetric bending of the functional group. There was a set of bands between 1009 and 1374 cm -1 that were associated with asymmetric vibrations of the C-C (=O)-O and O-C-C bonds. The high intensity peak located at 900 cm -1 correspond to deflections out of the plane of the C-O group and the one located at 1009 cm -1 was attributed to the symmetric angular deformation of the C-H bond of olefins. Gas chromatography-mass spectrometry (GC-MS) The chemical composition of the prepared biodiesel was determined by Gas chromatography. For this purposeGC-6890N model coupled with mass spectrometer; model MS-5973 MSD (mass selective detector) was used. A DB-5MS capillary column (30 × 0.2 m 2 ) was used to separate the different compounds present in the diesel. Helium was used as a carrier gas. The temperature of capillary column was raised from 120-300°C at a fixed rate of 10°C/min. Biodiesel in chloroform (0.1 µL) was injected as a sample using split mode with split ratio of 1:10. The scan rate of mass spectrometer was set from 50-550 m/z. Electron Impact (EI) mode of ionization was used. Fatty Acid Composition of Palm Oil Methyl Ester (POME) was determined using gas chromatography. The study of chemical properties of oils before the experimental work plays an essential role in understanding the behavior and the structure of materials. This study focused on using the Palm Oil Grade III (POG-III) as a renewable and sustainable raw material for biodiesel production. Usually, Palm Oil Grade III (POG-III) is traded based on the FFA content, moisture and impurities. Table 3 Illustrates fatty acid composition of Palm Oil Grade III (POG-III). The results showed that the prevalent fatty acids available were oleic, palmitic, lauric and stearic acid. Saturated fatty acids in palm oil grade III (POG-III) were 44.02 weight % while unsaturated fatty acids were 55.96 weight %. Due to its high percentage of saturated fatty acids and free fatty acids, Palm Oil Grade III (POG-III) exists in semi-solid phase at room temperature (28 ± 2°C). Hence, Palm Oil Grade III (POG-III) has higher pour and cloud points as compared to normal palm oil. Higher saturated fatty acids in oils give a higher cetane number, and the oil is less prone to oxidation. The fatty acid composition of biodiesel from the Palm oil grade III biodiesel is shown in Table 4. Fuel Properties of Biodiesel Biodiesel fuel characterization is done by the following parameterscetane number, cloud point, flash point, sulfur contents, density and acid value, pour point, ash contents, kinematics viscosity. The mentioned parameters are discussed below in detail [27]. Flash point Flash point shows the flammability hazards of the fuel while in the air presence. Flash point is important in a sense that it is useful for storage, handling and safety purposes. Flash point of the prepared biodiesel was 179.8°. It is a higher value than the ordinary petroleum fuels which shows that the prepared biodiesel is less volatile and it is for the storage and transportation purposes. Kinematic viscosity The flow rate of fuel is determined by the kinematic viscosity. Kinematic viscosity also controls the atomization of fuel. Viscosity is a factor that hinders the flow of fuel and the engine life is affected by the high levels of viscosity as it causes knocking and poor fuel atomization. Low levels of viscosity in the fuel are good because fuel will be easily propelled and fine droplets are formed. High level of viscosity results in the blockage of fuel injector as the fuel does not burn fully and deposits are formed. Prepared biodiesel has viscosity within the limits. Sulphur content Presence of sulphur in the biodiesel should also be known because if the sulphur is more in the diesel its burning will be effecting the environment. In the prepared biodiesel the sulfur content is much less as compared with the standard values hence the biodiesel made from the palm oil is environment friendly. Cloud point and pour The temperature at which wax crystal clouds are formed in the diesel fuel when it is cooled is said as cloud point and the temperature at which the fuel flows is called as pour point. Cloud and pour point of the prepared biodiesel are in the range of standards so the diesel can be used both in moderate and high temperature climates. Density Limit which is favorable: While designing a fuel injection system density is very important. Higher density causes the greater mass delivery of the fuel and fuel is consumed in a large quantity which is not good. Density of the prepared biodiesel is much less than the given standard values and it is favorable for use. Table 5 presents the biodiesel specifications following EN 14214 standard [28][29][30]. Conclusion The findings achieved in this work showed that the biodiesel produced from the Palm Oil Grade III (POG-III)met the European standard specifications but the ester content of the treated Palm Oil Grade III (POG-III) was slightly lower than the international standard limit. This can be rectified by optimising the transesterification reaction. The phosphorus value represents the phosphatide (gum) content in the fuel and it was found at a very low concentration. The flash-point and copper-strip corrosion tests were found to be highly suitable as operational safety properties for biodiesel compared with petroleum diesel. In general, the biodiesel produced achieved favourable scores with reference to the EN 14214 standard [28].	2019-04-30T13:05:14.769Z	2017-08-31T00:00:00.000	{ "year": 2017, "sha1": "fc40048a28d6fcf00ce0cdb5a23a7a00bbc5e6c8", "oa_license": "CCBY", "oa_url": "https://doi.org/10.4172/2576-1463.1000170", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "7a897714d8cd493130b6195aa31786b05966b0d5", "s2fieldsofstudy": [ "Environmental Science", "Chemistry", "Engineering" ], "extfieldsofstudy": [ "Environmental Science" ] }
236881600	pes2o/s2orc	v3-fos-license	Automatic recognition of suprasegmentals in speech This study reports our efforts to improve automatic recognition of suprasegmentals by fine-tuning wav2vec 2.0 with CTC, a method that has been successful in automatic speech recognition. We demonstrate that the method can improve the state-of-the-art on automatic recognition of syllables, tones, and pitch accents. Utilizing segmental information, by employing tonal finals or tonal syllables as recognition units, can significantly improve Mandarin tone recognition. Language models are helpful when tonal syllables are used as recognition units, but not helpful when tones are recognition units. Finally, Mandarin tone recognition can benefit from English phoneme recognition by combining the two tasks in fine-tuning wav2vec 2.0. INTRODUCTION Suprasegmentals are phonological units in speech that are larger than segments (i.e., consonants and vowels), such as syllables, lexical stress, tones, and intonation [1]. In this study, we propose using wav2vec 2.0 [2] fined-tuned with a Connectionist Temporal Classification (CTC) loss [3] for automatic recognition of suprasegmentals, similar to the approach used by [2] for phoneme recognition on TIMIT [4]. Speech segments and suprasegmental units are different not only in the domain of realization, but also in their features. "Suprasegmental" has often been used to refer to the phonetic features of suprasegmental units, and used interchangeably with prosodic features. It is well accepted that suprasegmentals are mainly distinguished by pitch, duration, and energy, whereas segments are distinguished by spectral information. There are studies, however, suggesting that spectral information may be also important for suprasegmentals. For example, [5] found that spectral balance is a reliable acoustic correlate of lexical stress, and [6,7] demonstrated that MFCCs outperform prosodic features for automatic recognition of Mandarin tones. With the advent of deep learning and end-to-end models, feature engineering has been largely abandoned with feature representations learned implicitly during training by the neu-ral network. In earlier work, these featured were learned in a supervised fashion from paired audio and transcripts [8]. However, more recent work has focused on unsupervised learning of representations using only audio [9,10,2], which are then used by downstream tasks such as speech-to-text. When compared to conventional acoustic features such as MFCCs, these representations substantially lower the amount of labeled data needed to train state-of-the-art speech-to-text systems for English [2] and low-resource languages [11]. Similarly encouraging results have been demonstrate for speaker recognition [9] and phone recognition [2]. However, it is not yet clear how well these representations perform for suprasegmental recognition, which requires the network to learn prosodic instead of, or in addition to, spectral features and representations. We conducted experiments with fine-tuning wav2vec 2.0 models using CTC for recognition of suprasegmentals, including syllables, tones, and pitch accents. We also made an effort to improve recognition of Mandarin tones by utilizing segmental information. The main results of our study are as follows: 1. We demonstrate that fine-tuning wav2vec 2.0 with a CTC loss can improve the state-of-the-art for automatic recognition of suprasegmentals, including syllables, tones, and pitch accents. Wav2vec 2.0 is a framework for self-supervised learning of speech representations. The speech signal is processed by a multilayer convolutional network to obtain latent representations every 25 ms, which are then fed into separate vector quantization and transformer networks. Training is performed using a noise contrastive estimation task in which consecutive sequences of frames of latent representations are masked and the network required to identify the correct quantized representation for each masked time step from a set of distractors sampled from the other masked time steps. This selection is made on the basis of cosine similarity between the quantized representations and the outputs of the transformr network. Pre-trained wav2vec models can be fine-tuned for speech recognition with labeled data and a CTC loss. [2] demonstrated that this approach achieved 1.8% word error rate on the test-clean set of Librispeech [12] with a Transformer language model, and 8.3% phone error rate on TIMIT test set without a language model. [11] applied wav2vec 2.0 to speech recognition in low-resource languages. The paper reported more then 20% relative improvements in six languages compared with previous work. It found that using coarse-grained modeling units, such as subwords and characters, achieved better results than fine-grained modeling units, such as phones and letters. Fine-tuning wav2vec 2.0 has also been used to perform other tasks such as speech emotion recognition by adding a classification head on the top of the network. [13] proposed a multi-task learning framework to simultaneously perform speech recognition and emotion classification with wav2vec 2.0, achieving the state-of-the-art performance on speech emotion recognition. Syllable detection Syllables play a crucial role in speech production and perception, and child language acquisition [14,15]. Automatic segmentation of speech into syllables has attracted research interests for decades. [16] proposed a method of syllable detection based on "assessment of the significance of a loudness minimum to be a potential syllabic boundary from the difference between the convex hull of the loudness function and the loudness function itself." [17] found that that even simpler methods, based on selecting peaks in a smoothed amplitude contour, also perform quite well on this task. Automatic detection of syllables has also been applied for speaking rate estimation. Motivated by the work of counting objects in an image, [18] proposed a more direct way of estimating the speaking rate that does not require segmentation, detection, or peak counting. Their approach achieved a correlation of 0.89 between estimated number of syllables and actual number of syllables on TIMIT test utterances. The SR error rate, i.e., the average of syllable recognition errors across TIMIT test utterances, was 12.2%. [19] proposed a signal processing pipeline for syllable detection and speaking rate estimation that is optimized based on the direct minimization of naturally arising task-specific objective functions. This approach achieved a correlation of 0.917 and SR error rate of 9.94% on TIMIT test set. Mandarin tone recognition Mandarin Chinese is a tone language. It has four lexical tones, Tone1 to Tone4, plus a neutral tone, Tone5. While the primary acoustic correlate of tones in Mandarin Chinese is fundamental frequency, i.e., F0, other phonetic parameters such as duration, amplitude, vowel quality, etc., are also involved in the production and perception of tones [20]. Automatic recognition of Mandarin tones in running speech has been a challenging task due to tonal coarticulation [21], tone sandhi [22], the interaction between tone and intonation [23], and speaker variation [24]. [25] presented an example, for example, showing that the second syllable ying4, which is a lexical falling tone, could be realized as rising in F0 in the phrase of "fan3 ying4 su4 du4" ("reaction time"). The interaction between tones and segments has also been documented in the literature [26]. The employment of deep learning models for Mandarin tone recognition has gained success in recent years. [6] built a deep neural network to classify tones in Mandarin Chinese using MFCCs. The system achieved a significant improvement compared to traditional methods using prosodic features on the task, despite the omission of F0 or other pitch-related features. [27] studied the effectiveness of articulatory information for Mandarin tone recognition in a DNN-HMM framework. The paper confirmed that the DNN model may be able to extract more useful information from the MFCC parameters for tone recognition. It also found that incorporating the articulatory information into tone modeling can further improve tone recognition, by either explicitly adding the articulatory features or building phone-dependent tonal models. [28] propose a method for tone recognition using a convolutional neural network with CTC. This method achieved a tone error rate of 11.7% on the Aishell-1 dataset. [29] proposed a multi-scale model which can gather information at multiple resolutions to better capture the characteristics of tone variations, achieving competitive results on the Chinese National Hi-Tech Project 863 corpus with TER of 10.5%. [30] reported that feeding both the Mel-spectrogram and the short term context segment features into an end-to-end model could significantly improve automatic speech recognition, improving the classification accuracy from 79.5% to 88.7% on the Aishell-3 database. Pitch accent detection In ToBI and ToBI-style intonation transcription, pitch accents and boundary tones are local intonational events and the basic units of intonation [31]. Speakers of English produce certain words in an utterance with special intonational prominence. These pitch-accented words are typically realized with increased duration, intensity, and fundamental frequency. The task of automatic pitch accent detection has attracted a considerable amount of research attention. A thorough review of early (prior to 2009) approaches using acoustic and lexical features and a variety of classification models can be found in [32]. In two more recent studies, [33] presented a CNN-based model for this task; and [34] extended the model to make greater use of context by using full utterances as input and adding an LSTM layer. The studies achieved 87.5% and 88.7% accuracy, respectively, on pitch accent detection on American English speech in the Boston University Radio News Corpus. Most reported studies of pitch accent detection were conducted on the Boston University Radio News Corpus. The corpus contains seven hours of speech from seven speakers, but only subsets of the corpus are labeled with phonetic alignments and intonation markers. It also does not provide a split of train and test sets. Because of these reasons, previous studies based on this corpus have trained and tested on different amounts of data and therefore cannot be easily compared on their performance. For example, Both [33] and [34] conducted 10-fold cross validation on the entire dataset, whereas [35] used 78% of the data from three female speakers only for training and the other 22% for testing. FINE-TUNING WAV2VEC 2.0 FOR AUTOMATIC RECOGNITION OF SUPRASEGMENTALS Out procedure for fine-tuning wav2vec 2.0 for suprasegmental recognition is illustrated in Figure 1. The framework is the same as phoneme recognition. In phoneme recognition, phonemes are used as recognition units and the model is fine-tuned using speech waveforms paired with phoneme sequences. In suprasegmental recognition, suprasegmental units are used to replace phonemes as recognition units. A randomly initialized linear projection is added on top of the contextual representations of wav2vec 2.0 to map the representations into suprasegmental units such as syllables, tones, and pitch accents, and the entire model is optimized by minimizing the CTC loss. Our experiments were conducted using fairseq. 1 In all experiments, the wav2vec 2.0 large model pre-trained on 960 hours of Librispeech audio (libri960 big.pt), was used for fine-tuning. For the first 10k updates only the output classifier is trained, after which the Transformer is also updated. The max tokens was set to 1.1 million (which is equivalent to 68.75-second audio with sampling rate of 16 kHz), the learning rate was 5e-5. Other details are described below. Syllables: The TIMIT dataset was used [4]. The model was trained on TIMIT train set and tested on TIMIT test set. The total number of fine-tuning updates was 20k. The vocabulary contained only one token, 'S', which represents a syllable (plus four special tokens that added by fairseq, <s>, </s>, <pad>, and <unk>). To generate target labels we simply replaced all vowels in the TIMIT phonetic transcriptions with "S" and ignored consonants and other symbols. For example, "h# sh ix hv eh dcl jh ih dcl d ah kcl k " → "S S S S". Two measures were used to evaluate the performance and compare with previous studies: the correlation between estimated number of syllables and actual number of syllables for utterances in the test set; and the syllable recognition error rate on the test set (because the inference output contains only one type of tokens, 'S', there are no substitution errors but only insertion and deletion errors). From the results listed in Table 1, we can see that our approach greatly improved previous results. The correlation was improved from 0.917 to 0.984, and the syllable recognition error rate was reduced by 70%, from 9.9% to 2.9%. Mandarin tones: Experiments of Mandarin tone recognition were conducted on three datasets: Hub-4 [36], Aishell-1 [37], and Aishell-3 [38]. The Hub-4 dataset is the same as used in [6], which contains 7549 utterances for training and 300 utterances for testing. The utterances were extracted from 20 news announcers in the 1997 Mandarin Broadcast News Speech corpus [36]. The Aishell-1 and Aishell-3 datasets were downloaded from openSLR. Aishell-1 2 contains 165 hours of read speech in Mandarin Chinese from 400 speakers. The speakers are from different dialect regions but most are from northern areas. The corpus includes training (150 hours), development (10 hours), and test (5 hours) sets. Aishell-3 3 contains 85 hours of Mandarin speech from 218 native Mandarin Chinese speakers. The corpus has a split of training and test sets, with 174 and 44 speakers, respectively. Aishell-3 provides pinyin transcripts. For Hub-4 and Aishell-1, however, only word transcripts are provided. We trained a forced aligner for each of the two datasets, with the Callhome Mandarin Chinese Lexicon [39] and the lexicon contained in Aishell-1, respectively, and ran forced alignment to obtain pinyin transcripts for these datasets. The tone marks in the pinyin transcripts were extracted and used as tone labels for the experiments. The vocabulary in the experiments contains five tokens, Tone1 to Tone5. For Aishell-1, its development set was used to find the optimal number of updates. For Hub-4 and Aishell-3, we randomly split the test set into two parts, with one part for development and the other part for testing. We then switched the development and test data to complete testing on the entire test set. The results are listed in Table 1. Our approach significantly outperformed previous studies, reducing tone recognition errors by 50% or more on all three datasets. Table 1 also lists results from two studies on Mandarin Tone recognition using the Chinese National Hi-Tech Project 863 corpus. We don't have access to this corpus. Pitch Accents: The Boston University Radio News Corpus was used for this experiment. In the corpus, pitch accents are labeled with time stamps but not on words or syllables. Preprocessing is needed to map pitch accent labels to words or syllables using phonetic alignment information. Because not all pitch accents are labeled within a word boundary in the corpus, researchers have used different practices. In [34] there are 28,489 total word tokens, and 15,544 (54.6%) of which carry pitch accents, whereas in [33] there are 26,742 total word tokens, and 13780 (51.5%) of which carry pitch accents. From our processing of the dataset, we got 30,330 word tokens, and 15,511 (51.1%) of which carry pitch accents. To compare with previous studies, we conducted 10-fold cross validation on the entire data. In every fold, 10% of the data was used for testing, 10% for development, and the remaining 80% for training. In the experiment we first mapped pitch accents to syllables. Every syllable token has a target label of either "1" (pitch accent) or "0" (no pitch accent). Wav2vec 2.0 was, therefore, fine-tuned to recognize two types of syllables, with a pitch accent or without a pitch accent. The inference consists of a sequence of "0"s and "1"s. If any frame within the boundaries of a word has an output of "1" from inference, the word is identified as bearing a pitch accent. The accuracy of pitch accent detection, i.e., the correct identifications divided by the total number of word tokens, is reported in Table 1. We can see that compared to previous studies, our method improved the accuracy from 88.4% to 89.5%. Units for tone recognition In addition to tones, we also tried two other types of units for recognition of Mandarin tones: initials & tonal finals (fi-nals+T), and tonal syllables (syllables+T). Examples of transcription using these units are illustrated below: • Sentence: 她的表现也更加全面 • Tones: T1 T5 T3 T4 T3 T4 T1 T2 T4 • Initials & finals+T: t a1 d e5 b iao3 x ian4 ii* ie3 g eng4 j ia1 q van2 m ian4 • Syllables+T: ta1 de5 biao3 xian4 ye3 geng4 jia1 quan2 mian4 (* "ii" represents a zero-initial as defined in the lexicon of aishell-1) For evaluation, the inference output of the model on tones was directly used as the result of tone recognition. The outputs of the other two models were converted to tones for calculating tone error rates. The units of initials in the output were ignored, and tonal finals and tonal syllables were changed to tones by discarding the segmental information, for example, "t a1 d e5" → "T1 T5", and "ta1 de5" → "T1 T5". Table 2 lists the results of the three models. The model using tones as recognition units had a tone error rate of 5.5%. Using initials and tonal finals as recognition units, the error rate was reduced to 5.0%. The best model used tonal syllables as recognition units, which achieved a tone error rate of 2.8% on the test set of Aishell-1. The effect of language model In the experiments above no language models were used in decoding. To study the effect of language models on Mandarin tone recognition, we trained language models using progressively increasing amounts of text data. Three text corpora were used for this purpose: Aishell-1 word transcriptions, Lancaster Corpus of Mandarin Chinese, and Chinese Gigaword Fifth Edition. We trained language models of both tones and tonal syllables, to use with wav2vec 2.0 and CTC trained on these units for decoding. To train language models of tonal syllables we converted the Chinese characters in the corpora to pinyin with tone marks, using a Python package called pypinyin 4 . The tonal syllables in pinyin were then converted to tones for training language models of tones. For example, "ta1 de5 biao3 xian4 ye3 geng4 jia1 quan2 mian4" is a sample of training data of tonal syllables and "T1 T5 T3 T4 T3 T4 T1 T2 T4" is a sample of training data of tones. 6-gram language models were trained using kenlm, and used for decoding with CTC. Table 3 lists the results in tone error rate for language models trained on different amount of text data, for tonal syllables and tones being recognition units, respectively. From Table 3, we can see that language models help tone recognition when tonal syllables are used as recognition units, but they don't help when the recognition units are tones. With a language model trained on 90M sentences, using tonal syllables achieved a tone error rate of 1.7% on the test set of Aishell-1, which is dramatically lower than using tones as recognition units (5.5%). Table 4 we can see that the tone error rate can be significantly reduced when combined with English phoneme recognition in fine-tuning. Trained on Hub-4 only, the tone error rate was 6.8% (after 20k updates) on its test set. When combined with TIMIT phoneme recognition, the error rate can be lowered to as low as 4.4%. From Table 4 we can also see that the TIMIT phoneme error rate was only 2.3% when canonical phonemes were used for recognition, which is greatly lower than the error rate of 8.3% from using transcribed phones (as reported in [2]). This difference is interesting. It may provide insight into why wav2vec 2.0 and CTC perform well on speech recognition, and may suggest a mismatch between speech production and perception, e.g., phonetic reduction vs. perceived deletion. CONCLUSIONS We demonstrate that fine-tuning wav2vec 2.0 with CTC can improve the state-of-the-art on automatic recognition of suprasegmentals, including syllables, tones, and pitch accents. Compared to previous studies, the method achieved 70% error reduction on syllable detection, 50% error reduction on Mandarin tone recognition, and 10% error reduction on pitch accent identification. Segmental information is helpful in Mandarin tone recognition. Employing tonal syllables as recognition units can significantly improve Mandarin tone recognition, compared to using tones as recognition units. Furthermore, language mod-	2021-08-04T01:16:08.456Z	2021-08-02T00:00:00.000	{ "year": 2021, "sha1": "6f31319545e5ec436c45e6142dff080795b78933", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "6f31319545e5ec436c45e6142dff080795b78933", "s2fieldsofstudy": [ "Computer Science", "Linguistics" ], "extfieldsofstudy": [ "Computer Science" ] }
235792866	pes2o/s2orc	v3-fos-license	Cultural Differences in People’s Psychological Response to COVID-19 The present research studied Chinese and Euro-Canadian students during the COVID-19 pandemic, focusing on their affect, optimism, well-being, and meaning in life. The results revealed both differences and similarities across cultures. As predicted, Chinese participants reported more positive affect and less negative affect, higher optimism, higher state psychological well-being, and higher meaning presence, compared to Euro-Canadian participants. The findings were replicated after a week’s delay. Analyses on longitudinal data showed that state optimism, state well-being, and meaning presence influenced one another over time. These variables also mediated the cultural differences in one another. These results are consistent with cultural work on naïve dialecticism and non-linear lay theory of change. Results also demonstrate underlying relationships among the constructs that are common to both cultural groups. Broadly, the present research highlights the impact of culture on people’s response to challenging life situations and the mechanisms underlying these cultural differences. Since December 2019, a new form of coronavirus has turned the world upside down. The pandemic has not only caused mass quarantines and deaths, but also generated high levels of stress due to fear of the virus and physical/social isolation. In Asia, divorce rates have skyrocketed unexpectedly, allegedly due to couples becoming fed up with each other from experiencing extended selfquarantine (Prasso, 2020;Sun, 2020). Travel bans, panic buying, and the closure of public spaces have all contributed to giving the world a harmful upsurge of stress and negativity, making the pandemic's effects worse than they already are. Despite all the difficulties and challenges presented by the pandemic, people are trying to react positively. Some people have taken the pandemic as an opportunity to bond with their families and friends, while others are reacting positively by offering help to their communities. These observations are in line with various ways of constructive coping, such as reacting positively to significant stressors (Dyer and McGuinness, 1996;Sinclair and Wallston, 2004), imbuing the future with a positive outlook (Scheier and Carver, 1985;Dember et al., 1989), and extracting positive meanings from bad experiences (Masten et al., 1990;O'Leary and Ickovics, 1995). The present research examines people's psychological responses during the pandemic from a cross-cultural perspective, comparing Chinese and Euro-Canadian students. This research question was motivated by prior work, which revealed the role of culture in the way people think about and understand the world (e.g., Peng and Nisbett, 1999;Ji et al., 2001;Lee et al., 2020), with implications for how they respond to highly aversive events. Applying such cultural differences to the context of suggests that compared to Euro-Canadians, Chinese may be more inclined to react to the pandemic with positivity in terms of optimism, well-being, and meaning in life, compared to Euro-Canadians. We discuss these constructs in turn. Optimism and Culture Under the threat of a life-changing event, such as a pandemic, how do people brighten the seemingly dim future? What mental processes are in operation when people manage to keep their hopes high? These questions are related to optimism, which is typically known as the positive expectations people have about the future (Scheier and Carver, 1985;Dember et al., 1989). In the literature, optimism has been conceptualized and measured as a stable trait assumed to change little over time (e.g., Carver, 1992, 2018). This assumption, however, has been challenged by recent research with a state view of optimism, which assumes that people's expectations about the future are fluid, subject to environmental cues, context-specific, and thus malleable in response to salient reminders (Millstein et al., 2019). In this view, state optimism may fluctuate in the context of the COVID-19 pandemic. As a psychological construct, optimism has been shown to play a key role in well-being. For example, people who are optimistic are less likely to use alcohol (Wray et al., 2013) or drugs (Carvajal et al., 1998) than people who are not optimistic. When times are tough, optimism has been shown to predict a host of favorable emotional and behavioral outcomes, including problem-focused coping (Friedman et al., 1992;Fournier et al., 2002), positive reappraisal (Slattery et al., 2017), active goal pursuit (e.g., Carver et al., 1983), and health-enhancing behaviors (Robbins et al., 1991). On the flipside, optimism is negatively associated with distress emotions (Aspinwall and Taylor, 1992), PTSD symptoms (Frazier et al., 2011) and suicidal behaviors (Fawcett et al., 1987). In principle, optimism has to do with positive anticipations when things go wrong. That is, people who are optimistic tend to have the anticipation that a situation, no matter how dark it feels at the moment, will eventually be replaced by a brighter outcome. Such a view is highly congruent with the non-linear theory of change (Ji et al., 2001;Ji, 2008) common in East Asian cultures, which assumes that everything in the world is in constant transformation between negativity and positivity, like the waxing and waning of the moon. Cultural differences in optimism are also compatible with naïve dialecticism (Peng and Nisbett, 1999), a mental pillar of reasoning in East Asian cultures which assumes that contradictions are the foundation of life. Through the window of naïve dialecticism, life events, no matter how bad, would always contain the seed of good, and vice versa. Both the non-linear theory of change and naïve dialecticism have been shown to be stronger among Chinese than among North Americans (see Spencer-Rodgers et al., 2010a for a review). In line with this claim, past work has shown that nonlinear thinking Chinese participants were more likely to appraise the SARS outbreak with optimism and positive thinking-for example, they reported more positive changes to their lives-compared to Euro-Canadian participants who assumed linearity in change (Ji et al., 2004). Furthermore, cultural differences in optimism may have to do with the fact that suffering in life is construed more positively by Chinese than by Euro-Canadians , corresponding to the themes of Buddhism and Taoism in East Asian cultures (Ji et al., 2010). Furthermore, Ji et al. (2021) have shown that Chinese participants were more optimistic than Euro-Canadians in response to negative events, traceable to cultural differences in the non-linear theory of change. In sum, optimism is associated with a wide range of mental, emotional, and behavioral outcomes, many of which are beneficial for people who are going through life adversities. Drawing from past research, we hypothesized that in the context of the COVID-19 pandemic, Chinese participants would feel more optimistic than would Euro-Canadian participants (Hypothesis 1). Optimism aside, would culture play a role in other responses to life adversities, such as the recognition of meaning in life and psychological well-being? Meaning in Life and Culture When people are faced with adversities, finding meaning in life can contribute to optimism. At a broad level, meaning in life has been discussed under the grand theme of purposefulness (e.g., Battista and Almond, 1973;Klinger, 1977). At a more concrete level, meaning is manifested in the awareness of purposes or significance (Ryff and Singer, 1998) from the environment that are otherwise hidden from view. For example, a person bound to a wheelchair may discover the presence of meaning by turning to art. Likewise, instead of dwelling on the bad side of the COVID-19 pandemic, some people may come to recognize new meaning in life through new hobbies, unexplored career paths, tighter bonds with loved ones, or the appreciation of nature. The recognition of meaning often involves the discovery of new perspectives. Life adversities may feel less painful when a person manages to take a step back from the problems at hand, understand them in a broader context, and integrate new perspectives with old ones. Deriving meaning through different perspectives is a key ingredient of wisdom, according to empirical scientists (e.g., Baltes and Staudinger, 2000;Grossmann et al., 2013) as well as philosophers. In particular, Taoism and Buddhism are known for their emphasis on perspectives as a source of wisdom (Yamamoto, 1998;Birren and Svensson, 2005). Knowledge makes people smart, but to be wise, one must go beyond the surface of things, attend to a broader context for underlying trends, and generate new interpretations from multiple perspectives, even if they contradict one another. The acquisition of new meaning through contexts, trends, and perspectives is consistent with naïve dialecticism and the nonlinear theory of change -both of which are more prevalent in East Asian than North American cultures (e.g., Peng and Nisbett, 1999;Ji et al., 2001;Ji, 2008). This claim is supported by empirical work. For example, studies have shown that compared to North Americans, East Asians tend to have a greater focus on contextual information (Morris and Peng, 1994;Ji et al., 2000), a wider mental frame for temporal patterns and trends (Ji et al., 2009(Ji et al., , 2019, and a stronger tendency for perspective-taking (e.g., Cohen et al., 2007;Wu and Keysar, 2007). The awareness for unapparent information, such as seeing positive meaning in a negative situation (e.g., fights between a couple can get them to know each other better) or finding insight in the mundane (e.g., the awe of nature through gardening), is also more common in East Asian than North American cultures (Ji et al., 2004Grossmann et al., 2014Grossmann et al., , 2016. All these findings seem to suggest cultural differences in finding meaning when life is troubled by adversities. Research has shown positive connections between trait optimism and meaning in life. For example, optimism has been shown to predict meaning in life (Kealy et al., 2020). Trait optimism facilitates subjective well-being and good health (Wrosch and Scheier, 2003). Likewise, finding meaning in life positively predicts optimism, which in turn increases psychological well-being (Ho et al., 2010). The association between meaning in life and optimism is found in Asian Americans and European Americans (Yu and Chang, 2019), and in young and older adults (Krause, 2003). Thus, the relationship between optimism (as a trait, at least) and meaning in life seems to be bidirectional, although most of these findings were based on cross-sectional research and trait measures of optimism. Optimism is also consistently associated with psychological well-being. In a longitudinal study, Daukantaite and Bergman (2005) examined participants' optimism at age 13 and their subjective well-being at age 43. Results showed that measures of optimism at an early age consistently predicted subjective well-being at later stages in life, suggesting that the role of optimism in well-being is robust and stable over time. Numerous other studies have revealed similar patterns (e.g., Scheier and Carver, 1992;Halama and Dedova, 2007;Ho et al., 2010), demonstrating associations between optimism and a host of benefits, including subjective well-being (Aglozo et al., 2019) and conceptually related constructs such as positive affect (Carver et al., 2010) and life satisfaction (Duffy et al., 2013). Wellbeing and its relationship with culture will be discussed in greater details below. In sum, the tendency to view the future in a positive light may promote people's well-being and a sense of meaning in their lives as the COVID-19 pandemic looms over the world. Success in finding meaning often stems from the tendency to examine an existing event from new perspectives. Such tendency may be more apparent among East Asians than among North Americans, as past findings suggest. Applying these findings to context, one may expect a stronger inclination to find meaning in the COVID-19 pandemic among Chinese participants than among Euro-Canadian participants (Hypothesis 2). Considering that optimism, meaning in life, and well-being are intertwined and that cultural differences are expected in optimistic response to the pandemic, one may also expect cultural differences in meaning and well-being. Psychological Well-Being and Culture People who feel hopeful when things go wrong, appreciate the good sides in bad, or see new meaning when there seems to be none, should, in principle, enjoy greater psychological wellbeing than people who do not. What does the literature say about this? For decades, psychological well-being has been recognized as one of the most crucial constructs of social life, studied by psychologists in multiple fields. In one approach, typically known as the hedonic approach, well-being concerns the extent to which one feels happy or satisfied with life. This approach essentially involves the attainment of pleasure and avoidance of pain, manifested in positive and negative emotions (Ryan and Deci, 2001;Diener, 2009). In another approach, typically known as the eudaimonic approach, well-being concerns the ability to strive for true potentials and life achievements. This approach essentially involves personal growth through the pursuits of wisdom and virtues, and is closely related to finding meaning in life (Ryff and Keyes, 1995;Ryan and Deci, 2001). Both approaches emphasize different aspects of well-being and, together, they represent a good and fulfilling life that is worth living. But what constitutes a good, fulfilling life? It depends on who is asking. For example, prior work has revealed cultural differences in the hedonic component of well-being, operationalized by the experience of positive affect. Results showed that while positive affect is generally appealing to everyone, culture does play a role in the kinds of positive affect people find as important or ideal for well-being. For example, North Americans are known for their emphasis on self-centered affect (e.g., pride, anger), or affect that concerns the attributes of the self, whereas East Asians tend to emphasize other-centered affect (e.g., respectful, shame), or affect that stems from significant others (Chow and Berenbaum, 2012). Hedonic differences in well-being are typically linked to the awareness for social contexts, more prevalent in East Asian than North American cultures (Nisbett, 2003). From other work, we also learn that affective experiences and well-being may have to do with the cultural assumptions people have about the world. For instance, unlike many North Americans, East Asians tend to perceive affective experiences and well-being as fluid, impermanent, and always changing, compatible with naïve dialecticism and a non-linear view of the world (Uchida and Kitayama, 2009). Similar observations have emerged from other studies (e.g., Miyamoto and Ma, 2011; see also Spencer-Rodgers et al., 2010a). These perspectives suggest that positive and negative affect need not be on the two ends of a continuum; the presence of one does not imply the absence of the other. Consequently, compared to North Americans whose tend to have polarized experience of affect (e.g., feeling happy with not sad), East Asians are more inclined to experience the co-occurance of opposing affect, such as feeling happy and unhappy at the same time (e.g., Bagozzi et al., 1999;Kitayama et al., 2000;Napa Scollon et al., 2005;Spencer-Rodgers et al., 2010b). This body of work is consistent with cultural differences in well-being, with East Asians more likely to perceive negativity as the seeds of positivity (e.g., Ji et al., 2001), respond to negative events with flexibility (Cheng, 2009), or, more generally, "finding the good in the bad" (Spencer-Rodgers et al., 2010a), relative to North Americans. Collectively, these findings forge links with other research showing that the thinking style of East Asians may dilute the negative impacts of stressful events and contribute to how pleasant, positive, or satisfied they feel about the stressful events (Ji et al., 2001;Hou et al., 2003). Another common measure for psychological well-being focuses less on affective experiences per se and more broadly on the things that make a life good, satisfying, fulfilling, and worth living for. Is well-being nothing but a large bag of positivity? Or is it a delicate balance between happy and sad times? It depends. To many North Americans, life is satisfying when it is imbued by positive events. In contrast, to many East Asians, life is satisfying when it is a good mix of happy and sad times (Oishi, 2002). These findings resonate with other work (Ji et al., 2001), showing that Euro-Americans expected life happiness to follow a linear trend, whereas Chinese expected life happiness to change in a nonlinear way -to them, happiness and unhappiness transform to each other over time. Together, these findings highlight the impact of culture on well-being, in terms of what it constitutes and how it is manifested during challenging times. To the extent that optimism is a predictor of well-being, Chinese participants should, by implication, report greater wellbeing in response to the pandemic, compared to Euro-Canadian participants (Hypothesis 2). In addition, ample research has revealed links between optimism, meaning in life, and well-being, conceptually and empirically (e.g., Carvajal et al., 1998;Wrosch and Scheier, 2003;Ho et al., 2010;Wray et al., 2013;Kealy et al., 2020). Drawing on these findings, one might expect state optimism to predict psychological well-being and meaning in life (Hypothesis 3). Finally, if optimism can be a source of wellbeing and meaning, and if optimism varies across cultures (e.g., Ji et al., 2004Ji et al., , 2020Ji et al., , 2021, then state optimism may mediate cultural differences in psychological well-being and meaning in life (Hypothesis 4). The Present Research The present research aimed to unpack the connections among culture, optimism, psychological well-being, and meaning in life in the context of the COVID-19 pandemic. We conducted a cross-cultural study with over 500 Euro-Canadian and Chinese participants. In the study, all participants were instructed to complete the survey twice-about one week apart-but not everyone completed both surveys. The purpose of the longitudinal design is twofold: (1) to test the reliability of the findings, and (2) to investigate the relationships among variables across time and across cultures. Prior cultural work has shown that East Asian and North American cultures are characterized by distinct assumptions about the world. Dialecticism and non-linearity are more central to the thinking styles of East Asians, such as Chinese, relative to the thinking styles of North Americans, such as Euro-Canadians (e.g., Peng and Nisbett, 1999;Ji et al., 2001). These findings suggest that Chinese should be more likely to see the silver lining of the pandemic, compared to Euro-Canadians. If so, Chinese participants would be more likely to respond to the pandemic in a positive way (i.e., more optimistic, better psychological wellbeing and higher meaning in life), relative to Euro-Canadian participants. No specific predictions were made about trait optimism given the mixed evidence in the literature 1 . In summary, we aimed to examine the following predictions: Hypothesis 1: Chinese participants should have higher state optimism than Euro-Canadian participants. Hypothesis 2: Chinese participants would report better psychological well-being and higher meaning in life than Euro-Canadian participants. In addition, given the empirical links between state optimism, psychological well-being, and meaning in life, we also examined the following predictions: Hypothesis 3: State optimism may predict psychological well-being and meaning in life. Hypothesis 4: State optimism may mediate cultural differences in psychological well-being and meaning in life. While cross-sectional data can be used to examine these predictions, longitudinal data have the unique advantage of capturing whether, and to what extent, different variables may predict each other or mediate cultural differences over time. Thus, we will address the first two predictions with crosssectional data and the last two predictions with longitudinal data. METHOD Participants The present study was conducted in late March and early April 2020, a time when Canada and China were impacted by the pandemic. At Time 1, 293 Euro-Canadians (242 women, 50 men and one other; M age = 20.66, SD age = 3.72) from Queen's University in Kingston, Canada, and 266 Chinese (220 women and 46 men; M age = 19.88, SD age = 1.07) students from the Central China Normal university in Wuhan, China 2 , completed the study. A week later at Time 2, 243 Euro-Canadians (205 women, 37 men, and 1 other) and 240 Chinese (201 women and 39 men) 1 The empirical picture of cultural differences in trait optimism is less coherent. On the one hand, research using standard measures of trait optimism (e.g., Extended Life Orientation Test; Chang et al., 1997) have shown that Euro-Americans are more optimistic compared to East Asians (Chang, 1996). Similar conclusions (Heine and Lehman, 1995;Lee and Seligman, 1997) appeared in other work with different measurements (e.g., attributional styles, scenarios). On the other hand, no cultural difference in optimism was observed in some studies (Ji et al., 2004;see Fischer and Chalmers, 2008 for a meta-analysis). Possible explanations have been proposed, including the cultural meanings of optimism (Lai and Yue, 2000), multifaceted nature of the construct (Ji et al., 2004), presence of confounds (Chang, 1996), and the impacts of contextual information (Ji et al., 2004(Ji et al., , 2021. In light of these discussions, we were unable to make specific predictions about cultural differences in trait optimism but decided to include it for the sake of completion. 2 During the time of the study, both locations were under quarantine. completed similar measures. Among the participants, 223 Euro-Canadians (188 women, 34 men, and 1 other) and 235 Chinese (196 women and 39 men) did the study at both times 3 . All participants completed the study in their native language. The study material was first developed in English, and then translated into Chinese. Two bilingual researchers verified the translation to ensure its equivalence across language. Measures and Procedure Participants completed the study online via Qualtrics. At each time, they reported their current affect, psychological well-being, optimism, and meaning in life. Due to time constraints, fewer measures were included at Time 2. Time 1 testing included the following measures 4 : (1) Current affect: Participants reported (0 = not at all, 9 = very) how distressed, scared, anxious, worried, angry, depressed, nervous, hopeless, relaxed, and happy they were feeling "overall these days" during the pandemic. Six of these items were selected from the affect measures used in Bruehlman-Senecal et al. (2016), in addition to four items we added (distressed, hopeless, worries, relaxed). These items were chosen because of their relevance to people's responses to the pandemic. Current affect was measured so that we could gain a general picture of participants' psychological state or a proxy of their well-being. Scheier et al., 1994). The SOM-7 contains 7 items that captured participants' tendency to feel positive about the future (e.g., "At the moment, I expect more to go right than wrong when it comes to my future"). The LOT-R includes 6 test items that capture participants' general expectations about the future (e.g., "In uncertain times I usually expect the best"). For both scales, participants indicated (1 = strongly disagree, 5 = strongly agree) their endorsement of each item. (3) Well-being: Participants completed two measures of wellbeing, one being the adapted WHO-Ten Well-Being index (Bech et al., 1996) and the other being the Satisfaction with Life Scale (SWLS; Diener et al., 1985). The adapted WHO-Ten index measured state-like well-being with 10 items, focusing on "the absence of negative symptoms (i.e., anxiety, depression) and the presence of positive symptoms (e.g., energy)" (Cooke et al., 2016, p. 743). Participants rated (0 = not at all, 7 = very much) how they felt at the moment (e.g., "I feel happy, satisfied or pleased with my personal life."). The measure aligns with the hedonic component of well-being, centering around positive and negative feelings. SWLS measured the global perception of life with 5 items, with participants rating (1 = strongly disagree, 7 = strongly agree) the extent to which they were satisfied with their lives in general (e.g., "In most ways my life is close to my ideal"). In sum, the WHO index measured participants' state well-being while SWLS measured their general perception of well-being. (4) Meaning in life: Participants rated (1 = absolutely untrue, 7 = absolutely true) the extent to which they perceived meaning in life with the Meaning in Life Questionnaire (Steger et al., 2006). The scale includes 10 items, measuring the presence of meaning (e.g., "My life has a clear sense of purpose") and search for meaning (e.g., "I am seeking a purpose or mission for my life") respectively. About a week later at Time 2, participants completed the same measures as at Time 1, except LOT-R. RESULTS We included all participants for cross-sectional analyses. For cross-time analyses, we included only participants who provided data at both time points. As seen in Table 1, the zero-order correlations across cultural groups are consistent with prior work. For example, when all participants were analyzed regardless of culture, state optimism, well-being, and meaning in life were all positively correlated with one another. Next, we examined cultural differences in current affect, optimism, psychological well-being, and meaning in life. Results at Time 2 fully replicated results at Time 1. Then we tested how the key variables of interest (e.g., optimism, psychological wellbeing, and meaning in life) were related to one another over time. Degrees of freedom varied due to occasional missing data. We have conducted measurement invariance tests and established partial measurement scalar invariance for state optimism, state well-being, and meaning presence (see details in the Supplementary Material). Cross-cultural comparisons based on invariant items showed similar patterns of results as those based on full scale items. The latter are reported in the paper, while the former can be found in Supplementary Material. Culture and Optimism Was optimism higher among Chinese than Euro-Canadian participants (Hypothesis 1)? State optimism was computed by averaging the ratings of all items on the State Optimism Measure at Time 1 (α CA = 0.91 and α CH = 0.87) and at Time 2 (α CA = 0.91 and α CH = 0.89), respectively. Culture, Psychological Well-Being, and Meaning Were psychological well-being and meaning in life higher among Chinese than Euro-Canadian participants (Hypothesis 2)? As a measure of state well-being, ratings of adapted WHO items were Longitudinal Effects Did state optimism predict psychological well-being and meaning in life (Hypothesis 3)? We examined the relationships across time. We investigated how variables at Time 1 may predict variables at Time 2, and how such relationships may vary across cultures. The cross-time analyses were done based on the data from participants who did the study at both times. We ran a series of regressions, in the following format: y2 ∼ y1 + x1 + culture + x1 * culture In the model, y2 was the outcome variable at Time 2; y1 was the same outcome variable measured at time 1 and served as a covariate; x1 was the predictor variable at Time 1, whose interaction effect with culture was x1 * culture. All continuous variables were centered. Culture was coded as Canada = -0.5 and China = +0.5. We reported only significant effects in the following analyses. Although not part of our predictions, we also examined the other combinations of the relationships among the three variables (state optimism, state well-being and meaning presence) and report them below. Together, these results revealed bi-directional, temporal relationships among state optimism, well-being, and meaning presence. For example, state optimism at Time 1 predicted subsequent well-being and meaning presence at Time 2; meaning presence and well-being at Time 1 predicted subsequent state optimism at Time 2. Cross-Time Mediation Analyses Digging into underlying pathways, did state optimism mediate cultural differences in psychological well-being and meaning in life (Hypothesis 4)? Given the longitudinal nature of our data, we conducted cross-time mediation analyses to investigate this. That is, we examined whether state optimism at Time 1 would mediate cultural differences in well-being or meaning presence at Time 2. We conducted the following mediation analyses using the lavaan package (Rosseel, 2012) in R (R Core Team, 2019). Culture (China = 0.5, Canada = -0.5) was the independent variable. The dependent variable was either state well-being or meaning presence at Time 2. The mediator was state optimism at Time 1. In each analysis, we also controlled for the same measure of the dependent variable at Time 1. As seen in Table 2, based on joint-significance tests (Yzerbyt et al., 2018), state optimism at Time 1 mediated cultural differences in state well-being and meaning presence at Time 2, respectively. Consistent with the conclusion from the jointsignificance tests, the 95% percentile bootstrap confidence intervals for both indirect effects did not contain 0. Next, we conducted similar mediation analyses with state optimism at Time 2 as the dependent variable, and state well-being and meaning presence at Time 1 as the mediator, respectively. We found that meaning presence (but not state wellbeing) at Time 1 mediated cultural differences in state optimism at Time 2 (see Table 2 for the respective 95% percentile bootstrap confidence intervals of the indirect effects). For completeness, we also examined and found that (a) state well-being at Time 1 mediated the cultural differences in meaning presence at Time 2 and (b) meaning presence at Time 1 mediated the cultural differences in state well-being at Time 2 (see the last two rows in Table 2). We elaborate on the implications of these findings in Discussion. DISCUSSION The present research found that during the pandemic (March 2020), Chinese participants reported more positive affect and Optimism refers to state optimism. Well-being refers to state well-being. Meaning refers to meaning presence. less negative affect, higher optimism, higher state well-being, and higher meaning presence, compared to Euro-Canadian participants. Chinese reported lower levels of general well-being than did Euro-Canadians, compatible with some prior studies with similar measures (e.g., Oishi, 2002). With a week's interval between the two tests, the results were generally stable across time. Indeed, for each variable measured at both times, the test-retest correlation coefficients ranged between 0.67 and 0.76 (see Table 1). Furthermore, the relationships among different variables were stable, as similar patterns of results were observed at both times. As expected, we found that state optimism predicted, and mediated cultural differences in, subsequent state well-being and meaning presence. In addition, we found that state well-being and meaning presence also predicted subsequent state optimism, and that meaning presence (but not state wellbeing) mediated cultural differences in state optimism. The present research shows that Chinese participants, while reporting lower life satisfaction in general, experienced more positive affect and less negative affect under the threat of the pandemic, compared to Euro-Canadian participants. These findings are consistent with prior work, which indicates that overall life satisfaction is influenced by cultural beliefs that are stable and chronic, whereas specific, online responses are subject to experiential and immediate contextual influences (e.g., the ups and downs that people are going through in their lives, Robinson and Clore, 2002). Our results also draw links with the literature on affective experiences (Uchida and Kitayama, 2009;Miyamoto and Ma, 2011) and the assumptions (Ji et al., 2001;Oishi, 2002) people have about life, satisfaction, and happiness, all of which can vary considerably across cultures. More broadly, our mediation analyses with the longitudinal data ( Table 2) revealed the temporal nature of key variables. That is, each of the three variables -optimism, well-being, meaning -at Time 1 mediated cultural differences in the other two variables at Time 2 (except that state well-being at Time 1 did not significantly mediate cultural differences in state optimism at Time 2). These results, compatible with past findings (Scheier and Carver, 1992;Wrosch and Scheier, 2003;Ho et al., 2010), have theoretical (e.g., incorporating time as an independent variable in theory-building) and practical (e.g., strategies for dealing with challenging events) implications for research on culture, coping, and health, as discussed below. Theoretical and Practical Implications One feature that stands out in this research is its longitudinal design, which allows us to examine relationships among variables in temporal sequence. In growing fields such as cultural psychology where questions are as numerous as answers, longitudinal designs may provide insights that may otherwise be hidden. For example, longitudinal designs allow researchers to model time as an independent variable (Wright, 2007). Time, while assumed to play a role in many cultural processes (e.g., acculturation, lay theories of change, temporal focus, acquisition of norms), seldom gets integrated into research designs (see Barlett et al., 2014 for an exception). With a longitudinal view, researchers can systematically examine time as a causal, mediating, or moderating variable in cultural phenomena. This approach takes a step forward from standard studies in which cross-sectional data often capture a thin slice of fluid processes. Furthermore, the present research reveals the mutual influence of state optimism, well-being, and meaning presence such that they predict one another over time. For example, state optimism at Time 1 accounted for cultural differences in state well-being or meaning presence at a Time 2, as state well-being or meaning presence at Time 1 accounted for cultural differences in state optimism at Time 2. The temporal impacts of these constructs on one another would have fallen out of our view if we had not collected data from the same participants at two different time points. The two waves of data collection were about one week apart during the pandemic. It is unclear to what extent the results would hold with a bigger temporal gap, which can be examined in future research. Our mediational results suggest that optimism can help people go through challenges in life, leading to joy and meaning. Likewise, in difficult times, finding joy in small things and imbuing old routines with new purposes may result in a brighter outlook on life. Both scenarios resonate with the literature on health (Aspinwall and Taylor, 1992;Scheier and Carver, 1992;Slattery et al., 2017) and the non-linear theory of change (Ji et al., 2001(Ji et al., , 2021. Furthermore, such reciprocal positive relationships can potentially lead to an upward spiral of empowerment, where state optimism, well-being, and sense of meaning perpetuate one another for good over time. Gradually, this cycle may become internalized, and people may be motivated to stay optimistic and imbue themselves with wellness and meaning for a positive, happy, and fulfilling life. Unpacking Cultural Differences: Conceptual Basis and Theoretical Links The present research highlights differences in the responses to COVID-19 among Chinese and Euro-Canadians. Observed cultural patterns can be attributed to various cognitive and motivational processes, which were not examined directly due to the lack of resources during the pandemic. This is a limitation of the present research. Empirical demonstrations would have been more complete if we had the chance to measure naïve dialecticism, lay theories of change, cultural tightness-looseness, influence-adjustment motivations, and their respective roles in our results. These processes forge a theoretical ground for the present findings, as we discuss below. Cross-cultural differences in optimism and well-being may be driven by naïve dialecticism (Peng and Nisbett, 1999), which assumes that the basis of life is full of contradictions, with good embedded in bad and bad embedded in good. Past research has shown that East Asians hold stronger beliefs in naïve dialecticism than North Americans (e.g., Nisbett, 2003;Spencer-Rodgers et al., 2010a). In particular, East Asians tend to embrace contradictions with high tolerance, whereas North Americans tend to view contradictions as something they should avoid in reasoning. Consistent with naïve dialecticism, prior work has shown that Chinese are more likely to infer the reality of things in a way that contradicts their public appearance, compared to Euro-Canadians Lee et al., 2020). Related to naïve dialecticism, the non-linear theory of change (Ji, 2005) refers to the belief that the universe consists of opposing states, with everything in it constantly shifting from one state to another in a nonlinear fashion. For example, prosperity can change into poverty, and poverty can turn to prosperity. East Asians tend to hold a stronger belief than North Americans in the non-linear development of events. When predicting the future given past trends, Chinese participants tend to make predictions that deviate from the original propensity of the trend (e.g., a decreasing trend would go up), reflecting a non-linear theory of change. In contrast, Euro-American participants tend to make predictions that follow the propensity of the trend (e.g., a decreasing trend would keep going down), reflecting a linear theory of change (Ji et al., 2001. Both naïve dialecticism and non-linear theory of change have implications when dealing with life adversities. These theories and beliefs, which people often take for granted thanks to cultural learning (e.g., Hirschfeld, 1996), exemplify how things in the world may not appear as they seem and how things may be opposite of what they appear to be. Within good there is evil, and beneath the surface of crisis there is opportunity for growth. Applying naïve dialecticism and non-linear theory of change to the context of COVID-19, one may predict that relative to Euro-Canadians, Chinese would be more inclined to react to the pandemic with positivity in terms of optimism, well-being, and meaning in life. This is indeed what our data show. Another potential factor underlying the present findings is cultural tightness-looseness (Pelto, 1968;Gelfand et al., 2011), or the extent to which cultures vary in their tolerance for norm deviations and in their punishments for them. China, for instance, is considered as a tight culture, where social norms are closely followed and deviations from norms can easily result in sanctions by the group (Gelfand et al., 2011;Uz, 2015). In contrast, Canada and the U.S. are loose cultures, where most people do not expect sanctions by the group for not following social and cultural norms closely (Gelfand et al., 2011;Uz, 2015). Tightness and looseness across cultures, when applied to the current context, may provide another perspective as to why Chinese participants responded to the pandemic more positively than Euro-Canadian participants, as our results have shown. Rigid norms that stemmed from the pandemic-such as lockdown orders, masks, social distancing, and travel bans-are undeniably inconvenient to people. But these new norms, when viewed through the lens of tight cultures, are not as big of an issue because people in tight cultures, such as China, are strict followers of social norms on a regular basis. In contrast, in loose cultures, such as Canada, where norms are guides and deviations are common, people may have trouble adjusting to the new norms imposed abruptly onto their lives, especially the rigid norms in the COVID-19 pandemic that cannot be challenged. Cultural manifestations of tightness and looseness echo past work, which showed that East Asians are motivated to adjust themselves to the environment outside of them, whereas North Americans are motivated to influence the environment to fit them (Morling et al., 2002;Morling and Evered, 2006;Tsai et al., 2007). Naïve dialecticism, lay theories of change, tightness-looseness, and adjustment-influence motivations are dimensions of culture that may contribute to how people react to the pandemic and the numerous safety rules that come with it. The respective and collective roles of these variables in pandemic-related reactions deserve a close look in future research, along with a broader and more gender-balanced sample. Beyond Negative Contexts? The present research examined people's responses in the context of a negative life experience. What would happen in a positive context? Applying cultural differences in reasoning styles, the opposite prediction might be made for positive life experience. For example, gratitude involves a positive life experience, as it represents "a felt sense of wonder, thankfulness and appreciation for life" (Emmons and Shelton, 2002, p. 460). Due to naïve dialecticism and non-linear theory of change, East Asians may generate negative responses while feeling gratitude. This prediction corresponds to cultural work on emotional complexity (Goetz et al., 2008;Miyamoto et al., 2010;An et al., 2017). It also contrasts with how gratitude is typically experienced by North Americans, which is overwhelmingly positive (Emmons and Shelton, 2002). The unconditional love of a friend can make people feel grateful, but also very guilty in some cultures, as some work has shown (e.g., Naito and Sakata, 2010). Likewise, Zhang et al. (2018) have shown that receiving verbal thanks may lead to stronger negative affective experiences among Chinese than among Euro-Canadians. In sum, the assumptions of dialecticism and lay theories of change may manifest in both negative and positive contexts. By examining this possibility, future research can enhance positive psychology with a cultural perspective. Limitations and Alternative Explanations The present research would have been more complete in the presence of other measures that capture the way the pandemic was experienced by the participants, such as the level of threat perceived by our participants and possibly perceived knowledge of the virus. Due to limited time and resources, no such information was collected as the narrow window of opportunity was closing on us. Including these measures, and statistically controlling for them in our analyses, would have strengthened the present findings. The present research was conducted during the pandemic. At the time of data collection, China (82,100 confirmed cases and 3,304 deaths on March 29, 2020) 5 had more positive cases than Canada (6,258 cases and 63 deaths on March 29, 2020) 6 , but the trend was more concerning for Canada as cases there were on the rise while cases in China had reached a plateau. In addition, there may be differences between the two locations in terms of local policies imposed and the medical challenges faced at the time of this study. Thus, one may say that the two cultural groups were not exposed to the same levels of threat, which might have contributed to the results in some ways beyond our control. Our Chinese data were collected at a university in Wuhan, the city where the outbreak originated. Taking the viral impact at ground zero without prior warning, one may reasonably expect the looming terror of the pandemic to persist in Wuhan, even when the situation was somewhat under control by that point. The city was completely shut down for 2 months. In late March, city public transportations in Wuhan started to resume and some stores started to reopen. On April 8 th , people in Wuhan were finally allowed to travel outside of the city, provided that they could show that they were virus-free with proper medical documents after 14 days of isolation. Still, residents were advised to stay home unless going out was absolutely necessary. Leaving and returning to their home compounds required examinations and documents. Students were learning online, while instructors were working off campus as well. Regardless of all the challenges, the situation was getting better overall. Meanwhile, in Kingston where the Canadian data were collected, the first 3 positive cases were identified on March 17, 2020, and were all travel related. The total positive cases were 43 on March 31, and 55 on April 14. Following the provincial declaration of a state of emergency on March 17, the city declared a city state of emergency on March 26. Students switched to online learning, and people were asked to work from home. Public transportation kept running. People could still travel, although the government encouraged people not to. Those who did travel were asked to self-quarantine for 14 days without any reinforcement by the government. Although the population density was low in Kingston and people were naturally more spread out against the threat of viral transmission, people had to change their behaviors and lifestyles, drastically and unprecedentedly, in anticipation of all kinds of uncertainties ahead. With different facts and realities in view, it is difficult to conclude which test location took a harder hit at the time of our study, though few would deny that both places were in bad shape. Still, we acknowledge the possibility that our cultural samples were experiencing different levels of threat from the pandemic, which could be a potential limitation of the present research. CONCLUSION The present research examined the way people respond to the COVID-19 pandemic across cultures. Systematic cultural differences emerged in positive and negative affect, optimism, psychological well-being, and meaning presence. State optimism, well-being, and meaning presence not only reinforced each other, but also mediated cultural differences in one another over time. These findings may shed new light on theoretical development and generate practical implications for psychological health and well-being in real life. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ETHICS STATEMENT The studies involving human participants were reviewed and approved by the General Research Ethics Board, Queen's University. The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS L-JJ and SY conceived the research idea, designed the study, and analyzed the data. SY, YL, and YD collected the data. AL did the literature review and wrote the manuscript with L-JJ. All authors undertook final clarification and agreed on the version of the manuscript for submission.	2021-07-12T13:24:33.339Z	2021-07-12T00:00:00.000	{ "year": 2021, "sha1": "fda87c85bea0032f37284a9a173b0bbd85fcedb0", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fpsyg.2021.636062/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "fda87c85bea0032f37284a9a173b0bbd85fcedb0", "s2fieldsofstudy": [ "Psychology", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
149908162	pes2o/s2orc	v3-fos-license	Anti-Niemann Pick C1 Single-Stranded Oligonucleotides with Locked Nucleic Acids Potently Reduce Ebola Virus Infection In Vitro Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC50) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection. INTRODUCTION Ebola virus (EBOV), as well as four other filoviruses-Bundibugyo virus (BDBV), Sudan virus (SUDV), Marburg virus (MARV), and Ravn virus (RAVV)-are causative agents of severe disease in humans, such as severe hemorrhagic fever, and are often associated with high morbidity and mortality rates. [1][2][3] These viruses belong to the family Filoviridae of non-segmented negative-strand RNA viruses and are biosafety level 4 pathogens transmitted by contact with body fluids, fomites, and droplets from infected patients. Filoviruses are considered a significant threat to public health and global security because of their pandemic potential and the risk of being used as a bioweapon. 1,[4][5][6] Therefore, accelerated efforts in the development of therapeutics is a key objective in the filovirus research community, especially since the 2013-2016 EBOV disease (EVD) epidemic in Western Africa. No vaccines or therapeutic agents with final US Food and Drug Administration (FDA) approval are currently available, and supportive care remains the standard for Ebola virus disease treatment. However, to reduce EBOV spread and the pandemic risk of the current outbreak in Democratic Republic of the Congo (750 confirmed cases and 449 confirmed deaths, as of February 9, 2019) (https://www.who.int/ebola/situation-reports/drc-2018/en/) use of rVSV-ZEBOV Ebola vaccine, as well as antiviral drugs and antibodies against EBOV, have been temporarily approved (https:// www.who.int/ebola/drc-2018/faq-vaccine/en/, https://www.who.int/ ebola/drc-2018/treatments-approved-for-compassionate-use/en/). Filovirus particles have a uniform diameter of 80 nm and variable lengths. A single transmembrane glycoprotein (GP), consisting of two subunits, GP1 and -2, is inserted into the virus envelope as a trimeric complex. GP mediates cell attachment and endocytosis by binding to attachment proteins of the host cell. 7,8 In late endosomes, the host cysteine proteases cathepsin-B and -L cleave and remove large C-terminal regions of the GP1 subunit, 8,9 thereby unmasking a binding site for the host factor Niemann-Pick C1 (NPC1). This cholesterol transport protein has been shown to be an essential host factor 10,11 and endosomal entry receptor for filoviruses. 12,13 In cooperation with Niemann-Pick C2 (NPC2), NPC1 is an endosomal transmembrane protein that mediates transport of luminal cholesterol across the endosomal and lysosomal membrane for dispersal to other cellular compartments. 14,15 Loss-of-function mutations in NPC1 or NPC2 cause a rare and often fatal hereditary neurovisceral disorder in humans. 16,17 Over time, patients with NPC disease accumulate cholesterol and glycosphingolipids in various tissues and organs, leading to neurological dysfunction and organ failure. Herbert et al. 18 demonstrated that Npc1-deficient mice are completely protected from EBOV infection and free of replicating virus. These results strongly implicate the NPC1 protein as a direct mediator of filovirus infection in vivo. It has been reported that NPC1 inhibition by U18666A, an amphipathic steroid, as well as the EBOV-specific antiviral compound 3.47 significantly inhibit filovirus replication by interfering with viral entry 10,11 in vitro and in vivo. 18 However, both compounds have not yet reached clinical trials. Besides small molecules and therapeutic antibodies, oligonucleotidebased gene expression inhibitors have developed into fully accepted therapeutics. The majority of compounds progressing through clinical trials [19][20][21] are either antisense oligonucleotides (ASOs), comprising single-stranded DNA-like molecules that recruit endogenous RNase H for target mRNA degradation or small-interfering RNAs (siRNAs) that work through the RNA-induced silencing complex (RISC). ASOs typically have a length of 12-21 nucleotides (nt). In our study, nucleotides were joined via phosphorothioate (PTO) linkages. The phosphorothioate linkage substitutes a sulfur atom for a non-bridging oxygen. This modification renders the internucleotide bond resistant to nuclease degradation and enhances plasmaprotein binding while retaining the ability to direct RNase H activity in the cell. 22,23 In addition, ribose moieties in the flanks of the oligonucleotide are modified by an extra bridge connecting the 2 0 oxygen and 4 0 carbon. This modification locks the conformation of the ribose, conferring high affinity to the RNA. Therefore, this modification is termed the "locked nucleic acid (LNA) modification." 24,25 Here, we demonstrate the potency of LNA-containing ASO (LNA-ASO) molecules targeting mRNA of the host factor NPC1, which is essential for filovirus replication. We show that NPC1-specific ASOs efficiently reduce viral replication in cultured cells without affecting cell viability or inducing immune-stimulatory responses. Selection of ASOs Targeting Host Factor Niemann-Pick C1 NPC1-specific 15-, 16-and 17-mer ASOs were selected based on human NCBI reference sequence (accession number GenBank: NM_000271.4) ( Table S1). The main criterion for sequence selection was selectivity, to avoid undesired off-target effects. Several sequences were completely cross-reactive to murine Npc1, several had one or more mismatches. ASO length, LNA modification pattern, and localization of ASO binding sequence on human NPC1 mRNA are depicted in Tables 1 and S1 and Figure 1. The in vitro activity of the 36 NPC1-specific ASOs was evaluated in two human and one murine cell line endogenously expressing NPC1 mRNA. After treating these cells with LNA-ASO without using a transfection reagent, 26 the level of NPC1 mRNA was measured after 3 days of treatment. Human HeLa and THP-1 cells were used as cell lines for screening, as both cell lines are susceptible to EBOV infec-tion. NPC1-specific ASOs led to reduced NPC1 mRNA expression levels in both human cell lines with correlating efficacies (Figure 2A). As expected, cross-reactive ASOs having full complementarity to both human and murine NPC1 mRNA were more efficient in murine 4T1 cells than ASOs that are human-specific and have mismatches to the murine target ( Figure 2B). Therefore, an increased number of mismatches of the human-specific ASOs to the murine Npc1 sequence resulted in decreased efficacy in murine 4T1 cells ( Figure 2B). In all three cell lines, the human-mouse cross-reactive ASO 05HM was the most efficient candidate with 95% (HeLa), 79% (THP-1), and 98% (4T1) NPC1 mRNA knockdown, while the human-specific ASOs 28H and 29H were among the most potent ASOs in human cells, but had poor activity in murine cells ( Figure 2). To test dosedependence of effects, HeLa and 4T1 cells were exposed to increasing concentrations of ASO 05HM and 28H. Endogenous mRNA levels were evaluated after 3 days of treatment with ASOs, and the 50% inhibitory concentration (IC 50 ) for the inhibition of NPC1 expression was determined ( Figures 3A-3C). As already indicated by the aforementioned screening results, ASO 05HM (IC 50 = 668 nM) was more potent in the HeLa cells than was ASO 28H (IC 50 = 2,781 nM; Figures 3A and 3B). In the murine cell line 4T1, the cross-reactive ASO 05HM was even more effective (IC 50 = 457 nM; Figure 3C). Notably, treatment with ASOs did not affect cell viability at any concentration (Figure 3D). Using immunoblot analysis, knockdown efficacy on protein level was evaluated and confirmed in HeLa cells, treated twice for 3 days with ASO 05HM and 28H ( Figure 3E). Both ASOs clearly reduced NPC1 protein levels compared with untreated cells or with cells treated with control Neg1 that is not complementary to any human or murine RNA ( Figure 3E). 27 Again, treatment of HeLa cells with ASO 05HM resulted in a more pronounced NPC1 knockdown than incubation with ASO 28H ( Figure 3E). Taken together, these data demonstrate the potential of the NPC1specific LNA-ASOs for efficient knockdown of NPC1 gene expression in human and murine cell lines. Immune activation leading to cytokine release is characteristic of therapeutic oligonucleotides, either as an unwanted side effect or intended pharmacology. This immune activation is mediated by pattern recognition receptors, such as the Toll-like receptors (TLRs). Binding of immune stimulatory ligands, e.g., bacterial DNA or immune stimulatory oligonucleotides, with or without nonmethylated CpG +G+C+AATTCCACACTC+T+C+C 28H AGCGCGAACGGCTTCTA 4,086 17 +T+A+GAAGCCGTTCGC+G+C+T Neg1 N/A N/A 18 +C+G+TTTAGGCTATGTA+C+T+T Depicted are name of ASO, mRNA binding sequence, position on mRNA, ASO length as well as ASO sequence and modification: LNA (+) and/or phosphorothioate (). Humanspecific ASOs (H) as well as cross-reactive ASOs targeting both, human and murine NPC1 (HM), were selected. www.moleculartherapy.org dinucleotides, 28 results in TLR activation. As immune activation can lead to a severe, possibly life-threatening, condition of excessive cytokine release, 29 the selected LNA-ASOs 05HM and 28H were analyzed for their potential to activate TLR9 and to induce cytokine release in human cells, enabling a safety assessment for future clinical studies. To assess the potential of NPC1-specific ASOs to activate TLR9-mediated signaling, an HEK-Blue hTLR9 SEAP reporter assay was used to measure activation of nuclear factor-kappa light-chain enhancer of activated B cells (NF-lB) induced by TLR9. In contrast to the human TLR9 agonist CpG ODN2006, neither ASO 05HM nor ASO 28H triggered human NF-lB activation ( Figure 4A). Murine Nf-lb was also not induced by treatment with cross-reactive ASO 05HM tested in stably transfected HEK-mTlr9_Nf-lb-LUC cells ( Figure 4B), whereas the murine Tlr9 agonist ODN1668 induced a considerable dosedependent response. TLRs are expressed by numerous cells of the immune system, such as B lymphocytes, monocytes, natural killer (NK) cells, keratinocytes, melanocytes, and plasmacytoid dendritic cells (pDCs). The peripheral blood mononuclear cell (PBMC)-based assay is well established for determination of immune activation by different drugs, including TLR ligands and oligonucleotides. 28,30,31 Here, PBMC isolated from leukocytes preparations of three different donors were used. In contrast to pattern recognition receptor agonists, ODN2006, lipopolysaccharide (LPS), or immune stimulatory CD3/CD28/CD2, the ASO 05HM failed to trigger a cytokine response ( Figure 5). These data support that the most potent ASOs, 05HM and 28H, do not trigger a host innate immune response. Treatment with phosphorothioate-modified non-targeting oligonucleotides reduced EBOV replication by 62-70% in vitro. Furthermore, oligonucleotides specifically targeting NPC1, an essential receptor of EBOV cell entry, led to a more potent inhibition of EBOV replication-up to 99%. DISCUSSION EBOV causes a severe, often fatal illness in humans, is transmitted to people from wild animals, and spreads in the human population through human-to-human transmission. 32 Case fatality rates of Ebola virus disease have varied from 25% to 90% in past outbreaks, with an average of around 50%. 33,34 There are two distinct paths for potential EBOV treatments: post-exposure prophylaxis and treatment of symptomatic patients. Both have different challenges, but a common strategy may be to limit virus replication, to allow the adaptive and innate immune systems time to fight the infection. 35,36 A limitation for ASO-based antiviral strategies directly targeting the virus may be the RNase H1-dependent mode of action of gapmer ASOs. Filoviruses replicate in the host cytoplasm and do not require the nucleus. 37 However, the cellular compartment of ASO-mediated RNA degradation is a controversial issue within the ASO community. [38][39][40][41] We and others showed that ASOs targeting EBOV mRNA could efficiently block viral transcription or replication in cell-free 42 and reporter gene assays (Figures S1A and S1B). In reporter-based assays, transcription of the reporter genes takes place in the nucleus. However, EBOV-specific ASOs were not capable of inhibiting viral propagation in Huh7 cells, with or without the use of a transfection reagent (Figures S1C and S1D). These results indicate, that the predominant location of RNase H1-mediated target RNA degradation is the nucleus that is, as previously mentioned, bypassed during EBOV replication. The use of ASO approaches aimed at sterically blocking viral translation in the cytoplasm could avoid this issue. However, translation-blocking approaches are much less efficient compared to RNase-H1-dependent mechanisms. Whereas one translation-blocking ASO-molecule is needed to repress translation of one target mRNA molecule, one RNase H1-depending ASO could mediate degradation of many individual RNA molecules. Therefore, much higher doses would be necessary for a translation-blocking approach in the cytoplasm. The antiviral efficacy of translation-blocking approaches was suggested by Chery et al. 42 indirectly by use of reporter-based assays. However, direct evidence for antiviral effects such as copy number determination of an EBOV isolate after ASO treatment is missing. A fundamental disadvantage of approaches targeting EBOV directly is the error-prone viral polymerase of RNA viruses that enables incorporation of the mutations that facilitate resistance against antiviral drugs. Furthermore, antiviral approaches have not been capable of overcoming strain-specific differences, so far. Although the efficacy of antibody cocktails, such as ZMapp, for EBOV in nonhuman primates is clear, a future challenge will be to identify similar treatments for other filoviruses. 43 For the aforementioned reasons, targeting an essential human host factor for viral replication may be one of the most promising approaches for fighting EBOV. An essential host factor for filovirus replication in vitro and in vivo is the cholesterol transport protein NPC1. This endosomal entry receptor has been shown to mediate cytoplasmic release of viral ribonucleoproteins, 10,13 making it an ideal target for blocking filovirus replication. Various small molecule therapeutics against host factor NPC1 have been described, some with demonstrated efficacy against filovirus replication in mice. 43 However, those molecules targeting NPC1 did not reach clinical trials, so far, probably because of the described toxic effects 44 or the need for further mechanistic characterization, as well as improvement of compounds. 43 The limited success of the available NPC1 inhibitors in protecting mice from EBOV challenge highlights the need for new molecules or approaches to target NPC1 in vivo. 18 A unique advantage of ASOs targeting essential host factors for filovirus replication, particularly in the context of emergency prophylactic or post-exposure therapeutics, is their target specificity, cost-effectiveness, and fast generation. Mode of action, uptake, biodistribution, pharmacokinetics, and safety issues have been extensively studied. 24,26,[45][46][47][48][49][50] In the present study, a total of 36 ASOs was designed for the initial screen broadly covering the NPC1 mRNA sequence. Fifteen-, 16-, and 17-mer ASOs were tested, and two ASOs, comprising 17 nt, were selected for further experiments. As recommended by the Oligonucleotide Safety Working Group, 51 an extensive in silico approach was used to avoid suppression of off-target genes. The bioinformatic analysis revealed no perfect match to any exonic or intronic off-target sequence. Even allowing one mismatch, still no off-target hit was detected in the NCBI RefSeq data base. This off-target characteristic is well in the range of the characteristic of other ASOs recently published in the field. 42,52 This should also decrease risk of generation of ASO-generated RNA fragments, which, as Dieckmann et al. 45 speculated, could cause toxicity. ASOs have been repeatedly described to stimulate immune activation. 22,[53][54][55][56] Reasons for immune stimulatory activities were reported to be nonmethylated CpG dinucleotides within the oligonucleotide sequence, as well as the stabilizing phosphorothioate backbone. [57][58][59] Studies using Tlr9-deficient mice demonstrated that this Tlr subtype is essential for the effects that are mediated by bacterial DNA or CpG oligonucleotides. 60 Since expression, ligand preference, and function of pattern-recognition receptors is highly species specific, 61 cytokine release in humans is hard to predict in animal studies. The human TLR9 is expressed in B cells and pDCs. [61][62][63] Both cell types are stimulated by CpG oligonucleotides to upregulate cell surface costimulatory molecules and to secrete a variety of cytokines. 64,65 These effects can lead to indirect activation of other cell populations, such as monocytes and NK or T cells. 65 The in vitro TLR9 assays as well as the PBMC ex vivo test used in this study are therefore helpful in deselecting ASOs with immune stimulatory potential early in the screening and compound characterization process, to prevent unexpected harmful effects in clinical development. In contrast to the CpG oligonucleotides ODN2006 and ODN1668, which were used as positive controls, both of the selected NPC1-targeting ASOs 05HM and 28H activated neither human nor murine TLR9 (Figure 4), nor did ASO 05HM stimulate cytokine release in treated PBMCs (Figure 5). These results are in line with the findings of Vollmer and colleagues, 28 who demonstrated that LNA modification of ASOs significantly decreases the immune stimulatory effects of ASOs. Our bioinformatics analysis enabled the selection of cross-reactive ASOs that target human as well as murine NPC1 mRNA for screening in hu-man and murine cell lines. Again, the benefit of our rational ASO design was confirmed, as cross-reactive ASOs were effective in both species in vitro ( Figure 2). As expected, a decreased knockdown of murine Npc1 by human-specific ASOs with no 100% complementarity to the murine mRNA was observed in murine cells. Thereby, a higher number of mismatches to the murine Npc1 sequence was associated with decreased activity in murine cells (Figure 2B). In Npc1 À/À mice, it was previously demonstrated that the Npc1 protein acts as a direct mediator for EBOV infection in vivo. 18 Since ASO 05HM targets human and murine NPC1, it is also a suitable tool for testing in vivo efficacy against different filovirus strains in mice. NPC1 ASO screening in two different human cell lines resulted in strong correlation of ASO activity, which enabled reliable selection of candidate ASOs. Consistently, ASO 05HM was the most effective candidate during single-dose screens in one murine and two human and cell lines (Figure 2), during IC 50 determination ( Figures 3A-3C), protein knockdown ( Figure 3E), and the infection assay in HeLa cells ( Figure 6). All experiments were performed by using a method called "gymnosis" without using a transfection reagent. 26 The pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing and is therefore of particular significance for drug discovery. 26 Recently, Chery and colleagues 42 also reported on efficient knockdown of NPC1 by use of target-specific ASOs. However, these ASOs were delivered into cells using lipofection and may have divergent characteristics in vivo. Macrophages, monocytes, and dendritic cells are the primary target cells during acute EBOV infection, and several organs, such as liver, kidneys, and spleen, show high viral loads during the course of infection, [66][67][68][69] These cell types and organs are also good targets for phosphorothioate-modified ASOs which have a preferred biodistribution to kidney, liver, and immune cells and are not capable of crossing the blood-brain barrier. 46 Therefore they cannot affect the transport of cholesterol in neurons within the CNS, which could cause a Niemann-Pick disease-like phenotype. Treatment with NPC1-specific ASO resulted in significantly decreased EBOV replication (>99%; Figure 6B). Similar results were obtained by Chery and colleagues 42 by use of a VSVluc-EboV GP reporter virus. Reduced luciferase expression up to 80% was detected after transfection of HeLa cells with an NPC1-specific ASO. However, neither was the immune stimulatory potential of the selected ASO tested nor was a negative control ASO included to test for potential unspecific effects. Therefore, whether the observed decrease in reporter virus replication is a backbone-mediated or a specific effect caused by NPC1 knockdown cannot be distinguised. 42 In the present study, the control oligonucleotides clearly diminished the amount of EBOV genome copies 24 h after infection, though to a lesser extent ( Figure 6B; Figure S2B). In addition to their sequence-specific functionality, single-stranded oligonucleotides are polymers with polyanionic characteristics that are largely conserved, regardless of their nucleotide sequence. Phosphorothioation of oligonucleotides confers increased hydrophobicity and has been shown to specifically mediate antiviral activity in a manner independent of the increased nuclease stability present with this modification. 70,71 Phosphorothioates with broad-spectrum activity against viral and other infectious diseases are also called "nucleic acid polymers" (NAPs). 72 The structure-function relationship of the antiviral activity of NAPs, as well as their molecular mechanism of action, was first elucidated in a study describing the specific antiviral effects of NAPs during the entry of HIV-1. 71 In this study, the entry inhibition effect of NAPs was shown to be independent of sequence, but dependent on size. 71 A large amphipathic protein domain in the viral gp41 glycoprotein is required for interaction with NAPs and conserved in class I fusion GPs from many other viruses susceptible to antiviral polymers. Following on the findings in the initial study in HIV-1, NAPs were subsequently shown to have the same sequence-independent and phosphorothioate as well as length-dependent antiviral effects in other viruses with class I fusion GPs, including herpesviruses, cytomegalovirus, influenza virus, and lymphocytic choriomeningitis virus. [73][74][75][76][77] Meanwhile, the company Replicor develops antiviral drug candidates against hepatitis B and D viruses based on the mechanism by which NAPs inhibit viral propagation. Interestingly, EBOV GP, which is required for virus entry into the host cell, is also a class I fusion protein. 78 Keeping this fact in mind, it may be reasonable that, to some extent, phosphorothioated oligonucleotides inhibit EBOV replication, independent of sequence. These findings could explain the 62-70% reduction of viral replication detected with control phosphorothioates that did not reduce NPC1 mRNA levels (Figures 6A and 6B; Figures S2A and S2B). However, the specific oligonucleotides 05HM and 28H significantly reduced the EBOV titer after 24 h of infection by 97%-99% compared to non-treated cells ( Figure 6B; Figure S2B). This result strengthens our hypothesis that RNase H1-mediated targeting of an essential host factor for virus entry has an additional effect of solely backbone-mediated reduction of EBOV entry, thereby vastly improving therapeutic antiviral potential. Taken together, the findings in this study demonstrate that knockdown of intracellular receptor NPC1 by target-specific ASOs is a promising approach for treatment of EBOV infection. Our selected human-mouse cross-reactive NPC1-specific ASO may be used in future studies to further investigate the efficacy of Npc1-specific ASO against EBOV in mouse models. Selection of NPC1-Specific ASOs The human mRNA sequence of NPC1, as defined by NCBI accession number GenBank: NM_000271.4, was taken as a basis to design the ASOs. For this sequence (4,827 bp) the possible 15-, 16-, and 17-mer sequence fragments were extracted. All 14,442 fragments were tested for specificity against the complete NCBI RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) and ENSEMBL (http://www.ensembl.org/) databases, by using PatMaN. 79 Possible matches, including up to three mismatches against any mRNA (RefSeq) or any intron (ENSEMBL), were collected to enable a filtering of those fragments showing the highest specificity. As a cross-reactivity to mice was additionally desired, the sequences were tested against all mouse sequences of both databases, as well. The main criterion for the selection process was the absence of a perfect match to any human or mice off-target mRNA or intron sequence. In addition, only a limited number of offtarget matches allowing up to three mismatches were tolerated. As recommended by the distributor (https://www.qiagen.com/de/shop/ pcr/primer-sets/custom-lna-oligonucleotides/? akamai-feo=off&clear=true#productdetails), sequences containing a triple C or triple G pattern were avoided. The melting temperatures of the reverse complements of the remaining sequences were determined in silico, considering the effects of LNA modifications. The typical LNA-gapmer format of three modifications at each side 80,81 was optimized, if there were strong deviations from the internally defined range of an appropriate melting temperature. In the first screen, 23 sequences showing a perfect match to human and mice NPC1 mRNA and 13 sequences specific only to the human mRNA were used to design ASOs containing phosphorothioate bonds and the optimal LNA pattern. For screening, IC 50 determination and analysis of protein knockdown, 40 nmol of ASOs were synthesized by Eurogentec (Cologne, Germany). After selection of two of the most effective candidates, 5 mg of the ASOs 05HM and 28H were purchased from Exiqon (Copenhagen, Denmark) for in vitro infection assays. Control oligonucleotides Neg1 and S5 were synthesized by Exiqon (Copenhagen, Denmark), and negative control oligonucleotide Neg1b was purchased from Axolabs (Kulmbach, Germany). ASOs were purified by reverse-phase high-performance liquid chromatography (HPLC) and subsequently lyophilized. After receiving the ASOs, they were solubilized in diethyl-pyrocarbonate (DEPC)treated H 2 O to a concentration of 1 mM. The cells were detached using trypsin (25200056; Gibco), seeded into 96-well plates at 6,000 (HeLa), 20,000 (THP-1), and 5,000 (4T1) cells per well and treated with the NPC1 ASO compounds on day 0. Treatments were done at a final concentration of 10 mM with triplicate experiments for each compound. A control oligonucleotide, Neg1, was used at a final concentration of 10 mM with triplicate experiments. On day 3, cells were lysed, and mRNA levels were measured according to the manufacturers' instructions, using QuantiGene Singleplex Gene Expression Assay (QS0011; Life Technologies, Darmstadt, Germany) and the following probe sets: human probe sets specific for NPC1 (SA-10502; Life Technologies) and the housekeeping gene HPRT1 (SA-10030; Life Technologies) or probe sets specific for murine Npc1 (SB-12805; Life Technologies) and Hprt1 (SB-15463; Life Technologies). Residual NPC1 mRNA expression levels were calculated by comparing the NPC1 values normalized to HPRT1 in the ASO-treated samples with that measured in the untreated control. IC 50 Determination HeLa and 4T1 cells were used to generate dose-response curves by treating them with different concentrations (5,000 nM, 1,000 nM, 200 nM, 40 nM, 8 nM, and 1.6 nM) of ASO 05HM (4T1; HeLa) and 28H (HeLa). On day 3 after treatment, cell viability was determined using the CellTiter-Blue Assay (G8081; Promega, Mannheim, Germany) according to the manufacturer's instructions. Afterwards, the cells were lysed, and mRNA levels were measured according to the manufacturers' instructions, using the QuantiGene Singleplex Gene Expression Assay (QS0011; Life Technologies) and the following probe sets: human probe sets specific for NPC1 (SA-10502; Life Technologies) and the housekeeping gene HPRT1 (SA-10030; Life Technologies) or probe sets specific for murine Npc1 (SB-12805; Life Technologies) and Hprt1 (SB-15463; Life Technologies). Values were normalized to the housekeeping gene HPRT1 and cell control (untreated cells). IC 50 values were calculated using Prism 6 (GraphPad Software). Immunoblot Analysis HeLa cells were seeded into 12-well plates at 75,000 cells per well and treated twice with 10 mM ASO 05HM and 28H for 3 days each. On day 6, cells were lysed using 100 mL RIPA buffer (89900; Thermo Scientific, Life Technologies) per well supplemented with Halt Protease Inhibitor Cocktail (1861278; Thermo Scientific). Samples were prepared for SDS-PAGE using 4Â Laemmli sample buffer (161-0747; Bio-Rad Laboratories, München, Germany) and proteins were separated using Mini-Protean TGXÔ Precast Gels TLR9 Reporter Assay HEK-Blue hTLR9 cells, which were generated by co-transfection of the hTLR9 gene and an optimized SEAP reporter gene into HEK293 cells, were obtained from InvivoGen (hkb-htlr9; Toulouse, France). The SEAP reporter gene was placed under the control of the interferon-beta (IFNb) minimal promoter fused to five NF-lB and activator protein 1 (AP-1) binding sites. Stimulation with a TLR9 ligand activates NF-lB and AP-1, which induce the production of SEAP. Cells were cultivated in DMEM GlutaMAX (32430027; Gibco, Life Technologies) supplemented with 10% FCS (10270106; Gibco), 1 mM sodium pyruvate (11360070; Gibco), 1Â antibioticantimycotic (15240062; Gibco), 10 mg/mL Blasticidin (ant-bl-05; InvivoGen) and 100 mg/mL Zeocin (ant-zn-1; InvivoGen). Cells were seeded at 25,000 cells per well in 96-well plates and incubated Figure 6. NPC1-Specific ASOs 05HM and 28H Specifically Inhibit EBOV Replication (A and B) HeLa cells pretreated with the respective ASO were infected with EBOV at an MOI of 0.01. At 1 day after infection (p.i.), NPC1 (A) and EBOV (B) levels were quantified by RT-qPCR and normalized to the internal control a-tubulin. Shown is the fold change compared to untreated control (set at 100), which was calculated using the 2 ÀDDCt method. Error bars show SD (n = 3, each in duplicate). Duplicates are labeled with identical symbol shapes. ANOVA was used to test for significant differences and p values were determined using Dunnett's test, in GraphPad Prism 7.04 Software. See also Figure S2. for 20 h at 37 C and 5% CO 2 . Then, they were treated with ODN2006 (tlrl-2006; InvivoGen) or LNA-ASOs 05HM and 28H, with 5-fold serial dilutions, starting at a concentration of 5,000-1.6 nM. As a control, cells were treated with cell culture medium without addition of oligonucleotides. Each condition was performed in triplicate. Twenty hours after cell treatment, 100 mL Quanti-Blue (QB) Solution (repqbs; InvivoGen) was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water in a sterile glass bottle or flask. QB Solution was gently mixed and incubated for 10 min at room temperature before it was added to the sample. HEK-Blue-hTLR9 cell supernatants (20 mL per well) were harvested into a fresh 96-well plate and 180 mL QB solution was added to each well. Samples were incubated for 2 h at 37 C, and SEAP activity was determined by measurement of the optical density at 620 nm with a microplate reader. Stably transfected HEK cells expressing a mouse Tlr9 Nf-lb luciferase reporter plasmid, kindly provided by Prof. Holger Garn, University of Marburg, were used for the murine Tlr9 reporter gene assay. Stimulation with mouse Tlr9 ligands activate Nf-lb which induces the expression of firefly luciferase (Photinus pyralis). HEK-mTlr9_ Nflb-LUC cells (25,000/well) were plated in a white-walled, 96-well tissue-culture plate in DMEM GlutaMAX (32430027; Gibco, supplemented as described above). Twenty hours after cell seeding, the cells were treated with 5-fold serial dilutions of ODN1668 (tlrl-1668; In-vivoGen) or LNA-ASO 05HM, starting at a concentration of 5,000-1.6 nM and incubated for 20 h at 37 C in the incubator. As a control, cells were treated with cell culture medium without addition of oligonucleotides. Each condition was performed in triplicate. After the incubation period, the plates were centrifuged for 5 min at 500 g, and the cell supernatants were removed. ONE-Glo EX reagent (50 mL, E8110; Promega) was added to each well, and cells were lysed according to the manufacturer's instructions. Luminescence was immediately measured at 560 nm. Cytokine Release PBMCs were isolated from leukapheresis products donated by three healthy individuals (Klinikum rechts der Isar, TU München, ethics commission reference: 329/16 S), by density gradient centrifugation. The leukapheresis product was diluted 1:10 with PBS (10010-023; Gibco) and carefully loaded onto 15 mL Biocoll separating solution (L6115; Biochrom, Berlin, Germany) in 50 mL Falcon tubes. Density gradient centrifugation was performed at 800 g for 20 min at room temperature with the brake turned off. Afterward, the mononuclear cell layer was collected carefully and transferred into a new 50 mL Falcon tube, and PBS was added to a final volume of 50 mL. After centrifugation at 500 g for 5 min at room temperature (brake turned on) supernatants were discarded, and cell pellets were pooled in PBS in a total volume of 50 mL. Cells were counted, divided into aliquots, and stored on liquid nitrogen for further use. PBMCs (400,000) were seeded in RPMI-1640 medium (72400-047; Gibco) supplemented with 10% FCS, 1 mM sodium pyruvate, 1Â antibiotic-antimycotic, and 50 U/mL Benzonase (70746-3; Merck, Darmstadt, Germany) per well of a 96-well plate. Afterwards, the cells were treated with RPMI containing either oligonucleotides (5 or 20 mM ASO 05HM or 20 mM negative control Neg1) or immune stimulatory agents: 20 mM ODN2006 (tlrl-2006; InvivoGen), 10 ng/mL LPS (L4391; Sigma Aldrich, Taufkirchen, Germany), or 25 mL/mL CD3/ CD28/CD2 (10970; StemCell Technologies, Inc., Grenoble, France). Each condition was performed in triplicate. Treated cells were then rested for 72 h at 37 C and 5% CO 2 . The third day after treatment, 96-well plates were subjected to centrifugation at 2,000 rpm for 10 min at 4 C. Cell supernatants were collected in 96-well plates. For the measurement of IFNg secretion, 50 mL of cell supernatant was diluted with 50 mL ELISA/ELISPOT diluent. IFNg measurement was performed with IFNg Human Uncoated ELISA Kit from eBioscience (88-7316-88; Thermo Fisher Scientific) according to the manufacturer's protocol. For the measurement of IL6 and TNFa secretion, 50 mL of cell supernatant was diluted with 50 mL Assay Diluent A. IL-6 and TNFa measurements were performed with IL-6 and TNFa ELISA Kits, respectively, from BioLegend (Koblenz, Germany [IL6; 430505] and [TNFa; 430201]), according to the manufacturer's protocols. EBOV Infection Assay All work with infectious EBOV was performed in compliance with national regulations at the BSL4 Laboratory of the Institute of Virology, Philipps-University, Marburg. HeLa cells were pretreated in duplicate two times for 3 days by adding medium containing 10 mM of ASOs 05HM and 28H and negative control oligonucleotides Neg1, Neg1B, or S5 or medium only (mock). At 1 h prior to infection, ASOs were removed. Cells were then infected with EBOV Mayinga (NCBI accession number GenBank: AF086833.2) at an MOI of 0.01 for 3 h in the absence of ASOs. Subsequently, the cells were washed to remove unbound input virus and incubated in the presence of 10 mM of the respective ASO. At 24 h after infection, cellular RNA was isolated using the RNeasy Kit (74106; QIAGEN, Hilden, Germany). For virus quantification, EBOV L-or GP-specific primer sets and probes were used for qRT-PCR analysis. 82 Knockdown of NPC1 was analyzed via qRT-PCR with NPC1 specific primers (VHPS-6283; Real-Time Primers, Elkins Park, PA) using the QuantiTect SYBR Green RT-PCR Kit (204143; QIAGEN). Ct values were normalized to the internal control a-tubulin (2 ÀDCt ), and the fold change over mock was calculated using the 2 ÀDDCt method. 83 Statistical Analysis GraphPad Prism 7.04 Software was used for statistical calculations. The efficacy of the ASOs was assessed by one-way ANOVA followed by Dunnett's multiple-comparison test, to contrast the treatment groups (including control ASOs) with mock control. Differences were considered statistically significant when p < 0.05. AUTHOR CONTRIBUTIONS The study design was developed by	2019-05-12T13:47:35.945Z	2019-04-25T00:00:00.000	{ "year": 2019, "sha1": "529068fbd36d7b246b27851f39e5d15b98b7317f", "oa_license": "CCBY", "oa_url": "http://www.cell.com/article/S2162253119300915/pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "ac49eb0a42c24dc89bf37d34b9d515cd48f6b0d5", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
239002072	pes2o/s2orc	v3-fos-license	Long noncoding RNA VPS9D1-AS1 promotes esophageal squamous cell carcinoma progression via the Wnt/β-catenin signaling pathway The VPS9D1 antisense RNA1 (VPS9D1-AS1, lncRNA MYU) can act as an oncogene or an antioncogene in different malignancies. In the present study, we demonstrated that VPS9D1-AS1 is significantly upregulated in esophageal squamous cell carcinoma (ESCC) and assessed its biological function and clinical prognosis. RNA-sequencing was conducted in four pairs of ESCC tissues and normal adjacent tissues (NATs). Compared with controls, lncRNA VPS9D1-AS1 was highly expressed in ESCC tissues, cell lines and plasma. VPS9D1-AS1 upregulation significantly correlated with the histopathological grade and clinical stage of ESCC. Analyses revealed poor prognosis in ESCC patients with high VPS9D1-AS1 expression. VPS9D1-AS1 knockdown led to the inhibition of tumor proliferation, migration, and invasion in vivo and vitro. VPS9D1-AS1 silencing downregulated the Wnt/β-catenin signaling pathways by acting on key proteins such as β-catenin and c-Myc. However, the expressions of these proteins increased after the addition of pathway agonist CT99021. Therefore, taken together VPS9D1-AS1 is highly expressed in ESCC and its expression can lead to poor prognosis. In conclusion, this study suggested that VPS9D1-AS1 acts as a vital part in facilitating ESCC progression and can be a potential biomarker for the diagnosis of patients with ESCC. Introduction Esophageal cancer has become the primary cause of cancer related deaths in China owning to its high incidence and low survival rate. Esophageal squamous cell carcinoma (ESCC) and adenocarcinoma are the two primary types of esophageal cancer. In China, ESCC is more frequent than adenocarcinoma. Although advancements in medical technology have resulted in some progress in the diagnosis and treatment of ESCC, the 5-year survival rate has not been significantly improved [1][2]. Owning to increasing incidence and worse prognosis, researchers are beginning to focus on the early diagnosis and treatment of ESCC. More and more noncoding RNAs (ncRNAs) are generally considered as regulators of tumorigenesis. Long noncoding RNAs (lncRNAs) are a kind of ncRNAs that are longer than 200 nucleotides and that lack or have no open reading coding frame. The number of lncRNAs is much lower than that of mRNAs; they are generally expressed in low levels in tissues and body fluids and have higher tissue and organ specificity [3]. Previous studies have reported a series of aberrant lncRNA expressions in patients with ESCC; these events were related to certain biological characteristics such as proliferation and invasion of ESCC [4][5][6][7]. Therefore, it is important to identify Ivyspring International Publisher tumor related lncRNAs and elucidate the regulatory networks. This will result in a deeper understanding of the tumorigenic mechanisms as well as provide effective cancer diagnosis and development of cancer therapeutics. RNA-sequencing Four pairs of ESCC tissues and normal adjacent tissues (NATs) were obtained from patients during surgery at Yancheng First Hospital, Affiliated Hospital of Nanjing University Medical School, Yancheng, Jiangsu, China. All samples were immediately stored in liquid nitrogen and transported to the designated biotechnology company (Gminix, Shanghai, China). The RNA-sequencing was performed using the Illumina platform by Genminix Informatics co. Ltd in Shanghai. Tissue and plasma sample collection A total of 92 patients with ESCC who were admitted to Yancheng First Hospital, Affiliated Hospital of Nanjing University Medical School between January 2015 and June 2016 were included in this study. In particular, NATs were collected at least 5 cm away from the tumor tissues. Inclusion criteria of patients with ESCC: 1. Patients aged 18-70 years, diagnosed with primary ESCC by digestive endoscopy and pathology; 2. There are indications for radical resection of ESCC or palliative surgery but no obvious contraindications to surgery. Quantitative real-time polymerase chain recation (qRT-PCR) assay Total RNA was extracted from frozen tissues, plasma, and cell cultures using the TRIzol reagent (Thermo Fisher Scientific, USA) and then reverse-transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit 1622 (Thermo Fisher Scientific, USA). qRT-PCR was performed using the ABI® step one plus Real-time PCR system (Applied Biosystems Life Technologies, USA) with the SYBR Green Master Mix (Thermo Fisher Scientific, USA). The primers used are as follows: VPS9D1-AS1: AGC TTTCCTCCTTCATCGGA (forward) and TGGCTTGC AGGGAAAACAC (reverse); GAPDH: GAACGGGA AGCTCACTGG (forward) and GCCTGCTTCACCA CCTTCT (reverse) (RiboBio Co., Guangzhou, China). The relative expression of VPS9D1-AS1 was standardized with that of GAPDH using the 2 −ΔΔCt method. Cell Counting kit-8 (CCK-8) assay The transfected cells were seeded into 96-well plates (3×10 3 /well), followed by the addition of CCK-8 reagent (Beyotime, China) according to the manufacturer's instructions every 24 h for 5 days. The cell proliferation rate determined by measuring the optical density values at 450 nm using a microplate reader (Molecular Devices, BioTek, USA). Cell migration and invasion assays To evaluate the migration and invasion abilities of ESCC cells, the Transwell chamber (Corning, USA) was used. Matrigel (Invitrogen, USA) was diluted with RPMI-1640 at a 1:9 ratio. After being stably transfected, the cells were resuspended to a density of 10 6 /ml. Then, 100 μl of the resuspension was added into the upper chamber. After incubating for 24 h, the cells remaining on the upper membrane were removed, fixed, stained, and counted under an inverted fluorescence microscope (EVOS FL Auto; Invitrogen). Wound healing assay The confluent transfected cells were scratched with a 1 ml pipette tip, followed by the replacement of serum-free medium. Wound healing was observed and recorded under a fluorescence microscope (EVOS FL Auto; Invitrogen). The Image J software was used to analyze the migration ability. Flow cytometric analysis The prepared transfected cells were stored in 70% ethanol at −20 °C. After washing the cells with phosphate buffered saline, propidium iodide was added to the cells, followed by incubation for 40 min. Then, cell cycle analysis was performed using the BD FACSCalibur system (BD, USA). Xenograft assays in nude mice Four-week-old female BALB/C nude mice were purchased from the Animal Center of Nantong University and were used in the next study. TE-13 cells, which were stably infected with the lentivirus or negative control, were subcutaneously inoculated into experimental group and control group of nude mice. After 3 weeks the mice were sacrificed and the tumor tissues were stripped and weighed. Immunohistochemistry (IHC) analysis The ENVISION method was used for IHC analysis. Anti-Ki-67 (ab92742) was purchased from Abcam. First, the specimens from the transplanted tumors were fixed, embedded, and cut into 5 μm thick slices. Then, the slices were deparaffinized, hydrated, blocked with 3% H2O2, and sequentially incubated with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Finally, five low power fields and high power fields were randomly selected for scoring the staining results by two independent observers. The number of positive cancer cells expression <5% was negative (-); 5%~25% was weakly positive (+); 25%~50% was moderately positive (++) and ≥50% was strongly positive (+++). Colony formation assay Approximately 15 ml of the cell suspension containing 2,000 transfected cells was planted into 10 cm petri dishes. After 12 days of incubation, the fixed cells were stained with methyl violet. The number of colonies (≥50 cells) was counted with naked eyes. Statistical analysis The SPSS 23.0 and GraphPad Prism 7.0 software were used to conduct all statistical analyses. All data are expressed as mean ± standard deviation. Between-group variances were calculated using the t-test, chi-square test, and nonparametric test (Wilcoxon test). Overall survival (OS) and progression-free survival (PFS) were calculated by the Kaplan-Meier method using the log-rank test. A p-value of less than 0.05 was considered significant. Analysis of lncRNA expression via RNAsequencing Based on the sequencing results (p < 0.05 and log2 fold change >2 or < 0.5), 3,514 lncRNAs were differentially expressed in ESCC tumors and matched NATs, among which 2,079 were upregulated and 1,435 were downregulated. All different lncRNAs were ranked based on their log2 fold change values. First, we removed lncRNAs with longer than 4000 nucleotides and then eliminated the molecules that have already been studied in esophageal cancer such as H19, SNHG6, LUCAT1, HOXA11. Through the above screening, we obtained several candidate molecules, which were verified using TCGA and ENCORI databases. Finally, VPS9D1-AS1 was selected among these lncRNAs for further studies (Fig. 1A). It can be detected in nucleus, cytoplasm, exosomes, and plasma, according to the RNAlocate database. Upregulation of VPS9D1-AS1 expression in ESCC tissues, cells and plasma The expression levels of VPS9D1-AS1 were detected in 92 paired ESCC tissue samples and NATs by conducting a series of qRT-PCR assays. The assays revealed that VPS9D1-AS1 was markedly upregulated in tumor tissues compared with NATs ( Fig. 1B-D). We further detected VPS9D1-AS1 expression in ESCC cell lines, covering Eca109, Kyse150, TE-1, TE-13, and the esophageal epithelial cell line het-1A. The expression level of VPS9D1-AS1 was higher in all ESCC cell lines than that in het-1A (Fig. 1E). Among the cell lines, TE-13 and Eca109 with higher VPS9D1-AS1 expression levels were selected for further studies. Besides that, the expression of VPS9D1-AS1 in 60 plasma samples including 25 patients with ESCC and 35 patients with esophageal benign lesions was detected; then, ROC curve analysis was conducted. The area under the ROC curve was 0.7223 (95% CI: 0.5958-0.8488; p=0.0035) (Fig. 1F). As a good early diagnostic indicator, the detection of VPS9D1-AS1 in plasma is a noninvasive and convenient method. However, the expression of VPS9D1-AS1 in ESCC plasma is lower than that in ESCC tissues; therefore, further analysis is warranted to determine whether it is a potential diagnostic biomarker. Relevant clinicopathologic factors of VPS9D1-AS1 in ESCC The expression levels of VPS9D1-AS1 in tumor tissues were classified into low-expression and high-expression groups according to the median values. Furthermore, age, gender, smoking history, tumor location, histopathological grade, invasion depth, lymph node metastasis, and tumor-nodemetastasis (TNM) stages were recorded, and their correlation with VPS9D1-AS1 expression was determined (Tables 1 and 2). VPS9D1-AS1 expression level was an independent factor affects PFS and OS of patients with ESCC. High expression of VPS9D1-AS1 leads to poor prognosis The results showed that the patients in the high-VPS9D1-AS1 expression group had a shorter PFS and OS than those in the lower-expression group ( Fig. 2A,F). Further, VPS9D1-AS1 expression was strongly correlated with PFS and OS in advanced clinical stages (stages III and IV) (Fig. 2B,G). In patients with early clinical stage, no significant difference was observed in PFS and OS between the high and low expression groups (Fig. 2C,H). The short-term PFS and OS of patients without radiotherapy or chemotherapy after undergoing surgery were not significantly different; however, the long-term PFS and OS of those with high VPS9D1-AS1 expression significantly decreased (Fig. 2E,J). Tumor invasion depth and lymph node metastasis were the factors affecting PFS and OS. These clinical variables were considered as potential predictors of survival in univariate and multivariate analyses (Tables 3 and 4). In the CCK-8 assay, the proliferation of Eca109 and TE-13 cells were significantly impaired after VPS9D1-AS1 knockdown compared with the negative control (Fig. 3B). In the Transwell assay, the number of cells penetrating through the filter was remarkably decreased following VPS9D1-AS1 knockdown in Eca109. Similar results were observed in TE-13 cells. Consistent with the abovementioned findings, the invasiveness of these two cell lines following VPS9D1-AS1 knockdown was also respectively repressed (Fig. 3C). Furthermore, VPS9D1-AS1 depletion caused a delay in wound healing in Eca109 and TE-13 cells (Fig. 3D). Taken together, the results demonstrate that VPS9D1-AS1 downregulation remarkably restrains the proliferation, migration, and invasion of ESCC cells. VPS9D1-AS1 regulates the cell cycle Cell cycle progression following VPS9D1-AS1 downregulation was significantly impeded at the G0/G1 phase in Eca109 and TE-13 cell lines (Fig. 3E). Next, we examined cell cycle regulators, such as CDk4, CDk6, and cyclin D1. All of them were markedly suppressed by VPS9D1-AS1 silencing (Fig. 3F). Therefore, we speculate that VPS9D1-AS1 downregulation suppresses tumor growth and progression by inducing cell cycle stagnation. Downregulation of VPS9D1-AS1 inhibits ESCC tumorigenesis in vivo With the downregulation of VPS9D1-AS1, tumors that were stripped from the nude mice were remarkably smaller. Further, the index of proliferation, Ki-67, was significantly downregulated, as revealed by IHC analysis. In conclusion, the results suggest that VPS9D1-AS1 promotes tumor growth (Fig. 4A,B). VPS9D1-AS1 affects ESCC via the Wnt/ β-catenin signaling pathway Through the enrichment analysis of VPS9D1-AS1, we obtained the most likely related biological function of this molecule, the top five were ribosome biogenesis, RNA modification, translation, ribosome assembly and cell cycle. Among several common pathways associated with tumor invasion and migration, Wnt and c-myc, key molecules in the Wnt/β-catenin pathway, were involved in regulating ribosome biogenesis. Therefore, we speculated that VPS9D1-AS1 may affect the occurrence and development of ESCC via this pathway. To further verify this hypothesis, we detected the expressions of key proteins in this pathway by Western blot. The expressions of these proteins decreased significantly after the downregulation of VPS9D1-AS1, but increased subsequently after the addition of Wnt/β-catenin pathway agonist CT99021 (Fig. 5A). The results suggested that VPS9D1-AS1 may affect the proliferation of ESCC cells by regulating the Wnt/β-catenin signaling pathway. In the colony formation assay, VPS9D1-AS1 knockdown in Eca109 and TE-13 cells greatly suppressed colony growth compared with that in control cells, however, when CT99021 was added into shVPS9D1-AS1 group, it was found that the ability of clone formation and growth was significantly improved (Fig. 5B). Moreover, we found that the expression of VPS9D1-AS1 positively correlated with that of β-catenin and c-Myc in ESCC tissue samples. Western blotting revealed that the expressions of β-catenin and c-Myc were significantly higher in ESCC tissues than in NATs. Furthermore, in the high-VPS9D1-AS1 expression group, β-catenin and c-Myc expressions were higher in ESCC tissues compared with those in the low VPS9D1-AS1 expression group (Fig. 5C). Taken together, these results demonstrate that VPS9D1-AS1 might promote ESCC progression by regulating the Wnt/β-catenin signaling pathway. Discussion Previous studies have confirmed the number of lncRNAs associated with the cancer prognosis [9][10][11]. Those studies have indicated that lncRNAs have high potential as a biomarker for cancer diagnosis or prognosis. LncRNAs, miRNAs, and target genes form an interactive regulatory network, in which lncRNAs function as "sponges" or "ceRNAs" [12][13][14]. Although a part of the mechanism of ESCC occurrence and development has been discovered, researchers have found no specific and sensitive biomarkers for early diagnosis and treatment, which have a central impact on improving the survival rate of ESCC. Several studies have shown that lncRNAs play a carcinogenic or inhibitory role in the occurrence and development of ESCC [4][5][6][7]. They are usually differentially expressed in ESCC and play a central role in the regulation of epigenetics, as well as in the transcriptional regulation and post-translational modification of proteins [15]. LncRNAs have been detected in a variety of microenvironments. They are found not only in tissues but also in serum, plasma, body fluid, and the cerebrospinal fluid [16]. LncRNAs such as HOTAIR or XIST may function as a scaffold to recruit proteins involved in chromatin remodeling or histone modification [17][18]. LncRNA CCAT2 and ESCCAL-1 have been found to be highly expressed in ESCC tissues; their expression is closely related to the degree of lymph node metastasis and TNM stage. The high expression of these lncRNAs increases the risk of death and shortens survival time in patients [19][20]. VPS9D1-AS1, an antisense RNA that is 1,637 nucleotides long, was initially identified as a lncRNA regulated by c-Myc in colon cancer, as reported by Yoshihiro et al. Therefore, it was named lncRNA MYU (c-Myc-upregulated lncRNA) [21]. In patients with gastric cancer, the expression of VPS9D1-AS1 negatively correlates with tumor size and TNM stage [22]. The present study further indicated that patients with gastric tumor with low VPS9D1-AS1 expression are expected to show a shorter OS and disease-free survival than those with high VPS9D1-AS1 expression [22]. In contrast, VPS9D1-AS1 is overexpressed in prostate and colorectal cancers [23][24]. These conflicting findings prompted our interest in accelerating research on VPS9D1-AS1 in ESCC. The present study demonstrated that VPS9D1-AS1 was upregulated in ESCC tissues, plasma, and ESCC cell lines; the expression level of VPS9D1-AS1 was directly correlated with clinicopathologic features. The present study further showed that VPS9D1-AS1 downregulation inhibited the proliferation, colony formation, migration, invasion, and cell cycle of ESCC cells. In addition, VPS9D1-AS1 expression was positively correlated with β-catenin and c-Myc in ESCC samples. A previous study has suggested that VPS9D1-AS1 is the direct lncRNA target of the Wnt/c-Myc pathway and participates in the tumorigenicity of colon cancer cells [21]. Furthermore, Kawasaki has demonstrated that VPS9D1-AS1 associates with the RNA-binding protein heterogeneous nuclear ribonucleoprotein-K to stabilize CDK6 expression, thereby promoting the G1/S transition of the cell cycle. Is there a similar effect in ESCC? Previous studies have shown that tumor invasion and migration in ESCC are closely regulated by activating the Wnt/β-catenin pathway [25][26]. Through the enrichment analysis of VPS9D1-AS1, we obtained the most likely related biological function of VPS9D1-AS1; the top five biological functions were ribosome biogenesis, RNA modification, translation, ribosome assembly, and cell cycle. Wnt and c-Myc co-regulate ribosome biogenesis [27]. Previous studies have reported that Wnt signaling regulates c-Myc via Wnt/STOP and the Wnt/β-catenin pathway. The subsequent research thus established the key role of Wnt signaling in ribosome biogenesis via two routes. One is via c-Myc, which is regulated both via Wnt-driven c-Myc expression and via the Wnt/STOP pathway. The other route is c-Myc independent and is a downstream effect of Wnt signaling on the transcription of ribosomal genes [21]. In the present study, a significant decrease in the levels of c-Myc and cyclin D1 proteins were observed following VPS9D1-AS1 silencing in Eca109 and TE-13. This result indicates that VPS9D1-AS1 and c-Myc might form a feedback loop to regulate each other; by inference, we found that VPS9D1-AS1 displays carcinogenicity by regulating the expression of c-Myc in ESCC. It is well-known that Wnt/β-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc is one of the most commonly activated oncogenes and is reported to be involved in many human cancers. Thus, much effort has been devoted to explore the potential mechanisms with the role of c-Myc in the cell cycle. It has been reported that c-Myc induced cell proliferation is generally associate with an increase in CDK4 and CDK6 activities, which regulated G1 progression in colon cancer cells [21]. Furthermore, it has been reported that c-Myc induces the expression of CDK4 by directly binding to the CDK4 promoter region. Moreover, c-Myc also regulates the mRNA expression of CDK6. β-catenin is widely distributed in different cells as a multifunctional protein and plays a vital role in cell proliferation, migration, apoptosis, and tumorigenesis [28][29][30]. β-catenin expression was found to be visibly declined when VPS9D1-AS1 was downregulated in Eca109 and TE-13 cells. β-catenin is considered a key point in the regulation of intracellular signal transduction via the Wnt signaling pathway. In conclusion, the present study showed that with VPS9D1-AS1 silencing, the expression of CDK4 and 6 and cyclin D1 declined sharply, but increased subsequently after the addition of Wnt/β-catenin pathway agonist CT99021. However, our experiment has two drawbacks. First, the detection of VPS9D1-AS1 expression in clinical samples was not plenitude. Hence, more samples should be used in future studies to further validate our findings. Second, we speculate that VPS9D1-AS1 promotes tumor progression by regulating the cell cycle via Wnt/β-catenin signaling pathway. However, the specific relative gene and ceRNA network analyses have not been completed. Therefore, its specific mechanism still needs to be explored in the future. Conclusions The present study showed that the level of VPS9D1-AS1 plays a significant role in tumor progression. First, we proved that VPS9D1-AS1 is overexpressed in ESCC tissues, plasma, and cell lines, and that higher expression is often associated with worse prognosis. Furthermore, VPS9D1-AS1 promotes the proliferation and invasion of ESCC cells and also influences the cell cycle via the Wnt/βcatenin signaling pathway. Overall, this study demonstrates that VPS9D1-AS1 might be a good potential marker for the early diagnosis and prognosis of ESCC.	2021-10-17T05:14:49.572Z	2021-10-02T00:00:00.000	{ "year": 2021, "sha1": "fc17d2487e4ce14ad5729efa7faf09c3f417c1f4", "oa_license": "CCBY", "oa_url": "https://www.jcancer.org/v12p6894.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "fc17d2487e4ce14ad5729efa7faf09c3f417c1f4", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
9603650	pes2o/s2orc	v3-fos-license	Online bipartite matching with amortized $O(\log^2 n)$ replacements In the online bipartite matching problem with replacements, all the vertices on one side of the bipartition are given, and the vertices on the other side arrive one by one with all their incident edges. The goal is to maintain a maximum matching while minimizing the number of changes (replacements) to the matching. We show that the greedy algorithm that always takes the shortest augmenting path from the newly inserted vertex (denoted the SAP protocol) uses at most amortized $O(\log^2 n)$ replacements per insertion, where $n$ is the total number of vertices inserted. This is the first analysis to achieve a polylogarithmic number of replacements for \emph{any} replacement strategy, almost matching the $\Omega(\log n)$ lower bound. The previous best known strategy achieved amortized $O(\sqrt{n})$ replacements [Bosek, Leniowski, Sankowski, Zych, FOCS 2014]. For the SAP protocol in particular, nothing better than then trivial $O(n)$ bound was known except in special cases. Our analysis immediately implies the same upper bound of $O(\log^2 n)$ reassignments for the capacitated assignment problem, where each vertex on the static side of the bipartition is initialized with the capacity to serve a number of vertices. We also analyze the problem of minimizing the maximum server load. We show that if the final graph has maximum server load $L$, then the SAP protocol makes amortized $O( \min\{L \log^2 n , \sqrt{n}\log n\})$ reassignments. We also show that this is close to tight because $\Omega(\min\{L, \sqrt{n}\})$ reassignments can be necessary. Introduction In the online bipartite matching problem, the vertices on one side are given in advance (we call these the servers S), while the vertices on the other side (the clients C) arrive one at a time with all their incident edges.In the standard online model the arriving client can only be matched immediately upon arrival, and the matching cannot be changed later.Because of this irreversibility, the final matching might not be maximum; no algorithm can guarantee better than a (1 − 1/e)approximation [KVV90].But in many settings the irreversibility assumption is too strict: rematching a client is expensive but not impossible.This motivates the online bipartite matching problem with replacements, where the goal is to at all times match as many clients as possible, while minimizing the number of changes to the matching.Applications include hashing, job scheduling, web hosting, streaming content delivery, and data storage; see [CDKL09] for more details. In several of the applications above, a server can serve multiple clients, which raises the question of online bipartite assignment with reassignments.There are two ways of modeling this: Capacitated assignments.Each server s comes with the capacity to serve some number of clients u(s), where each u(s) is given in advance.Clients should be assigned to a server, and at no times should the capacity of a server be exceeded.There exists an easy reduction showing that this problem is equivalent to online matching with replacements [BKP + 17].A more formal description is given in Section 6.1. Minimize max load.There is no limit on the number of clients a server can serve, but we want to (at all times) distribute the clients as "fairly" as possible, while still serving all the clients.Defining the load on a server as the number of clients assigned to it, the task is to, at all times, minimize the maximum server load -with as few reassignments as possible.A more formal description is given in Section 6.2 While the primary goal is to minimize the number of replacements, special emphasis has been placed on analyzing the SAP protocol in particular, which always augments down a shortest augmenting path from the newly arrived client to a free server (breaking ties arbitrarily).This is the most natural replacement strategy, and shortest augmenting paths are already of great interest in graph algorithms: they occur in for example in Dinitz' and Edmonds and Karp's algorithm for maximum flow [Din70,EK72], and in Hopcroft and Karp's algorithm for maximum matching in bipartite graphs [HK73]. Throughout the rest of the paper, we let n be the number of clients in the final graph, and we consider the total number of replacements during the entire sequence of insertions; this is exactly n times the amortized number of replacements.The reason for studying the vertex-arrival model (where each client arrives with all its incident edges) instead of the (perhaps more natural) edge-arrival model is the existence of a trivial lower bound of Ω(n 2 ) total replacements in this model: Start with a single edge, and maintaining at all times that the current graph is a path, add edges to alternating sides of the path.Every pair of insertions cause the entire path to be augmented, leading to a total of n/2 i=1 i ∈ Ω(n 2 ) replacements. Previous work The problem of online bipartite matchings with replacements was introduced in 1995 by Grove, Kao, Krishnan, and Vitter [GKKV95], who showed matching upper and lower bounds of Θ(n log n) replacements for the case where each client has degree two.In 2009, Chadhuri, Daskalakis, Kleinberg, and Lin [CDKL09] showed that for any arbitrary underlying bipartite graph, if the client vertices arrive in a random order, the expected number of replacements (in their terminology, the switching cost) is Θ(n log n) using SAP, which they also show is tight.They also show that if the bipartite graph remains a forest, there exists an algorithm (not SAP) with O(n log n) replacements, and a matching lower bound.Bosek, Leniowski, Sankowski and Zyck later analyzed the SAP protocol for forests, giving an upper bound of O(n log 2 n) replacements [BLSZ15], later improved to the optimal O(n log n) total replacements [BLZS17].For general bipartite graphs, no analysis of SAP is known that shows better than the trivial O(n 2 ) total replacements.Bosek et al. [BLSZ14] showed a different algorithm that achieves a total of O(n √ n) replacements.They also show how to implement this algorithm in total time O(m √ n), which matches the best performing combinatorial algorithm for computing a maximum matching in a static bipartite graph (Hopcroft and Karp [HK73]). The lower bound of Ω(log n) by Grove et al. [GKKV95] has not been improved since, and is conjectured by Chadhuri et al. [CDKL09] to be tight, even for SAP, in the general case.We take a giant leap towards closing that conjecture. For the problem of minimizing maximum load, [GKS14] and [BKP + 17] showed an approximation solution: with only O(1) amortized changes per client insertion they maintain an assignment A such that at all times the maximum load is within a factor of 8 of optimum. The model of online algorithms with replacements -alternatively referred to as online algorithms with recourse -has also been studied for a variety of problems other than matching.The model is similar to that of online algorithms, except that instead of trying to maintain the best possible approximation without making any changes, the goal is to maintain an optimal solution while making as few changes to the solution as possible.This model encapsulates settings in which changes to the solution are possible but expensive.The model originally goes back to online Steiner trees [IW91], and there have been several recent improvements for online Steiner tree with recourse [MSVW16, GGK13, GK14, LOP + 15].There are many papers on online scheduling that try to minimize the number of job reassignments [PW98, Wes00, AGZ99, SSS09, SV10, EL11].The model has also been studied in the context of flows [Wes00,GKS14], and there is a very recent result on online set cover with recourse [GKKP17]. Our results Theorem 1. SAP makes at most O(n log 2 n) total replacements when n clients are added.This is a huge improvement of the O(n √ n) bound by [BLSZ14], and is only a log factor from the lower bound of Ω(n log n) by [GKKV95].It is also a huge improvement of the analysis of SAP; previously no better upper bound than O(n 2 ) replacements for SAP was known.To attain the result we develop a new tool for analyzing matching-related properties of graphs (the balanced flow in Sections 3 and 4) that is quite general, and that we believe may be of independent interest. Although SAP is an obvious way of serving the clients as they come, it does not immediately allow for an efficient implementation.Finding an augmenting path may take up to O(m) time, where m denotes the total number of edges in the final graph.Thus, the naive implementation takes O(mn) total time.However, short augmenting paths can be found substantially faster, and using the new analytical tools developed in this paper, we are able to exploit this in a data structure that finds the augmenting paths efficiently: Theorem 2. There is an implementation of the SAP protocol that runs in total time Note that this is only an O( √ log n) factor from the algorithm by Hopcroft and Karp [HK73], which is matched by Bosek et al. [BLSZ14] in the online setting. Extending our result to the case where each server can have multiple clients, we use that the capacitated assignment problem is equivalent to that of matching (see Section 6.1 to obtain: Theorem 3. SAP uses at most O(n log 2 n) reassignments for the capacitated assignment problem, where n is the number of clients. In the case where we wish to minimize the maximum load, such a small number of total reassignments is not possible.Let opt(G) denote the minimum possible maximum load in graph G.We present a lower bound showing that when opt(G) = L we may need as many as Ω(nL) reassignments, as well as a nearly matching upper bound. Theorem 4. For any positive integers n and L ≤ n/2 divisible by 4 there exists a graph G = (C ∪ S, E) with \|C\| = n and opt(G) = L, along with an ordering in which the clients in C are inserted, such that any algorithm for the exact online assignment problem requires a total of Ω(nL) changes.This lower bound holds even if the algorithm knows the entire graph G in advance, as well as the order in which the clients are inserted. Theorem 5. Let C be the set of all clients inserted, let n = \|C\|, and let L = opt(G) be the minimum possible maximum load in the final graph G = (C ∪ S, E).SAP at all times maintains an optimal assignment while making a total of O(n min {L log 2 n, √ n log n}) reassignments. High Level Overview of Techniques Consider the standard setting in which we are given the entire graph from the beginning and want to compute a maximum matching.The classic shortest-augmenting paths algorithm constructs a matching by at every step picking a shortest augmenting path in the graph.We now show a very simple argument that the total length of all these augmenting paths is O(n log n).Recall the well-known fact that if all augmenting paths in the matching have length ≥ h, then the current matching is at most 2n/h edges from optimal [HK73].Thus the algorithm augments down at most 2n/h augmenting paths of length ≥ h.Let P 1 , P 2 , ..., P k denote all the paths augmented down by the algorithm in decreasing order of \|P i \|; then k ≤ n, and In the online setting, the algorithm does not have access to the entire graph.It can only choose the shortest augmenting path from the arriving client c.We are nonetheless able to show a similar bound for this setting: Lemma 6.Consider the following protocol for constructing a matching: For each client c in arbitrary order, augment along the shortest augmenting path from c (if one exists).Given any h, this protocol augments down a total of at most 4n ln(n)/h augmenting paths of length > h. The proof of our main theorem then follows directly from the lemma. Proof of Theorem 1.Note that the SAP protocol exactly follows the condition of Lemma 6.Now, Given any 0 ≤ i ≤ log 2 (n) + 1, we say that an augmenting path is at level i if its length is in the interval [2 i , 2 i+1 ).By Lemma 6, the SAP protocol augments down at most 4n ln(n)/2 i paths of level i.Since each of those paths contains at most 2 i+1 edges, the total length of augmenting paths of level i is at most 8n ln(n).Summing over all levels yields the desired O(n log 2 n) bound. The entirety of Sections 3 and 4 is devoted to proving Lemma 6.Previous algorithms attempted to bound the total number of reassignments by tracking how some property of the matching M changes over time.For example, the analysis of Gupta et al. [GKS14] keeps track of changes to the "height" of vertices in M , while the algorithm with O(n √ n) reassignments [BLSZ14] takes a more direct approach, and uses a non-SAP protocol whose changes to M depend on how often each particular client has already been reassigned. Unfortunately such arguments have had limited success because the matching M can change quite erratically.This is especially true under the SAP protocol, which is why it has only been analyzed in very restrictive settings [CDKL09,GKKV95,BLSZ15].We overcome this difficulty by showing that it is enough to analyze how new clients change the structure of the graph G = (C ∪ S, E), without reference to any particular matching. Intuitively, our analysis keeps track of how "necessary" each server s is (denoted α(s) below).So for example, if there is a complete bipartite graph with 10 servers and 10 clients, then all servers are completely necessary.But if the complete graph has 20 servers and 10 clients, then while any matching has 10 matched servers and 10 unmatched ones, it is clear that if we abstract away from the particular matching every server is 1/2-necessary.Of course in more complicated graphs different servers might have different necessities, and some necessities might be very close to 1 (say 1 − 1/n 2/3 ).Note that server necessities depend only on the graph, not on any particular matching.Note also that our algorithm never computes the server necessities, as they are merely an analytical tool. We relate necessities to the number of reassignments with 2 crucial arguments.1. Server necessities only increase as clients are inserted, and once a server has α(s) = 1, then regardless of the current matching, no future augmenting path will go through s. 2. If, in any matching, the shortest augmenting path from a new client c is long, then the insertion of c will increase the necessity of servers that already had high necessity.We then argue that this cannot happen too many times before the servers involved have necessity 1, and thus do not partake in any future augmenting paths. Paper outline In Section 2, we introduce the terminology necessary to understand the paper.In Section 3, we introduce and reason about the abstraction of a balanced server flow, a number that reflects the necessity of each server.In Section 4, we use the balanced server flow to prove Lemma 6, which proves our main theorem that SAP makes a total of O(n log 2 n) replacements.In Section 5, we give an efficient implementation of SAP.Finally, in Section 6, we present our results on capacitated online assignment, and for minimizing maximum server load in the online assignment problem. Preliminaries and notation Let (C, S) be the vertices, and E be the edges of a bipartite graph.We call C the clients, and S the servers.Clients arrive, one at a time, and we must maintain an explicit maximum matching of the clients.For simplicity of notation, we assume for the rest of the paper that C = ∅.For any vertex v, let N (v) denote the neighborhood of v, and for any Theorem 7 (Halls Marriage Theorem [Hal35]). There is a matching of size \|C\| if and only if Definition 8. Given any matching in a graph G = (C ∪ S, E), an alternating path is one which alternates between unmatched and matched edges.An augmenting path is an alternating path that starts and ends with an unmatched vertex.Given any augmenting path P , "flipping" the matched status of every edge on P gives a new larger matching.We call this process augmenting down P . Denote by SAP the algorithm that upon the arrival of a new client c augments down the shortest augmenting path from c; ties can be broken arbitrarily, and if no augmenting path from c exists the algorithm does nothing.Chaudhuri et al. [CDKL09] showed that if the final graph contains a perfect matching, then the SAP protocol also returns a perfect matching.We now generalize this as follows Observation 9.Because of the nature of augmenting paths, once a client c or a server s is matched by the SAP protocol, it will remain matched during all future client insertions.On the other hand, if a client c arrives and there is no augmenting path from c to a free server, then during the entire sequence of client insertions c will never be matched by the SAP protocol; no alternating path can go through c because it is not incident to any matched edges. Lemma 10. The SAP protocol always maintains a maximum matching in the current graph G = (C ∪ S, E). Proof.Consider for contradiction the first client c such that after the insertion of c, the matching M maintained by the SAP protocol is not a maximum matching.Let C be the set of clients before But this contradicts the well known property of augmenting paths that the symmetric difference M ⊕ M contains an augmenting path in M from c to a free server. The Server Flow Abstraction We now formalize the notion of server necessities from Section 1.3 by using a flow-based notation. Definition 11.Given any graph G = (C ∪ S, E), define a server flow α as any map from S to the nonnegative reals such that there exist nonnegative (x e ) e∈E with: x cs = α(s) We say that such a set of x-values realize the server flow. Note that the same server flow may be realized in more than one way.Furthermore, if The following theorem can be seen as a generalization of Hall's Marriage Theorem: there exists a server flow where every server s ∈ S has α(s) ≤ p q .Proof.Let C * be the original set C but with q copies of each client.Similarly let S * contain p copies of each server, and let E * consist of all pq edges between copies of the endpoints of each edge in E. Now let K * ⊆ C * , and let K ⊆ C be the originals that the vertices in K * are copies of.Then so the graph (C * ∪ S * , E * ) satisfies Hall's theorem and thus it has some matching M in which every client in C * is matched.Now, for cs ∈ E let a copy of c and s * is a copy of s Since for each c ∈ C all q copies of c are matched, s∈N (c) x cs = q q = 1 for all c ∈ C. Similarly, since for each s ∈ S at most p copies of s are matched, c∈N (s) x cs ≤ p q .Thus, (x e ) e∈E realizes the desired server flow. Definition 13.We call the server flow α balanced, if additionally: where That is, if each client only sends flow to its least loaded neighbours.We call the set A(c) the active neighbors of c, and we call an edge cs active when s ∈ A(c).We extend the definition to sets of clients in the natural way, so for Note that while there may be more than one server flow, we will show that the balanced server flow α is unique, although there may be many possible x-values x cs that realize α.Lemma 14.If α is a balanced server flow, then The first inequality is true because each client in the first set contributes exactly one to the sum (but there may be other contributions).The second inequality is true because every client contributes exactly one to s∈S α(s), and the inequality counts every client that contributes anything to s∈T α(s) as contributing one. We now show that the generalization of Hall's Marriage Theorem from Lemma 12 is "tight" for a balanced server flow in the sense that there does indeed exist a set of p clients with neighbourhood of size q realizing the maximum α-value p q .In fact, the maximally necessary servers and their active neighbours (defined below) form such a pair of sets: Lemma 15.Let α be a balanced server flow, let α = max s∈S α(s) be the maximal necessity, let S = {s ∈ S \| α(s) = α} be the maximally necessary servers, and let and note that K ⊆ K.However, we also have K ⊆ K: By definition of S, and since we assume the server flow is balanced, K = ∅, and for every c ∈ K, N (c) = A(c) ⊆ S. Thus, K = K and N ( K) = S. Now, note that by Lemma 14 We can thus show that α exactly equals the maximal quotient \|K\| \|N (K)\| over subsets K of clients. Lemma 16.Let α be a balanced server flow, and let α = max s∈S α(s).Then Proof.By definition of server flow, for Let K be defined as in Lemma 15.Then Note that for any two nonempty By Corollary 17, G 1 has a unique balanced server flow α 1 with α 1 (s) = α for all s ∈ N ( C).By our induction hypothesis, G 2 also has a unique balanced server flow α 2 .We proceed to show that the combination of α 1 with α 2 constitutes a unique balanced flow α of the entire graph G, defined as follows: Note first that α is a balanced server flow for G, because both G 1 and G 2 have a set of x-values that realize them, and by construction these values (together with zeroes for each edge between C \ C and N ( C)) realize a balanced server flow for G. For uniqueness, note that by Lemma 16 any balanced server flow α for G must have α (s) = α = α 1 (s) for s ∈ N ( C).We now show that for any s ∈ S \ N ( C), any balanced server flow α must also have α (s) = α 2 (s); then, the uniqueness of α will follow from the uniqueness of α 1 and α 2 . Let S = {s ∈ S \| α (s) = α} be the set of maximally necessary servers, and let K = {c ∈ C \| A(c) ∩ S = ∅} be the set of clients with a maximally necessary server in their active neighborhood.We will show that K = C. From now on, let α denote the unique balanced server flow.We want to understand how the balanced server flow changes as vertices are added.For any server s, let α old (s) be the flow in s before the insertion of c, and let α new (s) be the flow after.Also, let ∆α(s) = α new (s) − α old (s). Lemma 19.When a new client c is added, ∆α(s) ≥ 0 for all s ∈ S. It is easy to see that these sets form a partition of S, and that ∅ = S ∆ ⊆ S * .Now, let C ∆ contain all clients with an active neighbor in S ∆ before the insertion of c.Since each client sends one unit of flow, s∈S ∆ α old (s) ≤ C ∆ .Now, because we had a balanced flow before the insertion of c there cannot be any edges in G from C ∆ to S − (any such edge would be from a client u ∈ C ∆ to a server v ∈ S − with α old (v) ≤ α * < α old (s) for s ∈ S ∆ contradicting that u had an active neighbor in S ∆ ).Moreover, in the balanced flow after the insertion of c, there are no active edges from C ∆ to S + (any such edge would be from a client u ∈ C ∆ to a server v ∈ S + with α new (v) > α * = α new (s) for all s ∈ S ∆ so is not active).Thus, all active edges incident to C ∆ go to S ∆ , so s∈S ∆ α new (s) ≥ C ∆ .This contradicts the earlier fact that s∈S ∆ α old (s) ≤ C ∆ , since by definition of S ∆ we have s∈S ∆ α new (s) < s∈S ∆ α old (s). Lemma 20.When a new client c is added , ∆α(s) = 0 for all s where α old (s) < min v∈N (c) α old (v). Proof.Let us first consider the balanced flow before the insertion of c. Let S + = s ∈ S α old (s) ≥ min v∈N (c) α old (v) and define S − = S \ S + .We want to show that ∆α(s) = 0 for all servers s in S − .Define C + to contain all client vertices incident to S + ; that is Note that because the flow is balanced there are no edges in G from C + to S − and there are no active edges from C − to S + before the insertion of c.Thus, s∈S − α old (s) = \|C − \|. Now consider the insertion of c.By definition of S − the new client c has no neighbors in S − , so it is still the case that only clients in C − have neighbors in S − .Thus, in the new balanced flow we still have have that s∈S − α new (s) ≤ \|C − \|.But this means that s∈S − ∆α(s) ≤ 0, so if ∆α(s 1 ) > 0 for some s 1 ∈ S − then ∆α(s 2 ) < 0 for some s 2 ∈ S − , which contradicts Lemma 19. Analyzing replacements in maximum matching We now consider how server flows relate to the length of augmenting paths. Lemma 21. The graph (C ∪ S, E) contains a matching of size \|C\|, if and only if α(s) ≤ 1 for all s ∈ S. Proof.Let α = max s∈S α(s).It follows directly from Lemma 16 that \|K\| ≤ \|N (K)\| for all K ⊆ C if and only if α ≤ 1.The corollary then follows from Hall's Theorem (Theorem 7) It is possible that in the original graph G = (C ∪ S, E), there are many clients that cannot be matched.But recall that by Observation 9, if a client cannot be matched when it is inserted, then it can be effectively ignored for the rest of the algorithm.This motivates the following definition: Definition 26.Define an augmenting tail from a vertex v to be an alternating path that starts in v and ends in an unmatched server.We call an augmenting tail active if all the edges that are not in the matching are active. Note that augmenting tails as defined above are an obvious extension of the concept of augmenting paths: Every augmenting path for a newly arrived client c consists of an edge (c, s), plus an augmenting tail from some server s ∈ N (c). Lemma 27 (Expansion Lemma).Suppose every client is matched, let s ∈ S, and suppose α M (s) = 1 − for some > 0. Then there is an active augmenting tail for s of length at most 2 ln(\|C M \|). Proof.By our definition of active edges, it is not hard to see that any server s reachable from s by an active augmenting tail has α M (s ) ≤ 1 − . For i ≥ 1, let K i be the set of clients c such that there is an active augmenting tail s 0 , c 0 , . . ., c k−1 , s k from s with c = c j for some j < i. At a high level, our algorithm is very similar to the standard O(m √ n) blocking flow algorithm.We will keep track of heights to find shortest paths of length at most √ n ln(n).We will find longer augmenting paths using the trivial O(m) algorithm, and use Lemma 6 to bound the number of such paths.Our analysis will also require the following lemma: Lemma 30.For any server s ∈ S, there is an augmenting tail from s to an unmatched server if and only if α M (s) < 1. Proof.If α M (s) < 1, then the existence of some tail follows directly from the Expansion Lemma 27.Now let us consider α M (s) = 1.Let S 1 = {s ∈ S \| α M (s) = 1}.Since 1 is the maximum possible value of α M (s) (Observation 25), Lemma 15 implies that there is a set of clients C 1 ∈ C M such that N (C 1 ) = S 1 and \|C 1 \| = \|S 1 \|.Now since every client in C 1 is matched, every server S 1 is matched to some client in C 1 .Every augmenting tail from some s ∈ S 1 must start with a matched edge, so it must go through C 1 , so it never reaches a server outside of N (C 1 ) = S 1 , so it can never reach a free server. We now turn to our implementation of the SAP protocol. Theorem 2. There is an implementation of the SAP protocol that runs in total time Proof.Every vertex will keep track of its height up to h = √ n √ log n.If its height is larger than h, it will simply mark itself as a high vertex.Each vertex v will also track the height of each of its neighbors with O(h) buckets: N 1 (v), N 2 (v), N 3 (v), . . ., N h (v), and N * (v) for the high neighbors.Whenever a neighbor u of v changes height, we will have to change the corresponding bucket of v. Every vertex v will also keep track of its lowest non-empty bucket. When a new client c arrives, it will use the height information to find a shortest augmenting path.First off, put all of the neighbors of c in their corresponding buckets: this takes O(deg(c)) time.Now to find the shortest augmenting path from c we consider two cases.The first is that c has a neighbor who is not high.In this case, take the edge from c to its neighbor with minimum height by picking an arbitrary neighbor from the lowest non-empty bucket.Now follow the backwards (matched) edge to a new vertex c 1 , and again, take the edge to the vertex of minimum height, breaking ties arbitrarily.This will obviously compute a shortest augmenting path. The second case is when all the neighbors of c are high.In this case we can just brute-force compute a shortest augmenting path in time O(m).If we find an augmenting path, we augment down it; if we do not find an augmenting path, then we remove all servers and clients encountered during the search for this augmenting path, and continue the algorithm in the graph with these vertices removed.correctness We want to show that our implementation chooses a shortest augmenting path at every step.Although our implementation clearly always chooses the shortest augmenting path in the remaining graph, there is the potential problem that this remaining graph might not be the original graph, since the algorithm sometimes deletes vertices from the graph due to a failed brute-force search.We show that this is not a problem because whenever our implementation deletes a vertex v at time t, then in the original graph there is never an augmenting path through v after time t.To see this, note that when our implementation deletes a server s ∈ S, there must have been no augmenting path through s at the time that s was deleted.By Lemma 30, this implies that α M (s) = 1.But then by Lemma 19 we have α M (s) = 1 for all future client insertions as well. (Recall that by Observation 25 we never have α M (s) > 1.) Thus by Lemma 30 there is never an augmenting path through s after this point, so s can safely be deleted from the graph.Similarly, if a client c is deleted from the graph, then all of its neighboring servers had no augmenting paths at that time, so they all have α M (s) = 1, so there will never be an augmenting path through c. Running time: There are three factors to consider 1. the time to maintain the height buckets 2. the time to brute-force compute augmenting paths when all the neighbors of an incoming client are high vertices. 3. the time to follow the augmenting paths given the height buckets. Item 3 takes O(n log 2 n) time because we need O(1) time to follow each edge in the path, and by Theorem 1 the total length of augmenting paths is O(n log 2 n).For Item 2 we consider two cases.The first is brute-force searches which result in finding an augmenting path.These take a total of O(mn log(n)/h) = O(m √ n √ log n) time because by Lemma 6 during the course of the entire algorithm there are at most O(n log(n)/h) augmenting paths of length ≥ h, and each such path requires O(m) time to find.The second case to consider is brute-force searches that do not result in an augmenting path.These take total time O(m) because once a vertex participates in such a search, it is deleted from the graph. Finally, For Item 1, we observe that if augmenting along a path causes a vertex v to change height, then either v belongs to that augmenting path, or v has a neighbor whose height changes.Thus, to maintain the height buckets it is enough for each vertex v to inform all neighbors w whenever the height of v changes.In particular each neighbor w changes the location of v in bucket structure in O(1) time and then checks in O(1) time whether its height changed by looking in its lowest non-empty bucket; the lowest non-empty bucket of w is easy to keep track of because heights never decrease (Observation 29), so vertices only move up the bucket structure of w.If the height of w did not change then w does no further work.Otherwise repeat from w, i.e. update the buckets of all of the neighbors of w.All in all we do O(deg(v)) work whenever the height of a vertex v changes.Since heights never decrease, this can happen at most h times per vertex v, leading to a total time of Extensions In many applications of online bipartite assignments, it is natural to consider the extension in which each server can serve multiple clients.Recall from the introduction that we examine two variants: capacitated assignment, where each server comes with a fixed capacity which we are not allowed to exceed, and minimizing maximum server load, in which there is no upper limit to the server capacity, but we wish to minimize the maximum number of clients served by any server.We show that there is a substantial difference between the number of reassignments: Capacitated assignment is equivalent to uncapacitated online matching with replacements, but for minimizing maximum load, we show a significantly higher lower bound. Capacitated Assignment We first consider the version of the problem where each server can be matched to multiple clients.Each server comes with a positive integer capacity u(s), which denotes how many clients can be matched to that server.The greedy algorithm is the same as before: when a new client is inserted, find the shortest augmenting path to a server s that currently has less than u(s) clients assigned. Theorem 3. SAP uses at most O(n log 2 n) reassignments for the capacitated assignment problem, where n is the number of clients. Proof.There is a trivial reduction from any instance of capacitated assignment to one of uncapacitated matching where each server can only be matched to one client: simple create u(s) copies of each server s.This reduction was previously used in [BKP + 17].When a client c is inserted, if there is an edge (c, s) in the original graph, then add edges from c to every copy of s.It is easy to see that the number of flips made by the greedy algorithm in the capacitated graph is exactly equal to the number made in the uncapacitated graph, which by Theorem 1 is O(n log 2 n).(Note that although the constructed uncapacitated graph has more servers than the original capacitated graph, the number of clients n is exactly the same in both graphs.) Minimizing Maximum Server Load In this section, we analyze the online assignment problem.Here, servers may have an unlimited load, but we wish to minimize maximum server load. Definition 31.Given a bipartite graph G = (C ∪ S, E), an assignment A : C → S assigns each client c to a server A(c) ∈ S. Given some assignment A, for any s ∈ S let the load of s, denoted A (s), be the number of clients assigned to s; when the assignment A is clear from context we just write (s).Let (A) = max s∈S A (s).Let opt(G) be the minimum load among all possible assignments from C to S. In the online assignment problem, clients are again inserted one by one with all their incident edges, and the goal is to maintain an assignment with minimum possible load.More formally, define G t = (C t ∪ S, E t ) to be the graph after exactly t clients have arrived, and let A t be the assignment at time t.Then we must have that for all t, (A t ) = opt(G t ).The goal is to make as few changes to the assignment as possible. [GKS14] and [BKP + 17] showed how to solve this problem with approximation: namely, with only O(1) amortized changes per client insertion they can maintain an assignment A such that for all t, (A t ) ≤ 8opt(G t ).Maintaining an approximate assignment is thus not much harder than maintaining an approximate maximum matching, so one might have hoped that the same analogy holds for the exact case, and that it is possible to maintain an optimal assignment with amortized O(log 2 n) changes per client insertion.We now present a lower bound disproving the existence of such an upper bound.The lower bound is not specific to the greedy algorithm, and applies to any algorithm for maintaining an assignment A of minimal load.In fact, the lower bound applies even if the algorithm knows the entire graph G in advance; by contrast, if the goal is only to maintain a maximum matching, then knowing G in advance trivially leads to an online matching algorithm that never has to rematch any vertex. Theorem 4. For any positive integers n and L ≤ n/2 divisible by 4 there exists a graph G = (C ∪ S, E) with \|C\| = n and opt(G) = L, along with an ordering in which the clients in C are inserted, such that any algorithm for the exact online assignment problem requires a total of Ω(nL) changes.This lower bound holds even if the algorithm knows the entire graph G in advance, as well as the order in which the clients are inserted. The main ingredient of the proof is the following lemma: Lemma 32.For any positive integer L divisible by 4, there exists a graph G = (C ∪ S, E) along with an ordering in which clients in C are inserted, such that \|C\| = L 2 , \|S\| = L, opt(G) = L, and any algorithm for maintaining an optimal assignment A requires Ω(L 3 ) changes to A. Proof.Let S = {s 1 , s 2 , ..., s L }.We partition the clients in C into L blocks C 1 , C 2 , ..., C L , where all the clients in a block have the same neighborhood.In particular, clients in C L only have a single edge to server s L , and clients in C i for i < L have an edge to s i and s i+1 . The online sequence of client insertions begins by adding L/2 clients to each block C i .The online sequence then proceeds to alternate between down-heavy epochs and up-heavy epochs, where a down-heavy epoch inserts 2 clients into blocks C 1 , C 2 , ..., C L/2 (in any order), while an up-heavy epoch inserts 2 clients into blocks C L/2+1 , ..., C L .The sequence then terminates after L/2 such epochs: L/4 up-heavy ones and L/4 down-heavy ones in alternation.Note that a down-heavy epoch followed by an up-heavy one simply adds two clients to each block.Thus the final graph has \|C i \| = L for each i, so the graph G = (C ∪ S, E) satisfies the desired conditions that \|C\| = L 2 and opt(G) = L.We complete the proof by showing that all the client insertions during a single down-heavy epoch cause the algorithm to make at least Ω(L 2 ) changes to the assignment; the same analysis applies to the up-heavy epochs as well.Consider the kth down-heavy epoch of client insertions.Let β = L/2 + 2(k − 1) and consider the graph G old = (C old ∪ S, E old ) before the down-heavy epoch: it is easy to see that every block C i has exactly β clients, that opt(G old ) = β, and that there is exactly one assignment A old that adheres to this maximum load: A old assigns all clients in block C i to server s i (see Figure 1).Now, consider the graph G new = (C new ∪ S, E new ) after the down-heavy epoch.Blocks C 1 , C 2 , ..., C L/2 now have β + 2 clients, while blocks C L/2+1 , ..., C L still only have β.We now show that opt(G new ) = β + 1.In particular, recall that β ≥ L/2 and consider the following assignment A new : for i ≤ L/2, A new assigns β + 2 − i ≥ 2 clients from C i to s i and i clients in C i to s i+1 ; for L/2 < i ≤ L, A new assigns β + i − L ≥ 0 clients in C i to s i , and L − i clients from C i to s i+1 .(In particular, all β clients in C L are assigned to s L , which is necessary as there is no server s L+1 ).It is easy to check that for every s ∈ S, A new (s) = β + 1 (see Figure 2).We now argue that A new is in fact the only assignment in G new with (A new ) = β + 1.Consider any assignment A for C new with (A) = β + 1. Observe that since the total number of clients in C new is exactly (β + 1)L, we must have that every server s ∈ S has (s) = β + 1 in A. We now argue by induction that for i ≤ β/2, A assigns assigns β + 2 − i clients from C i to s i and i clients in C i to s i+1 (exactly as A new does).The claim holds for i = 1 because the only way s 1 can end up with load β + 1 is if β + 1 clients from C 1 are assigned to it.Now say the claim is true for some i < β/2.By the induction hypothesis, s i+1 has i clients from C i assigned to it.Since s i+1 must have total load β + 1, and all clients assigned to it come from C i or C i+1 , s i+1 must have β + 1 − i = β + 2 − (i + 1) clients assigned to it from C i+1 . We now prove by induction that for all L/2 < i ≤ L, A assigns β + i − L clients in C i to s i , and L − i clients from C i to s i+1 , which proves that A = A new .The claim holds for i = L/2 + 1 because we have already shown that in the above paragraph that L/2 clients assigned to s i = s L/2+1 come from C L/2 , so since (s i ) = β + 1, it must have β + 1 − L/2 = β + i − L clients from C i assigned to it.Now, say that the claim is true for some i > L/2.Then by the induction step s i+1 has L − i clients assigned to it from C i , so since (s i+1 ) = β + 1, it has β + (i + 1) − L clients assigned to it from C i+1 , as desired.The remaining L − (i + 1) clients in C i+1 must then be assigned to s i+2 . We have thus shown that the online assignment algorithm is forced to have assignment A old before the down-heavy epoch, and assignment A new afterwards.We now consider how many changes the algorithm must make to go from one to another.Consider block C i for some L/2 < i ≤ L. Note that because the epoch of client insertions was down-heavy, \|C i \| = β before and after the epoch.Now, in A old all of the clients in C i are matched to s i .But in A new , L − i of them are matched to s i+i .Thus, the total number of reassignments to get from A old to A new is at least L/2<i≤L (L − i) = Ω(L 2 ).Since there are L/4 down-heavy epochs, the total number of reassignments over the entire sequence of client insertions is Ω(L 3 ). Proof of Theorem 4. Recall the assumption of the Theorem that n/2 ≥ L 2 .Simply let the graph G consist of n/L 2 separate instances of the graph in Lemma 32, together with sufficient copies of K 1,1 to make the total number of clients n.The algorithm will have to make Ω(L 3 ) changes in each such instance, leading to Ω(L 3 n/L 2 ) = Ω(nL) changes in total. We now show a nearly matching upper bound which is off by a log 2 n factor.As with the case of matching, this upper bound is achieved by the most natural SAP algorithm, which we now define in this setting.Since opt(G) may change as clients are inserted into C, whenever a new client is inserted, the greedy algorithm must first compute opt(G) for the next client set.Note that the algorithm does not do any reassignments at this stage, it simply figures out what the max load should be.opt(G) can easily be computed in polynomial time: for example we could just compute the maximum matching when every server has capacity b for every b = 1, 2, ..., \|C\|, and then opt(G) is the minimum b for which every client in C is matched; for a more efficient approach see [BKP + 17].Now, when a new client c is inserted, the algorithm first checks if opt(G) increases.If yes, the maximum allowable load on each server increases by 1 so c can just be matched to an arbitrary neighbor.Otherwise, SAP finds the shortest alternating path from c to a server s with (s) < opt(G): an augmenting path is defined exactly the same way as in Definition 8, though there may now be multiple matching edges incident to every server.The proof of the upper bound will rely on the following very simple observation: Observation 33.For the uncapacitated problem of online maximum matching with replacements, let us say that instead of starting with C = ∅, the algorithm starts with some initial set of clients C 0 ⊂ C already inserted, and an initial matching between C 0 and S. Then the total number of replacement made during all future client insertions is still upper bounded by the same O(n log 2 n) as in Theorem 1, where n is the number of clients in the final graph (so n is \|C 0 \| plus the number of clients inserted). Proof.Intuitively, we could simply let our protocol start by unmatching all the clients in C 0 , and then rematching them according the SAP protocol, which would lead to O(n log 2 n) replacements.In fact this initial unmatching is not actually necessary.Recall that the proof of Theorem 1 follows directly from the key Lemma 6, which in term follows from the expansion argument in Lemma 27.The expansion argument only refers to server necessities, not to the particular matching maintained by the algorithm, so it will hold no matter what initial matching we start with. Theorem 5. Let C be the set of all clients inserted, let n = \|C\|, and let L = opt(G) be the minimum possible maximum load in the final graph G = (C ∪ S, E).SAP at all times maintains an optimal assignment while making a total of O(n min {L log 2 n, √ n log n}) reassignments. Proof.Let us define epoch i to contain all clients c such that after the insertion of c we have opt(G) = i.We now define n i as the total number of clients added by the end of epoch i (so n i counts clients from previous epochs as well).Extend the reduction in the proof of Theorem 3 from [BKP + 17] as follows: between any two epochs, add a new copy of each server, along with all of its edges.For the following epoch, say, the ith epoch, Observation 33 tells us that regardless of what matching we had at the beginning of the epoch, the total number of reassignments performed by SAP during the epoch will not exceed O(n i log 2 n i ) ⊆ O(n log 2 n).We thus make at most O(nL log 2 n) reassignments in total, which completes the proof if L < √ n/ log n.If L ≥ √ n/ log n, we make O(n √ n log n) reassignments during the first √ n/ log n epochs.In all future epochs, note that a server at its maximum allowable load has at least √ n/ log n clients assigned to it, so there are at most √ n log n such servers, and whenever a client is inserted the shortest augmenting path to a server below maximum load will have length O( √ n log n).This completes the proof because there are only n augmenting paths in total. Conclusion We showed that in the online matching problem with replacements, where vertices on one side of the bipartition are fixed (the servers), while those the other side arrive one at a time with all their incoming edges (the n clients), the shortest augmenting path protocol maintains a maximum matching while only making amortized O(log 2 n) changes to the matching per client insertion.This almost matches the Ω(log n) lower bound of Grove et al. [GKKV95].Ours is the first paper to achieve polylogarithmic changes per client; the previous best of Bosek et al. required O( √ n) changes, and used a non-SAP strategy [BLSZ14].The SAP protocol is especially interesting to analyze because it is the most natural greedy approach to maintaining the matching.However, despite the conjecture of Chaudhuri et al. [CDKL09] that the SAP protocol only requires O(log n) amortized changes per client, our analysis is the first to go beyond the trivial O(n) bound for general bipartite graphs; previous results were only able to analyze SAP in restricted settings.Using our new analysis technique, we were also able to show an implementation of the SAP protocol that requires total update time O(m √ n √ log n), which almost matches the classic offline O(m √ n) running time of Hopcroft and Karp [HK73]. The main open problem that remains is to close the gap between our O(log 2 n) upper bound and the Ω(log n) lower bound.This would be interesting for any replacement strategy, but it would also be interesting to know what the right bound is for the SAP protocol in particular.Another open question is to remove the √ log n factor in our implementation of the SAP protocol.Note that both of these open questions would be resolved if we managed to improve the bound in Lemma 6 from O(n ln(n)/h) to O(n/h).(In the implementation of Section 5 we would then set h = √ n instead of h = √ n √ log n.) Corollary 17 . If max ∅⊂K⊆C \|K\| \|N (K)\| = \|C\| \|S\| there is a unique balanced server flow.Proof.By Lemma 12 there exists a server flow with α(s) ≤ \|C\| \|S\| for all s ∈ S. Since s∈S α(s) = \|C\|, any such flow must actually have α(s) = \|C\| \|S\| for all s ∈ S, and be balanced.Uniqueness follows from Lemma 16.Lemma 18.A unique balanced server flow exists if and only if \|N (c)\| ≥ 1 for all c ∈ C. Proof.As already noted, \|N (c)\| ≥ 1 for all c ∈ C is a necessary condition.We will prove that it is sufficient by induction on i = \|S\|.If \|S\| = 1, the flow α(s) = \|C\| for s ∈ S is trivially the unique balanced server flow.Suppose now that i > 1 and that it holds for all \|S\| < i.Now let α = max ∅⊂K⊆C \|K\| \|N (K)\| and let and by Corollary 17 we are done, so suppose ∅ ⊂ N ( C) ⊂ S. Consider the subgraph G 1 induced by C ∪ N ( C) and the subgraph G 2 induced by (C \ C) ∪ (S \ N ( C)). " ⊆ " By Lemma 15, \| K\| = α \|N ( K)\| so by definition of C, K ⊆ C. "⊇" On the other hand, \| C\| = α \|N ( C)\| so by Lemma 16 we have N ( C) ⊆ S and in particular A(c) ⊆ S for c in C and thus C ⊆ K. Thus, by definition of K, A(c) ∩ S = ∅ for all c ∈ C \ C.And there are clearly no edges between C and S \ N ( C).But then, for any (x e ) e∈E realizing α , the subset (x cs ) c∈C\ C,s∈S\N ( C) realizes a balanced server flow in G 2 , so since α 2 is the unique balanced server flow in G 2 we have α (s) = α 2 (s) for s ∈ S \ N ( C). Definition 22 . We define the set C M ⊆ C as follows.When a client c is inserted, consider the set of clients C before c is inserted: then c ∈ C M if the maximum matching in (C ∪ {c} ∪ S, E) is greater than the maximum matching in (C ∪ S, E).Define G M = (C M ∪ S, E).Observation 23.When a client c ∈ C M is inserted the SAP algorithm finds an augmenting path from c to a free server; this follows from the fact that SAP always maintains a maximum matching (Lemma 10).By Observation 9, if c / ∈ C M then no augmenting path goes through c during the entire sequence of insertions.By the same observation, once a vertex c ∈ C M is inserted it remains matched through the entire sequence of insertions.Definition 24.Given any s ∈ S, let α M (s) be the flow into s in some balanced server flow in G M ; by Lemma 18 α M (s) is uniquely defined.Observation 25.By construction G M contains a matching of size \|C M \|, so by Lemma 21 α M (s) ≤ 1 for all s ∈ S. Finally, note that since C M ⊆ C, we clearly have α M (s) ≤ α(s) Figure 1 : Figure 1: Number of assignments of each type after first L/2 clients added to each block, and after each up-heavy phase.Each C i has β clients.Each server has β clients assigned. Figure 2 : Figure 2: Number of assignments of each type after each down-heavy phase.Each C i has β + 2 clients for 1 ≤ i ≤ L/2 and β clients for L/2 + 1 ≤ i ≤ L. Each server has β + 1 clients assigned.	2017-07-19T13:03:10.000Z	2017-07-19T00:00:00.000	{ "year": 2017, "sha1": "e198830d6141647e2d289578e3338a62769db0b4", "oa_license": null, "oa_url": "https://backend.orbit.dtu.dk/ws/files/210757891/hkkr_paper.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "e198830d6141647e2d289578e3338a62769db0b4", "s2fieldsofstudy": [ "Computer Science", "Mathematics" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
119125579	pes2o/s2orc	v3-fos-license	Fun from none: deformed symmetries and Fock space We give a pedagogical introduction to the basics of deformations of relativistic symmetries and the Hilbert spaces of free quantum fields built as their representations. We focus in particular on the example of a $\kappa$-deformed scalar quantum field for which the generators of spatial translations that label the field modes act according to a deformed Leibnitz rule. We explore the richer structure of the $\kappa$-Fock space and point out possible physical consequences of the deformation. I. INTRODUCTION In ordinary applications of quantum field theory (QFT) to high energy physics non-interacting fields are rather trivial entities which describe particles for which no scattering can take place. As soon as we leave our laboratory and let the quantum fields propagate on a non-trivial background or, alternatively, accelerate our experimental apparatus the free field comes alive and surprising phenomena of particle production take place. Ultimately these phenomena can be explained in terms of the existence of inequivalent field quantizations: each observer will define a notion of energy according to her own time evolution and this in turn determines the vacuum state of her own Fock space [1]. A less known context in which an ordinary free field exhibits non-trivial features upon quantization is provided by the multi-particle sector of a deformed Fock space where a n-particle state is no longer a (anti)symmetrized n-tensor product of one-particle states (for (fermions) bosons). Such deformed tensor products still provide a representation of the symmetric group but now the statistics becomes momentum dependent [2] in a way which will become clear in the following. In the present contribution we attempt to give a pedagogical introduction to such models and show how deformed Fock spaces naturally emerge when the phase space of the field carries a representation of certain quantum deformations of the (Hopf) algebra of relativistic symmetry generators. These deformed algebras have been proposed as emerging from the "no-gravity" limit of quantum gravity i.e. as to effectively represent the isometries of a "quantum version" of Minkowski space and are usually introduced to preserve covariance properties of certain non-commutative space-times (see e.g. [3,4,5,6,7,8,9]). In the next Section we recall the basics of free field quantization emphasizing the main ingredients and mathematical structures underlying the construction of bosonic Fock spaces. We then briefly comment on the simplest example of quantum deformation of relativistic symmetry generators, the "twisted" Poincaré algebra and the states of the related quantum scalar field. We then move our focus to κ-deformations of the Poincaré algebra and the quantization of a free scalar field. We conclude with a discussion on the possible "planckian" decoherence effects that the richer structure of the κ-Fock space can induce. II. FIELD QUANTIZATION The states of a system described by a classical scalar field are represented by points in its phase space. Usually the latter is given by the space Γ Σ of smooth initial data {ϕ, π} of compact support on a given Cauchy surface Σ. Observables are simply functions on Γ Σ the most important of them being the Hamiltonian which encodes the dynamics of the system. If we want to describe the states of a composite system given, for example, by two subsystems A and B we simply take the direct sum of the individual phase spaces Γ A∪B For a quantum field things are radically different. In this case the quantum states are rays in a complex Hilbert space H while observables are self-adjoint operators on H. At the level of composite systems the Hilbert space is now given by the tensor product of the subsystems Hilbert spaces H A∪B ≡ H A ⊗ H B . Field quantization is simply a recipe for obtaining the quantum description of the field system from its classical counterpart. It turns out that such a transition is better understood in terms of the so-called covariant phase space formalism [1,10]. In this framework the phase space of our scalar field is taken to be the space of solutions of the Klein-Gordon equation S KG . Indeed in Minkowski space (and, in general, for any globally hyperbolic space) each pair of initial data on a given space-like hyper-surface is uniquely associated to a solution of the equation of motion and this establishes an isomorphism between Γ Σ and S KG as vector spaces. The first step for obtaining a complex Hilbert space from S KG is, as expected, to complexify the latter i.e. consider S C KG ≃ S KG ⊗ C where C is the field of complex numbers. In order to turn such complex vector space into a Hilbert space we need to introduce an inner product. The vector space S KG has a natural symplectic structure i.e. a (conserved) non-degenerate anti-symmetric bilinear form given by the following integral over a Cauchy hypersurface Σ t at fixed time t which is nothing but the Wronskian for solutions of the Klein-Gordon equation. The hermitian inner product on S C KG will be given by where the bar indicates complex conjugation. To complete the Hilbert space construction we need to restrict to a subspace of S C KG on which the inner product above is positive definite, this will turn out to be the "positive energy" subspace S C+ KG . The "one-particle" Hilbert space of the theory H will be given by the Cauchy completion of S C+ KG with respect to the inner product (2). The last step will be to construct, using H as the building block, the full "multi-particle" sector of the theory. As we mentioned above composite quantum systems are represented by tensor products of the Hilbert spaces of their components. When we deal we systems of identical particles their quantum indistinguishability requires that we represent their states with combinations of tensor products of their Hilbert spaces which are symmetric under exchange of their labels. In other words such states must carry a representation of the symmetric group. For our quantum scalar field "n-particle states" will be given by "symmetrized" n-tensor products of the one-particle space H i.e. their Hilbert space can be written as where σ a permutation in the permutation group of n-elements P n and H ⊗n is the n-fold tensor product of n-copies of H. Given a "one-particle" observable O, i.e. a self adjoint operator on H, its action on multiparticle states is given by the second quantized operator [11] dΓ Such expression is simply telling us that the operator O acts on multiparticle states as a derivative i.e. following the Leibnitz rule. However this quite obvious expression has an important mathematical significance. Indeed the expression above can be re-written in terms of the coproduct where The coproduct introduced above is telling us that upon "second quantization" the (associative) algebra of observables acquires an additional structure which eventually turns it into a Hopf algebra. In an effort to keep our presentation more straightforward we will not comment further on the role of Hopf algebra at this point (the interested reader can refer to [12] for an introduction to the subject). The main point that should be kept in mind is that the coproduct encodes information on how observables act on a multi-component quantum system and, in our specific case, on the additivity properties of observables for the multiparticle sector of a quantum field Hilbert space. III. BEYOND LEIBNITZ What has been said so far seems to have little to do with deformations of space-time symmetries. This is not the case however. Symmetry generators are just a special kind of observables. Indeed, as we showed in the previous Section, the one-particle Hilbert space H is constructed from the space of solutions of the Klein-Gordon equation S and this in turn implies that H carries a unitary irreducible representation of the Poincaré algebra P. In particular we have a natural action of the generators of P as one-particle operators. Moreover one-particle states can be completely characterized by a commuting set of such operators, for example in the standard plane-wave representation the eigenvalues of spatial translation generators P will label the orthonormal set of kets \|p . The coproduct ∆P we introduced above extends the action of P to multiparticle states. In usual QFT the information carried by such object is simply a restatement of the Leibnitz rule for the action of symmetry generators (and general observables) on tensor product states. Symmetry deformation provides a generalization of the additivity of symmetry generators encoded in the Leibnitz rule 1 . A whole class of deformed symmetries can be obtained by introducing a "modulation" of the coproduct weighted by a certain deformation scale q in such a way that given a symmetry generator G: i.e. realize a non-symmetric Leibnitz rule for G acting on multiparticle states. From our point of view space-time symmetry deformation is a purely quantum (relativistic) phenomenon: we are not deforming relativistic kinematics tout court, we are simply changing the way classical relativistic symmetries are implemented in the multiparticle sector of (free) QFT. Before discussing in detail some explicit examples of symmetry deformation we briefly review the basics of Fock space formalism. We start with a plane wave basis {e k } for the one-particle Hilbert space H. In terms of such vectors a generic one-particle state ξ ∈ H can be written as Accordingly a n-particle state will read where \|k 1 ...k n indicates a symmetrized tensor product of basis elements \|k 1 ...k n = 1 √ n! σ∈Pn e k1 ⊗ ... ⊗ e kn . We write a generic Fock space element as Ψ = {ξ 0 , ξ 1 (k 1 ), ...ξ n (k 1 , ..., k n ), ...}. We can now introduce annihilation and creation operators whose action is defined respectively by a k and a † k : notice the symmetrization appearing in the last definition. From these two definitions it is easily seen that the operators a k and a † k obey the canonical commutation relations Having set the stage we proceed in the next subsection with a quick overview of a rather popular type of deformation, the twisted Poincaré algebra. A. Twisted Fock space The twisted Poincaré algebra provides a simple example which illustrates the basic features of deformed symmetries and Fock space. It was first introduced in [5,8] as the relevant algebraic structure to describe the relativistic symmetries of of a quantum field living on a specific type of non-commutative space-time, the Moyal plane For this particular type of deformation the modulation map is given by what is technically called a "Drinfeld twist" (for details see [8]) Such map obviously does not change the coproduct of translation generators since it commutes with them but it does change the coproduct of the generators of Lorentz rotations ∆ θ M µν = ∆M µν , again for details we refer the reader to the review [13]. The important point we would like to stress is that even such mild deformation has an effect on the definition of multiparticle states and the construction of the Fock space of the theory. Indeed now given two plane wave states e k1 , e k2 ∈ H θ the ordinary "flip map" τ which exchanges the left and right component of elements of H ⊗ H is no longer an intertwiner of representations of the twisted algbera [13]. Instead we must replace τ by a twisted flip Now in order to construct a generic bosonic n-particle state we need a rule to symmetrize tensor product states. For example for a two particle state we would normally take the superposition however in the twisted symmetry case since τ is no longer an intertwiner we must use the twisted flip and the new two-particle state will be given by This construction easily extends to n-particle states. The non-trivial structure of the twisted Fock space amounts to the appearance of phases depending on the modes of the states involved and on the deformation parameter θ. Introducing creation and annihilation operators, c k c † k one can see that such "momentum dependent" statistics is indeed encoded in the non-canonical commutation relations After this flash review of twisted Fock space we turn in the next subsection to a "stronger" form of deformation, the κ-deformation, and explore its possible physical consequences. B. κ-Fock space and hidden entanglement The first example of deformation of the algebra of relativistic symmetries, the κ-Poincaré algebra, was proposed more than fifteen years ago [14]. Our main interest in such algebra is due to the fact that it shares an important feature with the deformed symmetry algebras that have been shown to emerge in the context of three-dimensional quantum gravity [4]: the coproduct for translation generators (unlike the θ case) is now deformed. Indeed, in the specific variant of this deformation which is of interest to us, the coproduct is given by where the dimensionful deformation parameter κ is set at a given UV scale, usually the Planck energy E p . Other salient features of the κ-Poincaré algebra are the deformed positive and negative mass-shells (for simplicity we write it for the massless case) and a deformed action of boosts on linear momenta Note how at low energies (κ → ∞) the deformation switches off and we recover the usual Poincaré algebra and its representations (for more details on the κ-Poincaré algebra and related field theory see e.g. [15] and references therein). As noted in [2] (see also [16,17]) the deformed coproduct ∆ κ P can again be obtained from the undeformed one using a "modulation map" However in this case (unlike for the twisted coproduct discussed previously) such map is not a Drinfeld twist. This seems to raise certain mathematical inconsistencies when dealing with bosonic n-particle states for n ≥ 3 [18]. We will ignore such issues at this level and consider the features of κ-deformations listed above as the building blocks for a basic κ-Fock space construction which will allow us to explore the new physics of κ-deformed free fields. We start with the construction of the one-particle Hilbert space. The most straightforward way to characterize such space in this context is to consider functions on the deformed mass-shell 2 M κ given by (23) equipped with the inner product (see [19]) where ω is the ordinary symplectic product on the undeformed mass shell. The next step is to find a suitable subspace on which the inner product above is positive definite. While in the undeformed case it is enough to restrict to the positive mass shell in the present case one notices that for "transplanckian" (\| p\| > κ) modes (26) is no longer positive definite. In other words the κ-one-particle Hilbert space will be given by the positive energy mass-shell truncated at κ, M κ+ \|k\|≤κ ⊂ M κ equipped with the deformed inner product (26). Turning to the multiparticle sector of the theory we notice that, as in the twisted case, ordinary symmetrization can not be used to construct κ-multiparticle states. Indeed it is easily seen that, for example, if we consider the standard two-particle state this is not an eigenstate of the spatial translation generators P due to the non-trivial coproduct (22). Again in this case we have to resort to a non-trivial "momentum dependent symmetrization". One possibility is to proceed in analogy with the twisted case and make use of the "modulation map" (25). We will then construct a symmetrized state using the "modulated flip" operator τ κ = F κ τ F −1 κ whose action on a tensor product state is given by where ǫ i = \|ki\| κ . As an illustrative example consider the construction of a two-particle state. Given a set of two one-particle states \|k 1 and \|k 2 we will now have two distinct two particle states with same energy and different linear momentum given respectively by In general given n-different modes one has n! different n-particle states, one for each permutation of the n modes k 1 , k 2 ... k n . We see that the non-trivial algebraic structure of κ-translations endows the Fock space with a "fine structure". Indeed the different states (which in the absence of deformation would be degenerate) can in principle be distinguished by measuring their momentum splitting. For example for the two particle states above the momentum splitting is given by which is of order \|k i \| 2 /κ. In other words the 2-particle Hilbert (sub)space becomes H 2 κ ∼ = S 2 H 2 ⊗ C 2 , where S 2 H 2 is the ordinary symmetrized 2-particle Hilbert space. We can thus write our states as with ǫ = ǫ(k 1 ) + ǫ(k 2 ). Due to this additional structure we can now have states in which the macroscopic degrees of freedom of S 2 H 2 can be entagled with the "planckian" degrees of freedom C 2 as e.g. in the following state, superposition of two-particle states with total energies ǫ A = ǫ(k 1A ) + ǫ(k 2A ) and ǫ B = ǫ(k 1B ) + ǫ(k 2B ) The remarkable fact is that the possibility of this micro-macro entanglement renders possible interesting phenomena of decoherence [20]. To illustrate this consider the unitary evolution of a quantum system ρ(t) = U (t)ρ(0)U † (t) whose states are represented by our κ-Fock space. We start with a pure state ρ(0) factorized with respect to the bipartition in H n κ ∼ = S n H n ⊗ C n . If U (t) acts as an "entangling gate", the state ρ(t) will be entangled. A macroscopic observer who is not able to resolve the planckian degrees of freedom at the beginning will see the reduced system in a pure state ρ obs (0) = Tr P l ρ(0). As the system evolves she will see the system in the mixed state ρ obs (t) = Tr P l ρ(t) = Tr P l U (t)ρ(0)U † (t) . (37) For the macroscopic observer, the evolution is not unitary! This shows how κ-Fock space provides a simple example of a quantum system which exhibits decoherence due to the presence of hidden "planckian" degrees of freedom. A similar scenario has been advocated for certain models which could provide experimental signatures of quantum gravity (see Prof. Mavromatos contribution to these Proceedings). IV. CONCLUSION We have provided a brief overview of the interplay between deformations of relativistic symmetries and field quantization. In particular we discussed how the introduction of a deformation leads to non-trivial Fock space construction and a "momentum dependent" statistics. In the specific case of κ-deformations, where the Leibnitz rule for spatial translation generators is deformed, we showed that the new structure of κ-Fock space allows for decoherence due to entanglement between "macroscopic" and "planckian" degrees of freedom. It is important at this point to investigate whether this decoherence phenomena might lead to effects which could be checked in the real world. Work is in progress in order to answer such question.	2009-09-26T16:51:38.000Z	2009-09-26T00:00:00.000	{ "year": 2009, "sha1": "da7c3e955461753ed1ab314fcefef0a2fb31e506", "oa_license": null, "oa_url": "http://arxiv.org/pdf/0909.4873", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "da7c3e955461753ed1ab314fcefef0a2fb31e506", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
3850727	pes2o/s2orc	v3-fos-license	Pancreatic Adenocarcinoma: Current Therapies and Challenges Gastrointestinal (GI) cancers include the group of cancers that affect the digestive system such as the esophagus, gallbladder, liver, pancreas, stomach, small intestine, bowel colon and rectum, and anus. GI cancer is the most common form of cancer. In particular, pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of pancreatic cancers and is one of the leading causes of cancer-related deaths worldwide [1]. With minimal improvements in treatment outcomes over the last forty years, lack of effective screening and early detection methods prevent clinicians from identifying PDAC in a premalignant stage [2], resulting in a five-year survival rate of only 5% following diagnosis [3,4]. Current treatment options include surgical resection, neoadjuvant and adjuvant chemotherapy and radiation. Unfortunately, heterogeneous cancer cell populations found in primary tumors and secondary micrometastases renders them resistant to cytotoxic therapies. To overcome these limitations, future therapies must target and disable the multiple enabling hallmarks that drive PDAC progression, immune-derived promoters of tumor development and growth, acquired drug resistance mechanisms, and pro-metastatic signals in the tumor microenvironment that potentiate cancer cell dissemination and homing to distant organs. Here, we provide a brief review of the current treatment options available for pancreatic cancer and ongoing challenges that need to be overcome to improve cancer therapy. Introduction Gastrointestinal (GI) cancers include the group of cancers that affect the digestive system such as the esophagus, gallbladder, liver, pancreas, stomach, small intestine, bowel colon and rectum, and anus. GI cancer is the most common form of cancer. In particular, pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of pancreatic cancers and is one of the leading causes of cancer-related deaths worldwide [1]. With minimal improvements in treatment outcomes over the last forty years, lack of effective screening and early detection methods prevent clinicians from identifying PDAC in a premalignant stage [2], resulting in a five-year survival rate of only 5% following diagnosis [3,4]. Current treatment options include surgical resection, neoadjuvant and adjuvant chemotherapy and radiation. Unfortunately, heterogeneous cancer cell populations found in primary tumors and secondary micrometastases renders them resistant to cytotoxic therapies. To overcome these limitations, future therapies must target and disable the multiple enabling hallmarks that drive PDAC progression, immune-derived promoters of tumor development and growth, acquired drug resistance mechanisms, and pro-metastatic signals in the tumor microenvironment that potentiate cancer cell dissemination and homing to distant organs. Here, we provide a brief review of the current treatment options available for pancreatic cancer and ongoing challenges that need to be overcome to improve cancer therapy. Current Therapies and Limitations Although surgery remains one of the only curative treatment options, less than 20% of patients are surgical candidates [5]. Several poor predictors for successful resection are known: large tumor size [6], high tumor grade [7], positive tumor margins after surgical removal [5], elevated levels of CA 19-9 [8] and lymph node involvement [9]. Surgical PDAC patients remain at high risk for relapse and surgery has been shown to prolong survival by an average of only 10 months [10]. For patients with the advanced form of the disease that do not meet the criteria for surgery, radiation and chemotherapy are usually recommended as treatment options. Radiation therapy aims to eliminate rapidly proliferating cells in a specific area of the body by delivering a radioactive agent or high-energy rays that selectively induce DNA damage in dividing cells. Neoadjuvant radiation and chemotherapy therapy may be used to shrink an operable tumor prior to surgery. Adjuvant (post-surgery) radiation can also be used to treat the residual disease but the efficacy of this option is suboptimal due to its limited tolerance in normal tissue. The current standard of chemotherapy in the treatment of PDAC is gemcitabine (20,20-difluoro-20-deoxycytidine; dFdC), a nucleoside pyrimdine analog inhibiting DNA synthesis [5,11]. However, in non-surgical patients, gemcitabine treatment has been shown to prolong patient survival by only 4 months and is often used in palliation. Other neoadjuvant and adjuvant chemotherapeutics used today include flofirinox (5-fluorouracil, leucovorin, irinotecan, oxaliplatin) or a combination of gemcitabine with paclitaxel (abraxane), demonstrating tumor shrinkage in 20-30% of patients and slowing metastatic disease progression for approximately six months [12]. Acquired Gene Mutations and Resistance to Therapy One of the greatest difficulties in preventing metastatic disease is reversing the acquired resistance to therapies. The prevalence of chemoresistance in PDAC has been linked to the acquisition of tumor-promoting genetic mutations, such as K-ras, p53, CDKN2a and SMAD4/DPC4 [13]. 90% of PDAC patients also have KRAS2 point mutations, resulting in constitutively expressed Ras [14]. Once activated, Ras initiates a signaling cascade that activates cell proliferation and survival pathways, increasing cancer cell invasion [15]. Tumor-suppressor gene p53 is inactivated in approximately 80% of pancreatic tumors [16], resulting in impaired DNA damage recognition and repair, impaired apoptosis and deregulated mitosis [17]. Other tumorsuppressor genes encoded in the cdkn2a locus, including p16Ink4a and p15ARF, are present in about 90% of human pancreatic cancers [18] and are implicated in drug-resistance mechanisms. The deregulated activities of specific membrane drug transporters also play a vital role in treatment efficacy. For example, human equilibrative nucleoside transporter-1 (hENT1) is a membrane facilitative transporter that is used by hydrophilic gemcitabine in order to enter cancer cells [19]. Without hENT1 activity, the rate of gemcitabine entry through the hydrophobic plasma membrane is negligible, contributing to gemcitabineresistance. As major drivers of drug resistance, these genetic and cellular factors present as targets for drug development and patient-specific predictors of treatment response. Clinical Implications of Pancreatic Intratumoral Heterogeneity Pancreatic tumor heterogeneity refers to the presence of multiple subpopulations postulated to be derived from a unique lineage of origin, within a single neoplasm [20]. The occurrence of a tumor-initiating population with the capacity to self-renew and give rise to differentiated progeny has become an area of intense investigation. In a comprehensive study assessing 24 different pancreatic cancers, results revealed an average of 63 genetic mutations per cancer, spanning 12 signal transduction pathways [21]. Intratumoral heterogeneity at the molecular level within pancreatic tumors has become of increasing interest because identifying unique biomarkers can be used as prognostic tools. K-ras mutations, for example, have been heavily investigated for their prognostic value because they represent an early stage in driver gene alterations that are required for disease progression [22]. However, the presence of this mutation in over 90% of pancreatic cancers, and studies demonstrating its detection in late-stage cancers, its value as a prognostic tool is diminished [23]. Despite its ineffective as a prognostic tool, when in addition to CDKN2A, TP53, BRCA2 and SMAD4/DPC4 mutations, they represent key genetic events that are required in order for PDAC to progress into an aggressive malignancy [22]. These key driver mutations are consistent across the majority of PDAC, erroneously providing a homogenous genetic profile; however, subpopulations within the tumor acquire their own unique genetic profiles as the architectural arrangement of subpopulations can also vary. For example, two different subpopulations can intermix, or they can be separated by a physical barrier (such as blood vessels) or by a difference in their microenvironment, both of which may generate differences in how these subpopulations respond to therapy [24]. Theoretically, because subpopulations within a tumor can be under the selective pressure of their microenvironment and barriers, each unique tumor cell population can have its own repertoire of potential therapeutic targets. Targeting Pancreatic Cancer Stem Cells Cancer stem cells (CSC) present another major obstacle in the treatment of PDAC. These dedifferentiated cells have been shown to maintain long-term tumorigenic potential, and are able to regrow new micrometastases [25], under the regulation of the surrounding tumor microenvironment (TME) [26]. Li et al. [25] identified a small population of pancreatic cancer stem cells with a characteristic CD44+/CD24+/ESA+ phenotype and constitute 0.2-0.8% of the pancreatic cancer stem cell population [25]. The mammalian target of rapamycin (mTOR) has been a potential target of interest due to its involvement in the proliferation of CSCs. Phosphorylation of the s6 ribosomal protein (s6rp) by p70s6 kinase, a downstream target of mTOR previously shown in the literature to be a reliable marker for the mTOR signaling pathway was found in only a small subset of pancreatic cancer cells [27,28]. Rapamycin inhibition of mTOR resulted in a significant reduction of s6rp in pancreatic CSCs. However, the combination of cyclopamine (sonic hedgehog inhibitor), rapamycin, and gemcitabine (CRG) resulted in the elimination of virtually the entire CD133+ pancreatic CSC population [28]. This suggests that the combination of various therapeutic targets of CSC such as genes located in developmental pathways, such as hedgehog, Wnt, Notch, CXCR4 and Met, in combination with chemotherapy are capable of moderating the CSC pool. In addition, targeting apoptotic pathways such as DR5 and nodal-activin may also provide significant therapeutic benefit [25,29]. Nodal-activin are members of the TGF-k family, which are essential for embryonic stem cell (ESC) maintenance. Components of this signaling cascade are over expressed in pancreatic CSCs, with nodal highly expressed in pancreatic cancer tissue during the development and progression of PDAC, but not detectable in normal pancreatic tissue. Nodal inhibition was able to chemosensitize inherently chemoresistant pancreatic CSCs to therapy [30]. These findings support the notion that pancreatic intratumoral heterogeneity and CSCs are major enabling components that contribute to drug resistance and disease progression. A better understanding of CSC populations and how they interact with one another and the surrounding TME will enable further progress in the treatment of pancreatic cancer. Exosomes in Establishment of the Metastatic Niche PDAC is characterized by its late detection, which may be correlated with its high metastatic potential. Current screening methods lack the sensitivity to detect early onset with 80% of PDAC patients presenting with metastases at the time of diagnosis [31]. Metastatic disease is difficult to treat with conventional chemotherapeutic methods due to their unique genetic repertoire, size and location within a tissue relative to the primary tumor [32]. Therefore, it is important to elucidate the molecular events that mediate the development of metastatic disease. Exosomes are extracellular membrane-bound vesicles containing nucleic acids and proteins [31]. Exosomes have been implicated in the establishment of a premetastatic niche in liver metastases of patients with PDAC [33] and have been proposed as early detection tools as they circulate in the bloodstream, which make them an important area of study [31]. Exosome-mediated development of a metastatic TME is proposed to involve the release of PDAC exosomes containing macrophage migration inhibitor factor (MIF) to preferentially fuse with Kupffer Cells (KC) of the liver [33]. This leads to KC secreting TGF-β, which promotes the release of fibronectin by hepatic stellate cells, generating a pro-inflammatory microenvironment and recruiting macrophages [33]. Inhibition of MIF leads to the elimination of the liver premetastatic niche and prevents metastasis [31]. Recently glypican-1 (GPC1), a cancer exosome surface proteoglycan, has been investigated as an early detection biomarker as it is enriched on the surface of exosomes released from primary tumors. Its expression is also correlated with the presence of K-ras mutations found in pancreatic lesions that are undetectable by conventional means [31]. The efficacy of exosomes as mediators of metastasis has important implications on early detection strategies and possible therapeutic targets as they circulate the blood and are known mediators of intercellular communication. Paracrine signals from pancreatic tumor cells are able to induce a desmoplastic reaction, characterized by the extracellular proliferation of leukocytes, fibroblasts, endothelial cells, neuronal cells, and production of collagen type I and hyaluron [34]. Desmoplasia results in the formation of a thick stromal environment around pancreatic cancer cells [34] resulting in a mechanical barrier for drug delivery and is thought to contribute to the anti-angiogenic and hypoxic environment characteristic of PDAC, supporting tumor formation, progression and metastasis [35]. Studies have demonstrated that desmoplasia-promoting signals originate from the K-ras mutant oncogene in the tumor epithelium [36]. Sonic hedgehog (SHH) is also overexpressed in pancreatic cancer and results in the induction of extracellular fibroblasts, contributing to their growth and differentiation [36]. Furthermore, growth factors such as TGF-β, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) activate pancreatic stellate cells into myofibroblasts capable of secreting ECM components that further reduce the vascularization of the primary tumor [37]. Many proteins expressed by stromal cells have been correlated with poor prognosis and drug resistance, including COX-2, PDGF receptor, vascular endothelial growth factor (VEGF), stromal-derived factor (SDF), chemokines, integrins, secreted protein acidic and rich in cysteine (SPARC), and SHH elements [35,37]. Immune-Regulated Tumorigenesis Notably, the dense stroma is characterized by a tumorpromoting immunosuppressive environment. Using a CD40 antibody combined with gemcitabine therapy, researchers have attempted to reverse immune suppression and drive anti-tumor T-cell responses in patients with non-resectable pancreatic cancer. Studies have shown that this dual combination results in tumor regression by stimulating tumor-associated macrophages (TAM) to attack and degrade the stroma [38]. To date, PDAC treatment has proved most effective in patients with locally advanced disease, particularly in patients with wild-type DPC4 tumors, as they are known to be less prone to metastasis and possess higher stromal content. However, primary tumors that have already metastasized cannot be effectively treated with stromal-targeting agents because distant metastases arising from this cancer do not have a hypovascularized stroma [39]. Several cell types involved in the desmoplastic reaction, including TAMs, cancer associated fibroblasts, regulatory T-cells and myeloid derived suppressor cells contribute to tumorigenesis. K-ras-dependent signaling molecules have also been shown to up regulate granulocyte-macrophage colony stimulating factor (GM-CSF), thus promoting the maturation of myeloid progenitor cells into myeloid derived suppressor cells [40]. Unlike the stroma, the primary pancreatic tumor and/or distant micrometastases can be exposed to a highly inflammatory microenvironment. Cancer-associated inflammation can contribute to drug resistance, selection of CSCs and desmoplasia. Nuclear factor kappa B (NFkB) signaling, critical for the inducible expression of cellular and viral genes involved in inflammation, has been found to be constitutively activated in pancreatic cancer cells [41]. Inducible cyclooxygenase (COX)-2, a downstream target gene of NF-kB, is not normally expressed but can be up regulated by cytokines, growth factors and certain tumor-promoter genes. It is involved in prostaglandin synthesis, promotion of angiogenesis, immune evasion and inhibition of apoptosis. COX-2 has been found to be overexpressed in pancreatic adenocarcinomas and is localized to the cytoplasm of the tumor cells as opposed to the surrounding stromal or immune cells [41]. Conclusion Several clinical challenges remain in the treatment of pancreatic cancer. In addition to its advanced-stage detection, there have been no significant improvements in patient survival rates or in the development of effective therapies. These challenges arise from the understanding that the cancer cell program is adaptive, self-sustaining and invasive, and that despite therapy, aggressive phenotypes will survive and metastasize. Thus, future studies must not rely on targeting a single oncogenic or mitotic pathway, but must suppress the multiple stages of tumorigenesis of pancreatic cancer cells and their enabling microenvironment.	2019-04-02T13:12:09.438Z	2017-04-19T00:00:00.000	{ "year": 2017, "sha1": "ae92cf7031a838e510bc01260082574e571803e0", "oa_license": "CCBY", "oa_url": "https://juniperpublishers.com/argh/pdf/ARGH.MS.ID.555643.pdf", "oa_status": "HYBRID", "pdf_src": "MergedPDFExtraction", "pdf_hash": "8e786df02a8fcd846662453091d633182fdd9b68", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
42281947	pes2o/s2orc	v3-fos-license	Novel Class of Spider Toxin Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe. the genus Phoneutria (Ctenidae) (5), and the Australian funnelweb spiders from the genera Atrax and Hadronyche (Hexathelidae) (6). Today, however, spider venoms attract more research because they represent vast evolutionary-edited natural pharmacopoeias (7). These can be sources of indispensable tools for fundamental studies. For instance, -agatoxins from Agelenopsis aperta and hanatoxins from Grammostola rosea are used ubiquitously as specific ligands of voltage-gated calcium and potassium channels, respectively (8,9). At the very same time, spider toxins that specifically recognize different targets may serve as leads for drug and insecticide discovery and thus find application in medicine and green biotechnology (10 -13). Spider venoms are complex mixtures of compounds of diverse chemical nature varying from inorganic salts to high molecular weight proteins (7,14). However, preference is usually given to a certain chemical framework, and the major active constituents of spider venoms currently known can be allocated into four large structural groups. (a) Acylpolyamine toxins that block ionotropic receptors and ion channels with average low specificity are produced by several families of spiders, most notably the orb weavers (Araneidae), but also Nephilidae and Agelenidae (15,16). (b) Linear cytolytic peptides were found in venoms of spiders from the superfamily Lycosoidea (including Lycosidae, Ctenidae, and Oxyopidae) (17). More recently, Lachesana tarabaevi (Zodariidae) has been shown to be a unique "expert" in this type of toxin (18,19). Most of these linear peptides are disordered in solution but form ␣-helices upon contact with membranes. (c) Disulfide-containing neurotoxic peptides are the most typical molecules found in spider venoms (7,11,14,20). These peptides adopt compact folds with the inhibitor cystine knot (ICK) 2 motif being characteristic of the majority of spider toxins (21). (d) Large proteins are usually less abundant with the exception of the neurotoxic latrotoxins from the widow spiders (3) and the necrotizing phospholipases D (sphingomyelinases D) from the recluse spiders (4). Yellow sac spiders from the genus Cheiracanthium (Miturgidae), including the species Cheiracanthium punctorium common to Europe and Central Asia, have been erroneously suspected as inflicting skin lesions in humans similar to Loxosceles spp. In Europe in particular, C. punctorium is believed to be one of the few dangerous spider species. However, a systematic inspection of all available testimonies has proven these spiders virtually innocuous with bites causing local pain and sometimes swelling resembling bee or wasp stings with more severe effects occurring very rarely (22,23). The venom of Cheiracanthium spiders causes cytolytic effects. It lyses erythrocytes, but its venom does not contain sphingomyelinase D, as do Loxosceles spp., whereas phospholipase A 2 has been suspected to represent its active cytolytic component similar to other venomous animals, such as bees and snakes (24). In this study, we report the structure and activity of the major polypeptide toxin CpTx 1 from the venom of C. punctorium. This toxin is very unusual compared with all known animal toxins and includes two modules, each probably corresponding to a single domain ICK-type spider toxin. CpTx 1 was found to exhibit membrane-damaging and cytolytic effects, in sharp contrast to most other described disulfide-containing spider venom peptides that cause neurotoxicity. EXPERIMENTAL PROCEDURES Our detailed protocols on the general scheme for spider venom separation, toxin purifications, sequencing, and characterization can be found elsewhere (25). CpTx 1 Isolation-Crude venom of the spider C. punctorium was purchased from Fauna Laboratories, Ltd. (Republic of Kazakhstan) in the form of lyophilized powder. Venom (ϳ5 mg corresponding to 25 l of liquid venom) was redissolved in 100 l of 10% acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) and fractionated on a TSK 2000SW size-exclusion chromatography (SEC) column (7.5 ϫ 600 mm, 12.5-nm pore size, 10-m particle size; Toyo Soda Manufacturing Co., Tokyo, Japan) equipped with a guard column (7.5 ϫ 75 mm) in the same solvent at a flow rate of 1 ml/min. Fraction corresponding to the molecular mass range of 7-45 kDa was further separated by reverse-phase high performance liquid chromatography (RP-HPLC) on a Jupiter C 5 column (4.6 ϫ 150 mm, 30-nm pore size, 5-m particle size; Phenomenex) using a linear gradient of acetonitrile concentration (0 -20% in 10 min, 20 -60% in 60 min, and 60 -80% in 10 min) in 0.1% TFA at a flow rate of 1 ml/min. At each stage, eluate absorbance was monitored at 210 and 280 nm. Amino Acid Sequence Analysis-N-terminal sequencing was carried out by automated stepwise Edman degradation using a Procise model 492 protein sequencer (Applied Biosystems) according to the manufacturer's protocol. Reduction of Disulfide Bonds and Modification of Thiol Groups-Purified CpTx 1 (ϳ10 nmol) was dried and redissolved in 40 l of 0.5 M Tris-HCl (pH 8.5), 6 M guanidine hydrochloride, and 3 mM EDTA. A 150-fold molar excess of 1,4-dithiothreitol was added to the mixture. The probe was sealed and incubated at 40°C for 18 h. Then 4-vinylpyridine (a 3-fold molar excess with respect to dithiothreitol) was added. The tube was left at room temperature for 15 min in the dark. The modified polypeptide was purified by RP-HPLC on a Jupiter C 5 column (4.6 ϫ 150 mm). Cyanogen Bromide Cleavage-Reduced-alkylated CpTx 1 (ϳ1 nmol) was dried and redissolved in 80% TFA to a concentration of ϳ0.5 g/l. Cyanogen bromide was added with a ϳ100-fold molar excess, and the sample was incubated overnight at room temperature in the dark. Then 10 volumes of cool water were added, and the sample was dried and redissolved in 0.1% TFA. The mixture was analyzed directly by MALDI-MS, and the products were separated by means of RP-HPLC on a Jupiter C 5 column (2 ϫ 150 mm, 30-nm pore size, 5-m particle size; Phenomenex) at a flow rate of 0.3 ml/min. cDNA Library Construction, Sequencing, and Analysis-All steps were carried out in collaboration with DuPont Agriculture and Nutrition (Newark, DE) as described previously (26). Briefly, venom glands of C. punctorium were harvested and homogenized in liquid nitrogen, and cell lysis was induced by TRIzol (Invitrogen). Poly(A) ϩ RNA was purified on an oligo-(dT)-cellulose affinity column using the mRNA purification kit (Amersham Biosciences). First strand (using Superscript II from Invitrogen) and second strand cDNA synthesis, linker addition, cloning into pBlueScript SK ϩ (EcoRI and XhoI sites) were all carried out according to the Stratagene cDNA kit; cDNA was purified using a cDNA column (Invitrogen). Sequencing was performed using the M13 forward primer by the ABI PRISM BigDye Terminator Cycle Sequencing Ready reaction kit with AmpliTaq DNA polymerase, FS (PerkinElmer Life Sciences) on a model 373 DNA Sequencer (Applied Biosystems). UV Absorbance Spectroscopy-Absorption spectra were recorded on a Hitachi U-3210 spectrophotometer (Tokyo, Japan). CpTx 1 concentration was determined using the molar extinction coefficient at 280 nm (⑀ 280 ) of 17,460 M Ϫ1 cm Ϫ1 calculated by the GPMAW program (Lighthouse Data, Odense, Denmark). Circular Dichroism-For bicelle preparation, 100 mM 1,2dihexanoyl-sn-glycero-3-phosphocholine (DHPC) solution was prepared in phosphate-buffered saline (PBS: 1.47 mM KH 2 PO 4 , 4.29 mM Na 2 HPO 4 , 137 mM NaCl, 2.68 mM KCl). The appropriate amount of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was dissolved in the DHPC solution to obtain a molar DMPC/DHPC ratio of 2:1. Bicelle formation was accelerated by heating the mixture up to 45°C and cooling down to 0°C three times. The sample for CD measurements was prepared by mixing equal parts of bicelle suspension and 20 M CpTx 1 solution in PBS and incubating at 40°C for 30 min. The sample for base-line correction was obtained using a probe of DMPC/DHPC bicelles diluted 2-fold with PBS. Plasma Membrane Permeabilizing Activity-Sf9 cells were cultured as described above. Prior to the experiment, the cell suspension (10 6 cells/ml) was placed in a 12-well flexiPERM silicon chamber (Perbio Science, Erembodegem-Aalst, Belgium) attached to a cover glass, and cells were allowed to settle onto the bottom of the wells. Markers of plasma membrane permeability 5(6)-carboxyfluorescein (CF) (30) and PI were added at concentration of 50 and 10 M, respectively. CpTx 1 was added at 10 M corresponding to 20% cytotoxic effect. Confocal laser scanning microscopy was used to study CpTx 1-induced CF and PI entry into the cells. The silicon chamber was mounted on the stage of a FluoView FV1000 microscope. An UPLSAPO (60ϫO/1.35 NA) objective (Olympus) was used to visualize cells. Excitation light was provided at a wavelength of 488 nm (for CF) and 559 nm (for PI) by argon-ion and helium-neon lasers, respectively. The beam splitter DM 405/488/559/635 was used. Fluorescence emission was collected in the range of 497-558 nm (CF) or 579 -679 nm (PI). A transmitted light image was obtained using the 488 nm laser beam and recorded simultaneously with the fluorescent image. The scanning area size was 105 ϫ 105 m, and the pixel size was 0.066 ϫ 0.066 m. Pinhole diameter was 110 m corresponding to the optical slice thickness of Ͻ0.8 m. Sequential mode of scanning was used to collect CF and PI fluorescence unmixed. Insect Neuromuscular Preparations-Late third stage larvae of Calliphora vicina were used in all experiments (31,32). After dissection, the internal organs were removed so that the preparation consisted only of muscles attached to the cuticle. The ventral ganglion was removed, and the segmental nerves were stimulated through the suction electrode. Recordings of membrane potential were made by glass intracellular microelectrodes from ventral longitudinal fibers. To eliminate electrical contacts of the recorded fiber with its neighbors, the latter were dissected. This resulted in a significant increase of input resistance in the recorded cell. The preparation was perfused with saline as follows: 172 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl 2 , 8 mM MgCl 2 , 2.4 mM NaHCO 3 , 0.3 mM NaH 2 PO 4 , 52 mM sucrose (pH 7.2). Experiments were performed at room temperature (22°C). The excitatory postsynaptic currents were evoked by nerve stimulation and recorded by a conventional two-electrode voltage clamp using an Axoclamp-2B amplifier (Axon Instruments). The data were filtered at 2 kHz and stored on a PC. A series of experiments was performed under voltage clamp conditions to evaluate the effect of CpTx 1 on muscle cell input resistance and the corresponding current. Holding potential was set at Ϫ60 mV and periodically (each 20 s) shifted by small hyperpolarizing steps (5-mV amplitude and 300-ms duration). Changes of input resistance were estimated from the amplitudes of current response to these steps. Frog Neuromuscular Preparations-The following chemicals were used: amiloride, N-methyl-D-glucamine (NMDG) chloride, and ouabain (Sigma), and tetrodotoxin (TTX; Affinity Research Products). Experiments were performed on musculus sartorius neuromuscular preparations of the common frog Rana temporaria. The preparations were placed into a plastic chamber (1.5 ml) and superfused with solution of the following composition: 113 mM NaCl, 2.5 mM KCl, 0.6 mM CaCl 2 , 3 mM NaHCO 3 , 4 mM MgCl 2 (pH 7.3). In some experiments, 113 mM NaCl was replaced with 116 mM NMDG, 232 mM sucrose, 232 mM NaCl, or a combination of 113 mM NaCl and 232 mM sucrose. Solution containing a certain concentration of CpTx 1 and 0.01% bovine serum albumin (Sigma) was added to the bath. The temperature was kept at 22°C. Glass microelectrodes filled with KCl had a resistance of 10 -15 megohms. The resting membrane potential of muscle fibers was measured in 15-35 cells in control and after 30, 60, and 90 min of continuous perfusion with saline containing 50 nM of CpTx 1. Planar Lipid Bilayer Membrane Assay-CpTx 1 was tested for the ability to cause conductance changes in planar bilayer membranes (BLM). Virtually solvent-free membranes were formed from 1,2-diphytanoyl-sn-glycero-3-phosphatidyl-choline (Avanti Polar Lipids) as described by Montal and Mueller (36). Two symmetrical chambers of a Teflon cell each with a solution volume of 1.5 ml were separated by a 15-m-thick Teflon partition containing a round aperture of ϳ100-m diameter. Hexadecane was used for aperture pretreatment. BLM were formed from 0.1% lipid solution in pentane. The experimental cell was filled with 150 mM KCl solution buffered with 5 mM MOPS (pH 7.2). Electrical measurements were performed at room temperature (22 Ϯ 2°C) using a pair of Ag/AgCl electrodes connected to the cell chambers via 1.5% agarose bridges containing 2 M KCl. CpTx 1 was added to the cis-chamber 10 min after bilayer formation, and the resulting solution was mixed with a magnetic stirrer. Transmembrane currents were recorded with a custom-made amplifier and digitized with a pCLAMP-compatible board. Data collecting was performed with 2-kHz sampling frequency and low pass filtering at 200 Hz. Positive voltage means that the cis-chamber is positive with respect to the trans-chamber. Positive currents are those of cations flowing from the cis-to the trans-chamber. Clampfit 9.0 software (Molecular Devices) was used for data analysis, and Origin 7.0 (OriginLab) was used for statistical analyses. RESULTS Active Toxin Purification-Crude venom of the yellow sac spider C. punctorium (Miturgidae) 3 was found to possess insecticidal (LD 50 ϳ3 g/g in flesh fly larvae), hemolytic, cytotoxic, and membrane-damaging (33) activities. Our findings corroborate previous investigations of this and other Cheiracanthium species (24). To isolate the active principle of C. punctorium venom, we used a classically simple yet effective approach that combined two methods of liquid chromatography, i.e. SEC and RP-HPLC ( Fig. 1) (25). After SEC, the major insect toxicity resided in the two early eluting fractions (A, Ͼ30 kDa; B, 7-30 kDa). The high molecular weight fraction A contained several components with molecular masses of ϳ30 -60 kDa as estimated by electrophoresis (data not shown) and measured by MALDI-MS. In line with the previous report by Foradori et al. (24), we suspect that some of these components may exhibit phospholipase A 2 activity. Fraction B contained several polypeptides with molecular masses of ϳ15 kDa. RP-HPLC separation of this fraction yielded a number of peaks, the major of which were found toxic. According to analytical HPLC and MALDI-MS, the predominant peak (ϳ10% of crude venom protein) was represented by a single component (Ͼ95% purity) with a molecular mass of 15,100 Da that was named CpTx 1 (C. punctorium toxin 1). CpTx 1 Amino Acid Sequence-To determine the full amino acid sequence of the isolated toxin, we chose to follow the usual scheme of polypeptide structure analysis (25,34). The idea was to assemble the full sequence from overlapping peptide fragments generated through various types of selective proteolysis. First, the presence of disulfide bonds was assessed as follows. CpTx 1 was reduced with dithiothreitol, and thiol groups were subsequently modified with 4-vinylpyridine. The molecular mass of the toxin increased to 16,800 Da, indicating the presence of 16 cysteine residues. Because modification of unreduced CpTx 1 failed to increase its molecular mass, all cysteine residues were concluded to form intramolecular disulfide bridges. Sequencing of the reduced-alkylated CpTx 1 by Edman degradation identified the first 40 N-terminal amino acid residues (cysteine residues were determined as S-pyridylethylated derivatives). Next, the following reactions of selective proteolysis were performed: (i) cyanogen bromide, (ii) endoproteinase Glu-C, (iii) endoproteinase Arg-C cleavage of the reducedalkylated CpTx 1; and (iv) "limited" endoproteinase Glu-C digestion of the unreduced toxin. In each case, proteolytic fragments were separated by RP-HPLC; their masses were measured by MALDI-MS and their sequences established by Edman degradation (alternatively, but not exclusively, sequencing was performed by MS/MS). As a result, we were able to peptide-map the full amino acid sequence of CpTx 1 (134 residues; Fig. 2). In particular, CNBr cleavage resulted in two fragments, the larger fragment (ϳ12.5 kDa) corresponding to the N-terminal part of CpTx 1, and the smaller fragment (4312 Da) to its C-terminal part that was fully sequenced by Edman degradation (37 residues). Limited endoproteinase Glu-C digestion of the unreduced toxin produced four fragments as follows: N-terminal (ϳ5.9 kDa), C-terminal (1596 Da; 14 residues fully sequenced), and the full and C-terminally truncated forms of the central fragment (ϳ7.7 and ϳ7.1 kDa, respectively; 39 N-terminal residues sequenced). Digestion of the reduced-alkylated CpTx 1 with the same enzyme produced a number of fragments, most of which were fully sequenced. We note that at the alkaline pH value used in our experiment, endoproteinase Glu-C was not selective for glutamic acid but also cleaved at aspartic acid residues. Finally, endoproteinase Arg-C was found to preferably cleave at Arg-124, but minor cleavage events occurred at other arginine and lysine residues, and the corresponding fragments were unambiguously assigned (see Fig. 2B). CpTx 1 is a cationic polypeptide (charge ϩ8 at pH 7, pI ϳ10). It was also found to be C-terminally amidated; the measured molecular masses of the C-terminal fragments differed by 1 Da from the respective calculated values. The sequencing procedures revealed CpTx 1 microheterogeneity at two positions with minor substitutions N33S and D81E. In both cases, the minor forms represented ϳ30% of the total protein. However, at this point we could not unequivocally assign the sequences of CpTx 1 isoforms (either two additional isoforms that carry a single substitution or just one additional isoform with both substitutions simultaneously seemed equally plausible). This riddle was solved by cDNA sequencing (see below). The 16 cysteine residues of CpTx 1 were found to assemble in two distinct primary structure signatures, CX 6 CX 6 CCX 8 CXCX n CXC. These signatures contain both the principal structural motif CX 6 CX n CC and the extra structural motif (ESM) CXCX n CXC characteristic of spider neurotoxins (26), and CpTx 1 is therefore allocated to the structural class 2 of spider toxins according to Kozlov and Grishin (35). Moreover, the two signatures perfectly conform to the more general ICK motif CX 2-7 CX 3-11 CX 0 -7 CX 1-17 CX 1-19 C found in hundreds of peptides from various sources (37,38). As proven by Glu-C digestion of the native toxin (see above), S-S bridges are formed exclusively inside the two signatures. CpTx 1 may therefore be represented as a modular polypeptide containing two disulfide-rich (most probably ICK) domains. Interestingly, sequences of the two modules share low yet significant sequence homology with each other (ϳ28% identical residues) (Fig. 3). CpTx 1 exhibits a very low percent of identity with other known polypeptides, the only significant homology is found with toxins from the wolf spider Lycosa singoriensis (Lycosidae) identified from translated nucleotide sequences (39) and CSTX toxins from the venom of the wandering spider Cupiennius salei (Ctenidae) with ϳ30 -40% sequence identity and strict conservation of the cysteine residues (Fig. 3). Specifically, the insecticidal toxin CSTX-1 is known to block insect and mammalian calcium channels (40,41); CSTX-9 probably acts similarly (42), and CSTX-13 is a neurotoxic enhancer that possesses lower lethal activity compared with other mentioned CSTX toxins but synergistically augments their potency (43,44). Interestingly, the two modules of CpTx 1 cluster differentially with CSTX toxins; the C-terminal domain is more homologous to toxins CSTX-1 and CSTX-9, whereas the N-terminal part shows higher homology to the enhancer CSTX-13. The disulfide bridge arrangement has been determined for all CSTX toxins conforming to the ICK motif (42,43), and the same cysteine pairing is proposed for the two CpTx 1 domains. C. punctorium Venom Gland cDNA Analysis-A cDNA library was constructed for C. punctorium venom glands in collaboration with DuPont Agriculture and Nutrition (a separate paper dealing with all results in more detail is envisaged). Library analysis was performed in accordance with an earlier elaborated strategy and included not only the usual alignment and comparison with the nucleotide and polypeptide sequences deposited in public databases but also searches for structural elements and motifs specific for secreted polypeptide sequences and spider toxins (26,35,45,46). As a result, putative spider toxin sequences were deduced. CpTx 1 sequence established by protein chemistry and MS was then used to query the library. Clones coding for three isoforms of this toxin were thus identified. The major isoform was named CpTx 1a and the two minor isoforms with D81E and N33S substitutions were named CpTx 1b and CpTx 1c, respectively. CpTx 1a was found to be coded by two types of mRNA corresponding to two types of precursor protein (pCpTx 1a-1 and 1a-2) that differ by two amino acid substitutions (Fig. 4). In addition, two types of precursor for CpTx 1b (pCpTx 1b-1 and 1b-2) and a precursor for CpTx 1c (pCpTx 1c) were found. No sequence containing both substitutions was detected; therefore, CpTx 1 toxin is believed to be represented by three closely homologous isoforms differing by point amino acid substitutions. pCpTx 1 precursor proteins consist of three parts (Fig. 4). (i) N-terminal signal (pre-) peptide (leader sequence) of 20 amino acid residues (identified by the SignalP 3.0 program) is cleaved off cotranslationally, on entrance to the endoplasmic reticulum. (ii) An acidic prosequence (27 residues) is removed posttranslationally during the so-called limited proteolysis. The high negative charge of the prosequence (Ϫ10 at pH 7) cancels FIGURE 3. CpTx 1 sequence homologues. Two modules (domains) of CpTx 1 corresponding to amino acid residues 1-64 and 65-134 are aligned with sequences of toxins from C. salei CSTX-1, -9, and -13. Polypeptide chain lengths are given in the right column. Note that CSTX-13 is a two-chain molecule, with no covalent bond between Gln-34 and Ala-35. Similar residues are shaded gray. Residues more common to the N-terminal domain of CpTx 1 and CSTX-13 are highlighted red; those more common to the C-terminal domain of CpTx 1 and toxins CSTX-1 and 9 are highlighted green. Invariant cysteine residues are shaded black, and the disulfide bonds assigned for CSTX toxins are drawn above. The cysteine motifs the principal structural motif (PSM) and extra structural motif (ESM), each contributing to the ICK structural fold, are identified below. The putative C-terminal ␣-helical region is also indicated below. out the positive charge of the mature toxin. In spider toxins, propeptide cleavage most usually occurs at a specific site known as the processing quadruplet motif (PQM): X 1 X 2 X 3 R, where any X n ϭ E (26,45,46). In pCpTx 1, the PQM is 44 DESR 47 . (iii) The mature toxin chain is located C-terminally. All precursors were found to bear an additional C-terminal dipeptide -GR, a signature of C-terminal amidation, supporting our sequencing data. CpTx 1 Secondary Structure-Peptides exhibiting the ICK motif are typically ␤-structural with 2-3 antiparallel ␤-strands joined by a number of reverse turns and loops of variable size, stabilized by disulfide bridges. In line with this rule, CpTx 1 main chain was found to predominantly adopt ␤-sheet and ␤-turn conformation in aqueous solution, as suggested by the CD spectrum ( Fig. 5 and Table 1). In the presence of DMPC/ DHPC bicelles used to mimic phospholipid membranes, changes of the spectrum were observed suggesting molecular interactions that lead to structural perturbations of the toxin; a significant increase (ϳ2-fold) of ␣-helix with a simultaneous reduction of random coil content was noted. Application of a number of algorithms resulted in prediction of two putative ␣-helical segments into the cysteine-free C-terminal parts of both CpTx 1 domains (ϳ42-57 and ϳ111-134; Fig. 3). Plotted in a helical conformation, these sequences form amphiphilic structures with segregation of hydrophobic versus hydrophilic residues that may effectively interact with membranes. CpTx 1 Biological Activity-Purified toxin was subjected to different tests and was found to exhibit a number of activities. CpTx 1 caused lethal effects on flesh fly (S. carnaria) larvae (LD 50 of ϳ10 g/g); doses twice the LD 50 value and higher caused instant paralysis and death, and at doses close to and lower than the LD 50 value, reversible paralysis occurred. The toxin possessed concentration-dependent cytolytic activity on insect Sf9 cells as follows: marginal (ϳ5%) toxicity at 0.5 M and ϳ20% cell death at 10 M. CpTx 1 causes influx of organic cation (PI) as well as organic anion (CF) in the susceptible cells (Fig. 6). To visualize this effect accurately, confocal laser scanning microscopy was utilized, because the optical section must be thinner than the cell diameter to detect differences between intra-and extracellular marker concentrations. The peptide-induced membrane damage is accompanied by significant changes in cell morphology, including membrane blistering. At the concentration used (10 M), CpTx 1 affects 20% of the entire cell population and causes toxicity in 30 min. The paralytic effects were further studied on fly neuromuscular preparations. At 50 -200 nM, CpTx 1 caused stable and irreversible depolarization of fly muscle fibers eventually lead- 16 19 ing to contracture at higher toxin concentrations. Both the spontaneous and nerve stimulation-induced postsynaptic currents, however, were unaffected by the toxin. A similar depolarization induced by CpTx 1 was observed on frog neuromuscular preparations (Fig. 7, A and B). The average level of resting membrane potential in frog muscle fibers measured in control experiments was stable for more than 1 h (Ϫ65 to Ϫ90 mV in different cells). In the presence of 20 nM of CpTx 1, a moderate but statistically significant depolarization was observed (ϳ10 -20 mV in 30 -60 min). Increase of the toxin concentration up to 50 nM induced a much more pronounced effect that was not reversed by washing; the membrane potential drop gained ϳ50 mV. Some muscle fibers demonstrated transient contractures following 7-15 min of contact with CpTx 1. In the experiments done under voltage clamp conditions, the toxin induced the rise of transmembrane current and a corresponding drop of the cell input resistance (Fig. 7C). Removal from saline of the most parts of NaCl and its substitution by sucrose (232 mM) did not induce significant changes of the membrane potential in control. Interestingly, under these conditions of low Na ϩ content, the application of CpTx 1 (50 nM) for up to 90 min did not depolarize muscle fibers. To the contrary, a 2-fold increase of Na ϩ concentration (up to 232 mM) or addition of sucrose (232 mM, providing an equal increase of tonicity) intensified the depolarizing action of CpTx 1. Replacement of Na ϩ by NMDG ions, however, affected weakly the depolarizing action of the toxin (Fig. 7A). An increase of Ca 2ϩ concentration from 0.6 to 3.6 mM substantially damped the depolarizing action of CpTx 1. 60 min of toxin application resulted in a much smaller effect, the membrane potential gained only an ϳ15-mV drop compared with ϳ50 mV at low calcium concentration. Addition of 1 mM Co 2ϩ to saline (containing 0.6 mM Ca 2ϩ ) slightly inhibited the depolarizing effect of the toxin, but 5 mM diminished significantly the depolarization (Fig. 7B). A 15-min pretreatment of the preparations with 1 M TTX, 100 M ouabain, or 1 mM amiloride had no effect on the depolarizing action of CpTx 1. Toxin Action on Planar Bilayer Membranes-The membrane-tropic effects of CpTx 1 were explored in BLM. In control experiments, the membranes remained intact for over 100 min clamped at Ϯ100 mV. In the presence of 50 nM of CpTx 1, however, a number of effects were observed. In ϳ40% of cases, CpTx 1 induced BLM breakdown with or without prior appearance of conductance fluctuations. In the other ϳ40% of cases, the toxin did not lyse the membrane, but it induced conductance in the form of numerous temporal fluctuations of variable current amplitude (͉0.1͉Ϫ͉10͉ pA at Ϯ100 mV). All effects were equally pronounced at both potential polarities. In the remaining ϳ20% of experiments, the BLM remained stable as in the control. Increase of CpTx 1 concentration (Ն150 nM) resulted in a higher probability of both membrane rupture (ϳ60%) and temporal fluctuation occurrences. Very seldom were single channel-like events observed with current amplitudes in the range of 0.2-2 pA. DISCUSSION Principal Component of C. punctorium Venom-Two major toxic fractions were isolated from the venom of the spider C. OCTOBER 15, 2010 • VOLUME 285 • NUMBER 42 punctorium. The high molecular weight fraction probably contains a phospholipase (24), whereas the main component of the other fraction is a novel and unique 134-residue-long toxin CpTx 1 (represented by three closely related isoforms CpTx 1a, 1b and 1c, Fig. 2). CpTx 1 causes fast paralysis and mortality in flesh fly larvae, exerts pronounced membrane-tropic and cytolytic effects, reflecting the high insecticidal, cytotoxic, and membrane-damaging activities of the crude venom (24,33). Certain discrepancy was noted between the measured LD 50 values for the whole venom (ϳ3 g/g) and purified CpTx 1 (ϳ10 g/g), which either points to existence of additional powerful toxins, such as the presumed phospholipase, or may be result of synergy with other venom components, often noted for spiders (7,17,43,44). Interestingly, Cheiracanthium bites produce symptoms that closely resemble bee and wasp stings (23). This observation is corroborated by assigning cytolytic function to the principal components in both Cheiracanthium and hymenopteran (17) venoms. Novel Class of Spider Toxin CpTx 1 Mode of Action-The toxin was found to affect the resting membrane potential in both insect and vertebrate muscle fibers. This effect was concentration-dependent and eventually manifested in substantial depolarization leading to muscle contracture and fiber damage. Measured under voltage clamp, CpTx 1 induced a rise of the transmembrane current and a corresponding drop of the cell input resistance (Fig. 7C). This indicates appearance in the membrane of new paths for permeable ions. To explain the toxic effect of CpTx 1, we sought to characterize its probable target, and several possible causes of membrane depolarization were tested as follows. (a) Because substitution of sucrose for Na ϩ ions inhibited this effect, whereas increase of Na ϩ concentration potentiated it (Fig. 7A), CpTx 1 seems to trigger specifically direct entry of Na ϩ into the muscle cells. The same effect was exhibited when instead of an increase of Na ϩ the appropriate amount of sucrose was added. This may be interpreted as a result of an increase in the tonicity of the outer solution, which may also potentiate the Na ϩ influx. (b) Substitution of Na ϩ by NMDG ions much less permeable through voltage-gated Na ϩ channels (47) and epithelial Na ϩ channels (ENaC) (48) did not influence significantly the CpTx 1-induced depolarization. Neither TTX, a potent blocker of Na ϩ channels (49), nor Co 2ϩ (1 mM), a nonspecific blocker of Ca 2ϩ channels (50), nor amiloride, a blocker of the ENaC family and a subset of voltage-gated Ca 2ϩ channels (51,52), eliminated the depolarizing effect of CpTx 1. Therefore, activation of voltage-gated Na ϩ and Ca 2ϩ channels or ENaC by this toxin seems unlikely. (c) Phenomenologically, CpTx 1 effects resemble those of palytoxin from zoanthid corals, one of the most complex non-protein toxins in nature (53). Palytoxin targets the Na ϩ /K ϩ pump shifting its activity from active to passive transport of Na ϩ and K ϩ that eventually disrupts the ion gradient and abolishes the membrane potential (54). The blocker of the Na ϩ ,K ϩ -ATPase ouabain readily inhibits palytoxin activity (55) but fails to affect the action of CpTx 1. Therefore, the Na ϩ /K ϩ pump does not seem to be the primary target of CpTx 1. To sum up, we excluded a number of membrane ion transport proteins (voltage-gated Na ϩ and Ca 2ϩ channels, ENaC, and Na ϩ ,K ϩ -ATPase) as possible targets of the toxin in frog neuromuscular preparations. To the contrary, a considerable amount of evidence points to the cytoplasmic membrane lipid bilayer itself as the main target of CpTx 1. (i) Membrane environment induces structural changes of the polypeptide as determined by CD (Fig. 5 and Table 1) indicating toxin binding to the membrane. (ii) Artificial membranes are destabilized by CpTx 1, and the observed defects (numerous temporal current fluctuations of variable amplitude) are frequently seen in case of membrane-active peptides (MAPs) (56). (iii) Toxin-induced depolarization is independent of a number of membrane transport proteins and quite insensitive to the ionic composition of the extracellular medium (see above). (iv) Increase in concentration of divalent cations (3.6 mM Ca 2ϩ or 5 mM Co 2ϩ ) inhibits CpTx 1 effects (Fig. 7B), and a similar antagonism was noted in case of other MAPs; these cations compete with MAPs for binding to the membrane lipid phosphate groups (57). (v) The toxin was found to exhibit cytolytic activity characteristic of many MAPs. This activity was visualized directly by confocal laser scanning microscopy (Fig. 6). PI and CF in the ionized form are not able to penetrate the intact plasma membrane (29,30); therefore, their appearance in a cell indicates membrane lesion formation. PI is accumulated in the nuclei, where the dye fluorescence increases dramatically due to intercalation into DNA. Simultaneous use of two dyes with charges of opposite sign further provides evidence of little selectivity of the membrane defects induced by CpTx 1. The C-terminal parts of both domains of CpTx 1 (Fig. 3) are believed to assume ␣-helical conformation in the membrane environment, as suggested from secondary structure predictions and CD spectra measurements ( Fig. 5 and Table 1). The putative helices are amphipathic with clusters of hydrophilic and hydrophobic residues occupying opposite sides of the cylinders. Such structures are known to effectively interact with membranes and are a hallmark of MAPs (17,56,57). We suggest that these fragments serve as "membrane attachment devices" of the toxin. We also note that similar sequences are present in other toxins, such as CSTX toxins from C. salei (see Fig. 3), indicating their plausible membrane-active properties. In this regard, the "membrane-access" mode of action proposed for certain spider neurotoxins deserves special attention (58). These peptides adopt amphiphilic structures due to formation of a hydrophobic patch of residues located in specific loops and ␤-strands of the ICK fold corresponding to fixed positions in the amino acid sequence. These hydrophobic residues are missing in CpTx 1, CSTX, and some other toxins, the noted amphipathic ␣-helical segment of which thus represents a novel membrane-access motif. In conclusion, the lipid bilayer seems to represent the most probable target of CpTx 1. Most of the observed effects may be attributed to the cytolytic activity of the toxin. Existence of a specific receptor (acceptor) of this toxin cannot be totally ruled out at this point, however. Moreover, some evidence could testify in its favor; the low LD 50 value determined for CpTx 1 is typical of specific neurotoxins, whereas cytolytic membrane-active peptides are usually characterized by 1 to 2 orders of magnitude higher lethal doses; there is some discrepancy between the active toxin concentrations observed ex vivo in neuromuscular preparations and in vitro in artificial membranes. CpTx 1 Is a Unique Two-domain Modular Toxin-C. punctorium has shown profound difference from other studied spiders in terms of venom molecular composition. It is generally accepted that spider venoms may contain one of the three types of principal components: low molecular weight acylpolyamines, peptides (Ͻ10 kDa), or large proteins. The majority of investigated spider species produce venom rich in peptides having the "cystine knot" (ICK) fold (7,11,14,20). MS of crude venoms typically produces a "bimodal" distribution of molecular mass species with predominance of ϳ3-5and ϳ7-8-kDa species (59). In sharp contrast, C. punctorium whole venom gave a "unimodal" picture with an overwhelming dominance of ϳ15-kDa constituents. The major component of the venom, CpTx 1, was found to contain 134 residues determined by direct sequencing (Fig. 2) and further confirmed by cDNA cloning (Fig. 4). Its amino acid sequence may be considered as built up of two domains (modules), each corresponding to a putative "usual" ICK spider toxin of ϳ7-8 kDa. It was directly shown by endoproteinase digestion of the native CpTx 1 that the two parts indeed represent separate domains with disulfide bonds formed only inside them. The two modules are homologous, pointing to a possible gene exon duplication followed by mutation that eventually led to the modular toxin structure. Moreover, both domains have known single domain toxin homologues found in spiders of different families (wolf spider L. singoriensis, Lycosidae, and wandering spider C. salei, Ctenidae, see Fig. 3). Curiously, one of the CpTx 1 domains shows higher similarity with CSTX-1 and -9, the principal neurotoxins from C. salei venom, whereas the other domain is more like CSTX-13, the neurotoxic enhancer, the function of which is to potentiate CSTX-1 and -9 toxicity (40 -44). CpTx 1 may therefore represent a self-enhancing structure, which should be tested by comparison of the isolated modules toxicity with the full-sized polypeptide. CpTx 1 represents the first example of a class of modular toxins with two disulfide-rich domains, thereby expanding the toxin universe. Indeed, if we consider the folding patterns realized in arachnid peptide toxins (Table 2), the following types may be noted. (a) Single domain toxins are the most widespread. Disulfide-rich toxins usually contain the ICK motif in spiders (for instance, the famous -agatoxins IVA and IVB from A. aperta (8)) and the cysteine-stabilized ␣-helix/␤-sheet (CS␣␤) motif in scorpions (charybdotoxin from Leiurus quinquestriatus hebraeus is a classical example (60)). Most linear toxins adopt ␣-helical conformation in contact with membranes (for instance, latarcins from L. tarabaevi (18) or pandi-nins from the scorpion Pandinus imperator (61)). (b) Two-domain modular toxins have been subsequently described, widening the known arachnid toxic arsenal. The scorpine family of modular peptides from the venom of P. imperator and other scorpions includes an N-terminal linear cationic ␣-helical domain, showing similarities to many MAPs, including some ␣-helical antimicrobial peptides (AMPs), and a C-terminal CS␣␤ domain, common to a wealth of peptides, including scorpion neurotoxins and plant defensins (62). More recently, cyto-insectotoxins (CITs), giant linear peptides exhibiting equally potent antimicrobial, cytolytic, and insecticidal activities, were discovered in the venom of L. tarabaevi. CITs include two linear modules each corresponding to a usual ␣-helical AMP (19). Together with this study, the following three classes of modular arachnid toxins are now known (note that disulfide connectivities and three-dimensional structures have yet to be established): AMP ϩ AMP (CITs), AMP ϩ CS␣␤ (scorpines), and ICK ϩ ICK (CpTx 1). On the Evolution of Modular Toxins-It seems that in spiders the modular toxins evolved from the so-called "binary" toxin precursors that are processed into two separate mature chains due to limited proteolysis at two rather than one processing motif (18,45,46). In CITs, a cryptic processing motif PQM, which we believe once served for separation of the two modules, is evident; the key arginine residue was mutated (EEAR 3 EEAQ) (19). In CpTx 1, no such sequence is at once noticed between the two domains (Fig. 2). However, the exact linker peptide 61 PKPV 64 , immediately preceding the second domain, reverts to AETR (a typical PQM) with a "Ϫ1" frameshift in the coding DNA sequence (Fig. 4). We therefore propose that in CITs and CpTx 1 different mutation mechanisms, i.e. substitutions versus deletions/insertions, ensured the merging of two mature peptide chains of binary toxin precursors in a single toxin of modular structure. In conclusion, we believe that the discovered two-domain modular toxin CpTx 1 from C. punctorium venom enhances our knowledge of natural toxin biodiversity. We also anticipate that the known modular toxin variability will soon be expanded.	2018-04-03T05:17:51.344Z	2010-07-24T00:00:00.000	{ "year": 2010, "sha1": "aec23cf1209642e23788c0fd2a6f1df24436046b", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/285/42/32293.full.pdf", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "5178a32d49fc7cbd7aa9f11191d0705958086f19", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
259117920	pes2o/s2orc	v3-fos-license	Cerebral Autoregulation: Don’t go with the Flow, be the Flow; Author′s Response Dear editor, We would like to thank Ayasse et al. for their interest in our article “Blood Pressure Affects the Early CT Perfusion Imaging in Patients with aSAH Reflecting Early Disturbed Autoregulation” [1] and for providing their insights into cerebral autoregulation. We appreciate the opportunity to respond to the issues raised. Firstly, we would like to emphasize that our study primarily focused on the correlation between mean transit time (MTT) of early computed tomography perfusion (CTP) imaging and blood pressure during the early brain injury (EBI) phase, indeed on a population level. As Ayasse et al. alluded to, MTT was chosen as the parameter of interest because it is known to be the most relevant prognostic CTP parameter in patients with aneurysmal subarachnoid hemorrhage (aSAH), and a shorter MTT is associated with better outcomes and fewer complications [2–4]. Neither cerebral blood volume (CBV) nor cerebral blood flow (CBF) was shown to be a relevant factor in that regard. However, the quotient of these two parameters, the MTT, was found to apparently better reflect the relevant pathophysiology radiologically in the EBI phase in a clinical setting [2–4]. In part, this may be explained by the relative nature of CBV and CBF measurements in CTP imaging, and we agree with the statement by Ayasse et al. that it is essential to carefully choose the method for quantitatively establishing CBF. Both CBV and CBF measurements include a measure of mass of brain tissue in the respective units, which is an unknown during a CTP scan, thereby limiting comparability across patients and even across different imaging time points in the same patient. In MTT, however, as the quotient of CBV/CBF, all relative units are reduced from the results without any elaborate postprocessing, leaving only an absolute measurement: time. We acknowledge that CBF and CBV measurements are abundant in the literature, and different evaluation methods and software lead to variability in reported MTT values. However, when trying to find suitable surrogate parameters with potential for real-world clinical usage, we try to concentrate on “more comparable” parameters, such as MTT, where possible. Following the concepts of cerebral autoregulation laid out by Ayasse et al., a prolongation of MTT is indeed to be expected in patients with a decreased MAP, and we thank Ayasse et al. for contributing the valuable and clear explanations of the relationship between MAP, CBF, CBV, and MTT. Although both our title and the conclusion are arguably pointed, we feel that our discussion is a bit more nuanced and better explains our conclusions. Although CBV and CBF were not considered in our study, as explained previously, it has to be noted that at least some earlier observations have reported reduced CBF and constant CBV in CTP imaging in the EBI phase [3, 4] and that other studies have described impaired autoregulation in the EBI phase [5]. Instead, we would like to draw the reader’s attention to the increasingly stronger correlation between MAP and MTT as the patient’s neurological status worsens, as measured by the World Federation of Neurosurgical Societies grade. We found it remarkable to demonstrate, for the first time, that in less vigilant patients (a direct indicator of the severity of EBI), the correlation between MAP and MTT becomes stronger. We assumed that the most probable cause for this observation was the impairment of cerebral *Correspondence: bjoern.hofmann@med.uni-duesseldorf.de 1 Department of Neurosurgery, Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University, Düsseldorf, Germany Full list of author information is available at the end of the article Dear editor, We would like to thank Ayasse et al. for their interest in our article "Blood Pressure Affects the Early CT Perfusion Imaging in Patients with aSAH Reflecting Early Disturbed Autoregulation" [1] and for providing their insights into cerebral autoregulation. We appreciate the opportunity to respond to the issues raised. Firstly, we would like to emphasize that our study primarily focused on the correlation between mean transit time (MTT) of early computed tomography perfusion (CTP) imaging and blood pressure during the early brain injury (EBI) phase, indeed on a population level. As Ayasse et al. alluded to, MTT was chosen as the parameter of interest because it is known to be the most relevant prognostic CTP parameter in patients with aneurysmal subarachnoid hemorrhage (aSAH), and a shorter MTT is associated with better outcomes and fewer complications [2][3][4]. Neither cerebral blood volume (CBV) nor cerebral blood flow (CBF) was shown to be a relevant factor in that regard. However, the quotient of these two parameters, the MTT, was found to apparently better reflect the relevant pathophysiology radiologically in the EBI phase in a clinical setting [2][3][4]. In part, this may be explained by the relative nature of CBV and CBF measurements in CTP imaging, and we agree with the statement by Ayasse et al. that it is essential to carefully choose the method for quantitatively establishing CBF. Both CBV and CBF measurements include a measure of mass of brain tissue in the respective units, which is an unknown during a CTP scan, thereby limiting comparability across patients and even across different imaging time points in the same patient. In MTT, however, as the quotient of CBV/CBF, all relative units are reduced from the results without any elaborate postprocessing, leaving only an absolute measurement: time. We acknowledge that CBF and CBV measurements are abundant in the literature, and different evaluation methods and software lead to variability in reported MTT values. However, when trying to find suitable surrogate parameters with potential for real-world clinical usage, we try to concentrate on "more comparable" parameters, such as MTT, where possible. Following the concepts of cerebral autoregulation laid out by Ayasse et al., a prolongation of MTT is indeed to be expected in patients with a decreased MAP, and we thank Ayasse et al. for contributing the valuable and clear explanations of the relationship between MAP, CBF, CBV, and MTT. Although both our title and the conclusion are arguably pointed, we feel that our discussion is a bit more nuanced and better explains our conclusions. Although CBV and CBF were not considered in our study, as explained previously, it has to be noted that at least some earlier observations have reported reduced CBF and constant CBV in CTP imaging in the EBI phase [3,4] and that other studies have described impaired autoregulation in the EBI phase [5]. Instead, we would like to draw the reader's attention to the increasingly stronger correlation between MAP and MTT as the patient's neurological status worsens, as measured by the World Federation of Neurosurgical Societies grade. We found it remarkable to demonstrate, for the first time, that in less vigilant patients (a direct indicator of the severity of EBI), the correlation between MAP and MTT becomes stronger. We assumed that the most probable cause for this observation was the impairment of cerebral autoregulation due to the severity of EBI. We appreciate that our thought process should have been laid out more carefully, and we are thankful to Ayasse et al. for providing this opportunity. We remain convinced that disturbances in cerebral autoregulation play an important role in the EBI phase in patients with aSAH, and further efforts should be made to understand these relationships, especially in patients with poor-grade aSAH. Finally, we would like to extend our gratitude to Ayasse et al. for contributing to the discussion with clear and constructive comments. We hope that our response clarifies the issues raised in the letter and provides additional insight into the complexity of this topic. Source of support Open Access funding enabled and organized by Projekt DEAL. None. Conflicts of interest None. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	2023-06-10T06:17:25.779Z	2023-06-08T00:00:00.000	{ "year": 2023, "sha1": "4edcee0f6a5e804df686ceade454445eccb4e331", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s12028-023-01759-5.pdf", "oa_status": "HYBRID", "pdf_src": "Springer", "pdf_hash": "66123457e3c6596c425f7e3d9d0ac28147dee134", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
54820854	pes2o/s2orc	v3-fos-license	ELDERLY VISUAL TASK PERFORMANCE BY DIFFERENCES ON READING MEDIA AND LAMP POSITION IN A ROOM This research aimed to find out how lighting influence visual task performance of the elderly.The experiment was done on the table with different lamp position and by giving mark on the landolt ring chart printed on different reading media. Three position, the front, the middle and the back were the variables for lamp position, whilst different reading medias were the brown paper usually used for newspaper, the white silk uncoated paper or „HVS‟ paper and the white gloss paper. The lamp were TL5 LED with 4000 °K and 6500 °K color temperature. The illumination level maintained specificly at 250-325 lux range. The respondents were 12 elderly with 58 years as the mean age. The result findings, for newspaper, the best position for speed was the front with 1.15 second per stroke; followed by the back with 1.18 second per stroke; and the middle got the lowest result with 1.4 second per stroke. For the HVS and glossy paper with higher luminance, the best for speed was the middle position that was the darkest in illumination, with 1.25 second per stroke for HVS and 1.3 second per stroke for glossy. The worst result was the back with 1.4 second per stroke for HVS and 1.36 second per stroke for glossy paper. For accuracy, the best for newspaper was the front, 96.02%. For the HVS paper, the middle and the back position has got the same highest accuracy, 90.45%. For the glossy paper, the middle position has got the highest accuracy, 91.62%. The visual task performance result, for the newspaper, the front position has got the highest score, followed by the back position that have brighter in illumination, but the score was lowest for the middle position. Contrary, for white HVS and white gloss paper, which get almost identic result, the middle position which was rather dark in illumination was the best, while the front and the back position were worse in visual task performance score. So that can be concluded that the higher illumination level was not always give the best to visual task performance; but depending to the user and the kind of paper or reading media as well. INTRODUCTION Nowadays we found the growth of elderly people in their productive age still.In offices and universities, elderly people with visual activities such as reading books, writing articles, even making technical drawing are usual.Beside their capability in such kind of visual activities, their eyes as the media are decreasing toward ages.The cornea is not clear, but yellowish.The eyes accomodation getting slower as well.Through the normal ageing process, even in the absence of cataracts (clouding the lens), the lens becomes less transparent, blocking additional light from entering the eye, and changing the color of the light that does get through (Sekuler & Sekuler, 2000;Sekuler, 2010).This condition actually increases functioning difficulties, in the older age.And disability became an important feature as the demographics of societies change and more people live to an old age (Hartley et al., 2011).According to this background, this research aimed to find out how lighting influence visual task performance of the elderly.This research study how lamp position, different reading media and lamp color temperature influence the visual task of the elderly.Findings from this research can give suggestion to architect, interior designer and building owner to give the best table to lamp position according to media uses, especially for elderly occupants. AGING AND VISION As people age, their vision is affected by changes both to their eyes and their brains.The average pupil size decreases with age, so that less light enters the eye, and this reduction in light is compounded by changes in the structure of the lens.Through the normal ageing process, even in the absence of cataracts (clouding the lens), the lens becomes less transparent, blocking additional light from entering the eye, and changing color of the light that does get through.Changes to the lens and intraocular media also increase light scatter, reducing the contrast of the retinal image.These structural changes to the lens also make it more difficult to focus on close objects as we age; this change in accomodative ability-the ability to change the optical power of the eye to focus on objects at different distances-is known as presbyopia, and is normal part of the ageing process (Sekuler & Sekuler, 2000;Sekuler, 2010).Fiorentini, Porciatti, Morrone & Burr (1996), found that ageing causes a small and unspecific decline of the response of the visual system to luminance and colour contrast.Another research of Facchin, Daini & Zavagno (2017), found that luminosity thresholds of GE (Glare Effect) for elderly was higher than those for young (this result was contrary from their original expectations) and it was suggesting a non-direct relationship between luminosity perception and discomfort glare.Another research on retail store lighting for elderly consumers found that older adults have more difficulty with warmer lighting when value contrasts were reduced.Besides, the older adults perceived the higher color temperature light source as less cool than younger adults did.(Park & Farr, 2007) EFFECTS OF LIGHTING ON VISION The effects of lighting on vision is the most obvious impact of light on humans.The stimulus presented to the visual system by any task can be described by five parameters: visual size, luminance contrast, color difference, retinal image quality and retinal illumination.These five parameters imply that it is the interaction between the object to be seen, the background against which it it seen, and the lighting both object and background determine the stimulus the object presents to the visual system.It is the stimulus and the operating state of the visual system that determine the level of visual performance achieved.This visual performance then contribute to task performance.It is important to point out that visual performance and task performance are not necessarily the same.Visual performance is the performance of the visual component of the task.Task performance is the performance of the complete task.Tasks in which visual performance is large or critical will be more sensitive to changes in lighting conditions than tasks where the visual component is small or unimportant (Boyce, 2011).The visual task here was reading the Landolt ring chart printed on different papers.According to Boyce (1981), many different features of printing can be expected to influence the ease of reading; for example, the typeface of the print, its quality, its contrast, its size and its layout are likely to be important.Grimm, Rassow, Wesemann, Saur & Hilz (1994), stated that initial attempts to standardize the determination of visual acuity date back to Snellen"s work in 1862.Subsequently, Green (1868) and Monoyer (1875) examined letters of different typefaces to evaluate their suitability for visual acuity estimation.Landolt recognized the necessity of a standard optotype that displays smaller differences in legibility than the different letters of the alphabet.His "circle interromptu" was accepted 21 years later as a standard optotype at the International Ophthalmological Congress in Naples.As the reference optotypes, the Landolt ring or Landolt "C" is the most widely accepted reference optotype for use in the vision testing laboratory.It is an interrupted circle whose stroke width and gap width are one fifth of its outer diameter.It major advantage is that it contains only one, easy measured, element of critical detail that represents the only difference between its various presentations, usually 4 or 8 orientations.(International Council of Ophthalmology, 1988).The advantages of the Landolt ring are: (1) a single characteristic feature in all eight directions of presentation; (2) low directional dependency in fully corrected patients; and (3) the negligible influence of shape recognition compared with letters or numerals.(Grimm, et. al., 1994) That"s why used here the Landolt ring, in order to hinder the typeface variable. ILLUMINANCE, LUMINANCE CONTRAST, GLARE, COLOR TEMPERATURE, AND LAMP POSITION RECOMMENDATION In working spaces in Indonesia, a uniform illuminance at 250-350 lux and a relative color temperature of daytime white light at 3300°K to >5300°K was recommended (Badan Standardisasi Nasional, 2001).A study of mid-scale office buildings in Japan found that for illuminance, a range between 200 to 800 lux, and for the color temperature, a range between 3000°K to 5500°K was preferred (Ono, 2012).A field survey on actual conditons of light environment in mid-scale office buildings in Japan found the highest percentage of frequency of the ceiling illumination usage, 91.3% always use ceiling illumination; 4.6% often use ceiling illumination; while only 10% of the office occupants used task lighting (Mochizuki & Koike, 2010).The CIBSE and IESNA recommend that luminance contrast between light sources and adjacent areas, and anywhere within the normal field of view should be less than 20:1 and 40:1 respectively (CIBSE, 1994).Other recommendations suggest that luminance contrast between task surface, immediate surrounds, and distant areas should be less than the ratios 1:3:10 (Arup, 2007 in Amirkhani, Garcia-Hansen, Isoardi & Allan, 2017).Creating the luminance contrast between 11:1 and 12:1 on the window wall in an office room with 45% window to exterior wall ratio using supplementary LED light can improve subjective assessments of window appearance; and significantly reduce discomfort glare from windows (Amirkhani, Hansen, Isoardi, & Allan, 2017).Huebner et al. (2016) suggest the manipulation of the ambient light color as a tool for energy-saving in buildings; while temperatures could be lower under a reddish/yellowish illumination in the heating season, or, conversely, be kept higher under bluish illumination in the cooling season.According to the lamp position theory, the parallel and perpendicular lamp position influence the visual comfort of the visual task on the table below.The parallel position created a severe glare problem, while the perpendicular was much more diserable and effective (McGuinnes, Reynolds & Stein, 1980).From the preliminary measurement found that the critical was the table position persistently below the lamp that tend to reflected glare, that"s why this position was researched further. METHODOLOGY This research has done by field measurement by doing visual task on the table.The visual task size was the Landolt ring for 40 cm distance (reading distance on the table) at 4 orientations.The outer ring diameter was 2.92 cm and the stroke and the gap was one-fifth of this dimension.Used here three kinds of printed paper as reading media that has been widely used as book-printed paper.They were white silk uncoated paper widely known as the "houtvrij schrijfpapier" (HVS), the gloss paper, and the newspaper.The reflectance factor of HVS paper was 76%, the gloss paper was 77%, and the newspaper was 55%.The luminance of the HVS paper was 10 cd/m2, the gloss paper was 12.2 cd/m2, while the newspaper was 4.6 cd/m2.The reading table was from dark brown teakwood shiny polished, 0.75m height.The respondent were 12 elderly with 58 year as the mean age, in their comfortable reading behaviour, with or without glasses.The test room was a hall with table to lamp position shown in figure 3. The wall as the room boundaries were far away and without exterior wall, in order to make sure that the lamp gives the most influence to the table.The ceiling height was 3.40 m, white paint smooth finished.The maintained illumination ranging from 315 lux (front position), 255 lux (middle position), to 325 lux (back position).The measurement done by three respondent at the same time.While seating, each respondent asked to read through the chart on each kind of paper and give mark/stroke in same way all the rings that have a gap orientated in a specified direction.They started at the same time as instructors given; and when one task paper was finished, they raised their hands and the time was recorded by the instructor.Then they done the same way to two other papers.Each respondent must do the visual task for all position; the front, the middle and the back position, but with different order.After they finished all the position, they filled into the questionnaire. RESULTS and DISCUSSION Result from this research reported in speed aspect, accuracy aspect, visual task performance, and the color temperature preference as well.Speed is given by the number of rings correctly marked divided by the total time taken.Speed is then stated in stroke/second.Accuracy is given by the number of rings correctly marked divided by the total number of rings that could have been marked.Speed and accuracy are then multiplied to form what is called the performance score.The color temperature preference got from the questionnaire. Speed vs Paper From figure 4, found that for doing task on the newspaper that was brown color and rather textured, the result for the fastest speed was at the front with 0.52 stroke/second, meant it"s needed 1.15 second per stroke; then the back was 0.51 stroke/second or needed 1.18 second per stroke; whereas the middle position was the lowest with 0.43 stroke/second, meant that it"s needed 1.4 second for doing one stroke.This reading media need more light to read, so that the front and the back position was more suitable, while the middle position as the darkest was not. For the white HVS paper, the fastest speed was at the middle with 0.48 stroke/second, meant it"s needed 1.25 second for doing one stroke; followed by the front with 0.46 stroke/second or 1.3 second per stroke, whereas the back position got 0.43 stroke/ second or 1.4 second per stroke.Since this reading media had high luminance but diffuse, so the bright condition at the back can brought to reflected glare.The front and the middle position was more suitable for this paper. For the white gloss paper that has highest luminance, the middle position was the fastest speed with 0.46 stroke/seconds or meant that 1.3 second needed for doing one stroke; followed by the front with 0.45 stroke/ second or 1.33 second per stroke; while the back get the longest time, 0.44 stroke/ second or needed 1.36 second per stroke.Found that this highest luminance paper got the lowest result.By the result we could analyze that this paper might reflect more light from lamps to many direction and different angles, than two other papers, so that this paper was the most difficult to read. Speed vs Position According to the position, the result shown that the front and the back position have averagely the same character in speed.Whereas the easiest to the most difficult paper were the newspaper, the white HVS and the white gloss paper.But at the middle position that was the darkest, the newspaper was the most difficult paper to read; while the white gloss was easier, and the white HVS was the easiest paper to read.From figure 5, accuracy for reading newspaper get the highest percentage at the front, 96.02% and the middle 90.04%.While at back the accuracy was the lowest, 86.08%. Accuracy vs Paper White HVS paper that has high luminance but diffuse get the lowest in accuracy at the front 87.02%.At the middle and the back, the accuracy got the same point, 90.45%. White gloss paper with highest luminance has got 87.43% accuracy at the front, 91.62% at the middle, and 90.09% at the back.By the result, found that white gloss paper accuracy was the highest at the middle. Accuracy vs Position Averagely, at all position, accuracy in reading newspaper was the highest, 90.71%, followed by white gloss paper with 89.71% accuracy, while white HVS paper has got the lowest in accuracy, 89.31%. CONCLUSION From the speed and accuracy toward different lamp position and different reading media, can be summarized the visual task performance score for the elderly as Table 1. This result shown that for the elderly, visual task performance score of the newspaper was highest at the front position, followed by the back position that have brighter in illumination, but the score was lowest for the middle position.Contrary, for white HVS and white gloss paper, which get almost identic result, the middle position which was rather dark in illumination was the best, while the front and the back position were worse in visual task performance score. From the questionnaire, the front position chosen best in illumination by 5 of 12 respondents.The back chosen by 2 of 12 respondents.Two from 12 respondent chose the middle as best position as well.Whereas 3 from 12 respondents felt all position had the same illumination.Also stated by 6 from 12 respondents that the middle was too dark, and the back was too bright (5 of 12 respondents). By this visual task test and user preference result, can be concluded that the higher illumination was not always give the best to visual task performance; but depending to the user and the kind of paper or reading media as well. For the color temperature, 5 of 12 respondents chose cool daylight as the best ambience; only 3 of 12 respondents chose the neutral white as their favorite ambience.Other respondents abstain or feel no differences regarding this. This research still need to be continued on different age respondents to give recommendation for the best visual acuity according to occupant"s activity with considering to contrast sensitivity of the table to room ambience.	2018-12-12T05:09:38.171Z	2018-07-31T00:00:00.000	{ "year": 2018, "sha1": "89b6499c5949c884e38aa780d55700f324ffd000", "oa_license": "CCBY", "oa_url": "https://dimensi.petra.ac.id/index.php/ars/article/download/20946/19439", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "89b6499c5949c884e38aa780d55700f324ffd000", "s2fieldsofstudy": [ "Psychology" ], "extfieldsofstudy": [ "Mathematics" ] }
244782738	pes2o/s2orc	v3-fos-license	Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters. Introduction P-glycoprotein (P-gp) is an ATP-dependent multi-drug transporter of the ATP-binding cassette (ABC) family that is involved in the active efflux of many drugs and xenobiotics from cells [1]. Overexpression or activation of P-gp can lead to multi-drug resistance in cancer cells [2][3][4][5]. Drugs and inhibitors are thought to bind to the inward-facing conformation of P-gp [6][7][8][9], which is more favored in the post hydrolytic or nucleotide-free conditions [10][11][12]. The drug is then translocated across the plasma membrane by transition of the protein to the outward-facing state, which is more favored when ATP is bound [12]. The dynamic conformational nature of P-gp (and other ABC family members [13]) has challenged structural studies. Nevertheless, P-gp structures have been obtained by X-ray crystallography [14][15][16][17][18] and cryo-electron microscopy (cryo-EM) [7][8][9][19][20][21]. Stabilizing antibodies and ATP hydrolysis-inactivating mutations have been exploited to generatẽ 4 Å resolution structural data by cryo-EM [7,9,19]. Similarly, trapping the protein in a posthydrolytic state with vanadate and ATP yielded an~8 Å resolution structure [21]. However, under these experimental conditions, allosteric changes could be altered or inhibited. Recent studies have demonstrated that the novel cystic fibrosis (CF) therapeutic ivacaftor is a competitive inhibitor or transported substrate of P-gp [22,23]. This drug is a potentiator of the channel activity of an P-gp homolog, the cystic fibrosis transmembrane conductance regulator CFTR/ABCC7 [24], and ivacaftor is now widely used in the clinic, both on its own as well as in combination with CFTR-corrector compounds such that roughly 90% of CF patients may now be treatable [25,26]. A prior structure for ivacaftor bound to CFTR showed the binding mode of the drug, which was on the external surface of the membrane-spanning region and within the hydrophobic region of the detergent micelle [27]. The drug itself is highly hydrophobic and was used at a relatively high concentration, but mutagenesis studies and experiments with another potentiator drug with a slightly different structure were undertaken to provide further evidence that the position of the drug in CFTR represented a common binding mode. A further interesting observation in the study was that there was minimal conformational change upon drug binding, and because of the rigid nature of the drug, conformational selection (of the drug) may not occur. Structure-activity relationships (SAR) of ivacaftor homologs with potentiating and inhibiting properties identified the quinoline nitrogen as crucial for activity via hydrogen bonding. π-π stacking with nearby residues by the same quinoline group was also found to be important. The planar nature of the drug was also shown to be a significant factor in the SAR study. The hydroxyl group of the hydroxyphenyl moiety is polar with a computed pK a of 11 (CHEMBL2010601) and points towards the guanidino group of R933 in the structural model [24,27], implying polar interactions that are too distant (6 Å) for hydrogen bonding. In this study, we examined the P-gp structure in the presence of ivacaftor. In order to minimize non-specific binding of the hydrophobic drug, we employed a concentration likely to be encountered in vivo [28] and at a sub-stoichiometric ratio with the protein. This approach had the advantage of allowing both drug-bound and drug-free forms of the protein to coexist as a mixture in the protein/drug solution immediately prior to flash freezing and cryo-electron microscopy. We reasoned that if drug binding caused a conformational change in P-gp, we should be able to distinguish different conformations using 3D classification of the particles. If no major change in the overall 3D structure occurred (as was the case for CFTR [27,29]), we expected to obtain a single 3D structure but with fractional occupancy of the drug at its binding site, leading to weak density for ivacaftor in the experimental 3D density map. A third possibility existed: that drug binding would cause conformational changes, but that discrimination of these different conformations would not be possible by the image processing routines. In this case, we would expect a smeared, low-resolution 3D map for the protein with no information about drug binding. In order to minimize the chances of the latter scenario, we employed the Volta phase plate to collect very high signal:noise data with minimal under focus. There are difficulties in the use of the Volta phase plate that restrict its current wide application [29], and there are currently only a few examples of studies resulting in resolution better than 3 Å [29]. Nevertheless, previous studies with 3D maps of resolution similar to those reported here have allowed drugs and small ligands such as sterols and nucleotides to be positioned and their binding interactions modeled within the protein of interest [30][31][32][33][34][35][36][37][38][39][40][41][42]. In this report, we aim to compare apo and drug-bound states within a single cryo-EM specimen. If successful, the approach we describe should minimize specimen preparation and biological variation as well as allowing relatively physiological levels of drugs to be employed. Materials and Methods P. pastoris harboring the opti-mdr3 gene was kindly provided by Prof. Ina L. Urbatsch, Texas Tech University [43]. Cell culture was performed using the shake-flask method described by Beaudet and co-workers with minor modifications [44]. Overexpression of the gene product (abcb1a) was initiated by the addition of methanol, and the expression was prolonged following an optimized procedure [21]. Cell pellets were collected and stored at −80 • C. Drug binding and P-gp thermostability were assayed in 3 ways using an UNCLE (Unchained laboratories) instrument: concentrated P-gp protein (1 µg per sample) was diluted into 10 µL of buffer (100 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol, 0.1% DDM and 0.02% CHS) containing different concentrations of ivacaftor. The experiments were also repeated with the further addition of 100 ng of CPM dye (7-Diethylamino-3-(4 -Maleimidylphenyl)-4-Methylcoumarin), which acts as a reporter of solvent-exposed cysteine residues [22,[45][46][47]. The mixtures were loaded into capillaries at 4 • C, and the latter were inserted into pre-chilled 16 well UNCLE capillary cassettes. Fluorescence emission spectra were recorded between 200 nm and 700 nm with an excitation wavelength of 266 nm. Static light scattering was recorded simultaneously using the 266 nm laser source. Data were gathered between 16 • C and 90 • C with a stepwise increase in the temperature (2 • C increments) followed by the recording of spectra for all the samples. The total heating run required approximately 70 min. Raw data were exported and then analyzed for tryptophan fluorescence, static light scattering, and CPM fluorescence using the GraphPad Prism software package. P-gp was initially buffer-exchanged into a detergent/glycerol-free buffer. The protein was diluted to 1.1 mg/mL and incubated with 2 µM Ivacaftor (stock: 100 µM in DMSO) for 30 min on ice. Quantifoil 200 or 400 Au grids with a 1.3-micron spacing pattern were pre-treated prior to protein deposition, as described previously [21]. Briefly, grids were surface-cleansed by multiple chloroform washes to eliminate hydrophobic residues, then glow-discharged. An FEI Vitrobot MkIV was employed to facilitate sample vitrification where 3 µL of protein was gently loaded onto the middle of the grid, immediately blotted for 6 s, and flash-frozen in liquid ethane. Grids were assessed for specimen quality (i.e., ice thickness, particle distribution, etc.) using a Polara G30 before shipping to the eBIC UK National facility for high-resolution data acquisition on an FEI Titan Krios G2 microscope. Data were recorded at 300 kV using a 20 mV energy filter and a Gatan K2 electron detector. Images were acquired via the Volta phase plate [48] with a phase shift range between 0.15 and 0.8 radian and fixed defocus at −0.5 µm. A total of~64 e − Å −2 were used over 40 frames spanning 10 s of exposure, and early and late frames were discarded. The magnification was calibrated at 1.043 Å/pixel. A total of 2436 movie stacks were collected, and beam-induced image shift was eliminated using MotionCorr2 [49]. The data processing of the 2436 images was entirely performed with cisTEM [50]. A total of 1030 images were eliminated for possessing poor CTF fitting profiles and exceeding defocus values. The final set of 1406 images were divided into 2 similarly-populated subsets (696i and 710i) to independently assess the reproducibility of the 2D classification procedure. Ab-initio 3D reconstruction was performed employing~50,000 particles derived from several 2D classification runs of the 696i subsets. For each of the 5 classes that were generated ab-initio, a PDB model was built using Molecular Dynamics Flexible Fitting (MDFF) with NAMD 2.12, as detailed previously [21]. These models were used as a measure for comparing NBD separation in the five classes. Ab-initio 3D maps were then low pass filtered to 10 Å and employed as starting templates for 3D refinement using the full final set of 104,000 selected particles. After full 3D refinement of the maps, real-space refinement of atomic models against the maps was performed within the PHENIX package [51], with the MDFF-derived models used as the initial inputs. Half-maps and Fourier shell correlation curves were generated employing the cisTEM and calculate_fsc routine. Local resolution was also estimated using the program ResMap [52]. Rigid-docking of ivacaftor was performed with the fit-in-map routine of UCSF Chimera [53] with a 5 Å-resolution simulated map of ivacaftor atoms at 0.252 (7 × SD) and with optimization for overlap with the experimental (additional) density in the P-gp central cavity. Fitting was subsequently , concomitantly allowing the rotamer adjustments of neighboring residues. The final result was verified to maintain the occupancy of the cryo-EM density. Nucleotide-binding pocket volumes of NBD1 and NBD2 were estimated by summation of all voxels with lower than mean density values that were within 5 Å of a positioned nucleotide. This was carried out with the matchmaker, color zone/split map, and calculate volume and area functions of Chimera [52] and using the NBDs in the 6q81 P-gp atomic model for positioning [21]. Ivacaftor Binding Affinity of Murine P-gp Ivacaftor has been identified as a competitive inhibitor of Hoechst 33,342 and digoxin transport by human P-gp [22,23]. Ivacaftor stimulated human P-gp ATPase activity eightfold, suggesting it could also be considered as a transported allocrite, and its dissociation constant (K D ) was estimated at 0.3 µM using a fluorescent dye transport assay with the purified protein [22]. Similarly, a half-maximal inhibitory concentration (IC 50 ) of 0.2 µM was estimated using an in-vivo assay [23]. We tested ivacaftor binding to murine P-gp using various methods: Static light scattering and tryptophan fluorescence changes yielded K D values of 0.2 µM (static light scattering) and ∼ =1 µM (tryptophan fluorescence) (Supplementary Figure S1a,b). The binding of a sulfhydryl-reactive fluorescent dye (7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methyl-coumarin, CPM) to solvent-exposed P-gp cysteine residues detected an unfolding transition between 30-50 • C that was sensitive to ivacaftor with a K D estimated at 0.2 µM (Supplementary Figure S1c). Hence, we concluded that the affinity for ivacaftor of murine P-gp was similar to human P-gp. Moreover, the detection of light scattering changes upon ivacaftor binding implied conformational changes in P-gp (e.g., due to an induced fit). Cryo-EM studies were subsequently performed with a concentration of ivacaftor (2 µM) that was well above the K D , although at this level it was still at a sub-stoichiometric ratio versus protein (estimated at 1.1 mg/mL, or roughly 8 µM using a molecular mass of 150kDa for P-gp). Cryo-EM of P-gp in the Presence of Ivacaftor Images (2436) were recorded with the Volta phase plate, and after initial rejection of poor quality images by eye, the contrast transfer function (CTF) of each was estimated. A total of 1537 images gave CTF fits that were judged to be reliable whilst 868 were rejected at this stage. A second criterion was applied to eliminate all images affected by excessive defocus values, and a total of 131 further images were eliminated, with a final set of 1406 images (Supplementary Figure S2). As expected, the P-gp particles were readily identifiable in the images in the inward-facing conformation (Supplementary Figure S3), and automated particle picking was performed. Reference-free classification of the projections of the resulting picked particles allowed a further pruning of the dataset by deleting any non-P-gp contaminants as well as small P-gp aggregates (Supplementary Figure S4). Several iterations of 2D classification were completed to remove bad particles, and this was judged to be complete when consistent 2D classes were obtained. The reproducibility of the 2D classification was then assessed by splitting the dataset into two roughly equally populated sub-sets, which individually showed the same outcome (Supplementary Figure S2). Ab-initio 3D classes of the final P-gp dataset were generated from a subset of onlỹ 50,000 particles, and these are shown in Supplementary Figure S5. At this early stage, the 3D classes were relatively low resolution (~7 to 8 Å, as estimated by Resmap [54]). Nevertheless, the two procedures clearly identified only inward-facing conformations, even when five classes were allowed. Sets of particles displaying a wider separation of the NBDs (classes a and c) were also picked out by the image processing package. For each of the five low-resolution ab-initio maps, a PDB model was derived using Molecular Dynamics Flexible Fitting (see Methods), and results were compared. Utilizing the center of mass of the NBDs as a reference, classes b, d, and e showed a narrower separation of the NBDs (from 56 Å to 53 Å), while both classes a and c were found to have a wider distance (60 Å). A moderate low-pass filter was applied to the ab-initio maps to avoid overfitting effects. CTF parameters were also further refined at this stage with per-micrograph CTF fits using the dedicated cisTEM functionality for this. Refinement of the full dataset (104,000 particles) starting with each of the five cisTEM ab-initio 3D classes was carried out with a combination of auto-and manual-refinement, and an atomic model was generated for each resulting refined 3D map using the PHENIX real space refinement routine [51]. The 3D maps, compared at a density threshold yielding a map volume of 153,000 Å 3 are displayed in Figure 1. the 3D classes were relatively low resolution (~7 to 8 Å , as estimated by Resmap [54]). Nevertheless, the two procedures clearly identified only inward-facing conformations, even when five classes were allowed. Sets of particles displaying a wider separation of the NBDs (classes a and c) were also picked out by the image processing package. For each of the five low-resolution ab-initio maps, a PDB model was derived using Molecular Dynamics Flexible Fitting (see Methods), and results were compared. Utilizing the center of mass of the NBDs as a reference, classes b, d, and e showed a narrower separation of the NBDs (from 56 Å to 53 Å ), while both classes a and c were found to have a wider distance (60 Å ). A moderate low-pass filter was applied to the ab-initio maps to avoid over-fitting effects. CTF parameters were also further refined at this stage with per-micrograph CTF fits using the dedicated cisTEM functionality for this. Refinement of the full dataset (104,000 particles) starting with each of the five cisTEM ab-initio 3D classes was carried out with a combination of auto-and manual-refinement, and an atomic model was generated for each resulting refined 3D map using the PHENIX real space refinement routine [51]. The 3D maps, compared at a density threshold yielding a map volume of 153,000 Å 3 are displayed in Figure 1. (3) Density for the N-terminus (rearwards, vertical arrow) and the end of the NBD1-NBD2 linker (slanted arrow, front) is present in map (a), whilst other maps lack the N-terminus density (maps (b,d)) or lack the linker density (c). (4) The density for the extracellular loop (ECL) between TM helices 1 and 2 is weaker for maps (c,e). Lower panel: slices through the transmembrane regions of the maps (as indicated by the red dashed lines in the upper panel). Real-space refined atomic models that were generated are also displayed using ribbon representation in contrasting colors. The diameter of the micelles in maps (a,c) are larger; map (e) has the smallest micelle with some distortion from circular. Map (a) contains a central density (blue mesh, arrow) not accounted for by the real-space refined model. Other maps have only small features in the aqueous central cavity. Map (e) shows the most compact transmembrane organization. The resolution of each map was assessed by splitting each data set in half and separately refining each half dataset. The correspondence between half-maps was analyzed in reciprocal space using Fourier shell correlation (FSC) as well as using the Resmap routine, which calculates local resolution in the maps [54,55]. The resolution assessments as well as other data pertaining to the five refined maps and the five atomic models, are summarised in Table 1. Each 3D map could be generated from fewer particles than in (3) Density for the N-terminus (rearwards, vertical arrow) and the end of the NBD1-NBD2 linker (slanted arrow, front) is present in map (a), whilst other maps lack the N-terminus density (maps (b,d)) or lack the linker density (c). (4) The density for the extracellular loop (ECL) between TM helices 1 and 2 is weaker for maps (c,e). Lower panel: slices through the transmembrane regions of the maps (as indicated by the red dashed lines in the upper panel). Real-space refined atomic models that were generated are also displayed using ribbon representation in contrasting colors. The diameter of the micelles in maps (a,c) are larger; map (e) has the smallest micelle with some distortion from circular. Map (a) contains a central density (blue mesh, arrow) not accounted for by the real-space refined model. Other maps have only small features in the aqueous central cavity. Map (e) shows the most compact transmembrane organization. The resolution of each map was assessed by splitting each data set in half and separately refining each half dataset. The correspondence between half-maps was analyzed in reciprocal space using Fourier shell correlation (FSC) as well as using the Resmap routine, which calculates local resolution in the maps [54,55]. The resolution assessments as well as other data pertaining to the five refined maps and the five atomic models, are summarised in Table 1. Each 3D map could be generated from fewer particles than in prior studies of P-gp by cryo-EM [7,19,21] due to the high contrast of individual particle projections produced with the Volta phase plate. However, the resolution achieved was estimated at 4-6 Å for all the maps (Table 1, Supplementary Figures S6-S8). Map resolution was not correlated with particle numbers, consistent with the idea that, for structural studies of unstabilized P-gp, intrinsic protein flexibility is likely to be a major factor in determining the resolution achievable. Interestingly, map a displayed the lowest resolution of all the maps in the various tests employed, although the overall map-to-model correlation was better (Table 1, Supplementary Figure S8). The Resmap analysis of map a, implied that variation between the half-maps was mainly confined to the periphery of the NBDs and the micelle (Supplementary Figure S6). Conversely, map e, with the closest approach of the NBDs, was estimated to have the highest resolution by the Resmap algorithm [54] and by Fourier shell correlation [55] (Supplementary Figure S7). In general, the map-to-model correlation was lower for the NBDs compared to the TMDs in all maps (Supplementary Figure S8). Table 1. Summary of all the data for reciprocal space resolution assessment of the five maps as well as the real-space correlation coefficient (CC) between the atomic model and the relevant map. Interpretation of the Differences between the 3D Maps The various 3D maps showed the typical hinging of the two halves of the molecule, giving differences in NBD separation, but it was apparent that NBD1 flexing relative to the other P-gp domains was also being differentiated by the 3D classification algorithm: NBD1 appeared to progressively rotate upwards towards the TMDs when one examines maps a through e; (Figure 1, upper panel). Separation of NBD1 from NBD2 was greatest for maps a and c; was narrower in maps b and d; whilst map e had the closest approach of the NBDs (Figure 1, upper panel). As previously reported [21], densities assigned to the N-terminus and to the end of the NBD1-NBD2 linker on the opposite side of the molecule can be observed in the maps, especially map a (Figure 1 upper panel). Maps b and d showed additional density for the linker but lacked density for the N-terminal region. In contrast, map c showed the latter but appeared to lack the linker density. The density for the extracellular loop (ECL) between TM helices 1 and 2 was weaker for maps c and e, perhaps representing increased disorder in this loop, which may be core glycosylated in the expression system used. These symmetry-breaking features of P-gp likely aid the image processing via discrimination of projections of alternative 180 • -rotated orientations of particles (Supplementary Figure S9). The transmembrane regions (Figure 1, lower panel) also showed some subtle differentiation between the five 3D maps. The diameter of the micelles in maps a and c are noticeably larger; whilst map e has the smallest micelle, displaying some distortion from a circular annulus. These differences may be a reflection of the degree of separation of the two halves of the protein, which is greatest for maps a and c and least for map e. The latter map shows the most compaction of the transmembrane α-helices. The transmembrane regions of all five maps (Figure 1, lower panel) displayed the large central aqueous cavity that connects to the cytoplasm as well as to the lipid bilayer (via the two lateral gaps formed between TM helices 4 and 6 and between 10 and 12). These lateral gaps are a common feature of type IV ABC transporters such as Sav1866, MsbA, and P-gp [56]. In P-gp the central aqueous cavity has been found to house the binding sites for transported drugs and inhibitors [7,14,16]. We, therefore, closely examined this cavity for evidence of ivacaftor binding in the five maps. The criteria we used were that: (i) any features due to ivacaftor should be visible at the same density level used for the protein (ii) that at this density level, the feature(s) should be roughly rod-shaped, and (iii) have a continuous volume equivalent in size to that expected for the drug. Finally, (iv) once fitted, the ivacaftor molecule should not have serious clashes with nearby residues in a fitted P-gp atomic model (i.e., none that could not be remedied with rotamer adjustment). Only map a contained a single feature that satisfied all these criteria (blue mesh, arrow, Figure 1, lower panel). All the other maps did show small features in the aqueous central cavity (Figure 1 lower panel), especially at lower density thresholds, but none of these met the first three criteria. The additional density in the aqueous cavity of map a has a rocket-shape with two fin-like protrusions at one end. At the opposite end to the protrusions, the density is close to TM6, and in the fitted atomic model, it was close to F339 and Q343 (Figure 2). In accordance with the last criterion, a single ivacaftor molecule could be fitted into this density, and the overall rocket shape favored a fit with the 2,4 tert-butyl groups of the ivacaftor molecule corresponding to the two 'fins' of the rocket-shaped density (CC = 0.960 vs. CC = 0.938 for the 180 • rotated version). The density is surrounded by residues contributed by TM helices 6, 10, and 12, and hence is positioned more in one lobe of the aqueous cavity ( Figure 1). Residues F339 and Q343 within TM6 in the refined atomic model are close enough to form π-π (F339) and H-bonding (Q343) interactions with the quinoline group of the modeled ivacaftor molecule (Figure 2), and the phenolic hydroxyl group of the drug could also form H-bonding interactions with E871 within TM10 in this model. Small residues G868 and G985 in TM helices 10 and 12, respectively, allowed the fitting of the bulky tert-butyl groups of ivacaftor without steric clashes with the protein. Although the cavity is aqueous, I864, M872, L875 in TM10, and A981 in TM12 form a hydrophobic patch on its surface that appears to be compatible with the positioning of the hydrophobic drug ( Figure 2). K185 on the cytoplasmic side of TM3 in the aqueous cavity is within~10 Å of the additional density. This is somewhat reminiscent of R933 in human CFTR [27]. density level, the feature(s) should be roughly rod-shaped, and (iii) have a continuous volume equivalent in size to that expected for the drug. Finally, (iv) once fitted, the ivacaftor molecule should not have serious clashes with nearby residues in a fitted P-gp atomic model (i.e., none that could not be remedied with rotamer adjustment). Only map a contained a single feature that satisfied all these criteria (blue mesh, arrow, Figure 1, lower panel). All the other maps did show small features in the aqueous central cavity (Figure 1 lower panel), especially at lower density thresholds, but none of these met the first three criteria. The additional density in the aqueous cavity of map a has a rocket-shape with two fin-like protrusions at one end. At the opposite end to the protrusions, the density is close to TM6, and in the fitted atomic model, it was close to F339 and Q343 (Figure 2). In accordance with the last criterion, a single ivacaftor molecule could be fitted into this density, and the overall rocket shape favored a fit with the 2,4 tert-butyl groups of the ivacaftor molecule corresponding to the two 'fins' of the rocket-shaped density (CC = 0.960 vs. CC = 0.938 for the 180° rotated version). The density is surrounded by residues contributed by TM helices 6, 10, and 12, and hence is positioned more in one lobe of the aqueous cavity (Figure 1). Residues F339 and Q343 within TM6 in the refined atomic model are close enough to form π-π (F339) and H-bonding (Q343) interactions with the quinoline group of the modeled ivacaftor molecule (Figure 2), and the phenolic hydroxyl group of the drug could also form H-bonding interactions with E871 within TM10 in this model. Small residues G868 and G985 in TM helices 10 and 12, respectively, allowed the fitting of the bulky tert-butyl groups of ivacaftor without steric clashes with the protein. Although the cavity is aqueous, I864, M872, L875 in TM10, and A981 in TM12 form a hydrophobic patch on its surface that appears to be compatible with the positioning of the hydrophobic drug ( Figure 2). K185 on the cytoplasmic side of TM3 in the aqueous cavity is within ~10 Å of the additional density. This is somewhat reminiscent of R933 in human CFTR [27]. The drug (iva) is modeled into the additional central density. The density is surrounded by residues in TM helices 6, 10, and 12 in the real-space refined atomic model. The 2,4 tert-butyl groups at one end of the ivacaftor molecule favored its orientation as shown within the rocket-shaped density. In this model, F339 and Q343 within TM6 could form π-π and H-bonding interactions with the ivacaftor quinoline group. Small (glycine) residues at 868 and 985 in TM helices 10 and 12 may be important for accommodating the bulky tert-butyl groups at the other end of the drug, and A981 may also satisfy hydro- Figure 2. Additional density: Magnified, orthogonal views of additional density not accounted for by the fitted model in map a (blue mesh). The drug (iva) is modeled into the additional central density. The density is surrounded by residues in TM helices 6, 10, and 12 in the real-space refined atomic model. The 2,4 tert-butyl groups at one end of the ivacaftor molecule favored its orientation as shown within the rocket-shaped density. In this model, F339 and Q343 within TM6 could form π-π and H-bonding interactions with the ivacaftor quinoline group. Small (glycine) residues at 868 and 985 in TM helices 10 and 12 may be important for accommodating the bulky tert-butyl groups at the other end of the drug, and A981 may also satisfy hydrophobicity requirements for these groups. Similarly, H-bonding with E871 by the phenolic hydroxyl group of ivacaftor may occur in this fitting. K185 in TM3 extends up into the central cavity to within 10 Å of this group. Conformational Changes Associated with the Additional Density Assuming the additional density in map a is due to drug, then its presence is associated with a wider separation of the two halves of P-gp, similar to observations for the co-crystal structures of murine P-glycoprotein with the marine pollutant and P-gp inhibitor BDE-100 [15,16,57]. Similarly, widening of the structure of the TmrAB heterodimeric peptide exporter was associated with substrate-binding [13]. We, therefore, examined whether other local changes in individual domains could be identified as being specific to map a versus the other four maps. In this line of conjecture, we assumed that maps b to e were representative of multiple conformations of the apo-state. When map a's atomic model was compared to the one most closely matching it: map d model (rmsd =1.17 Å for 1018 atom pairs within 2 Å, 1.46 Å overall 1182 atom pairs), a local bulging outwards of the transmembrane helices were apparent (see Figure 3), similar to that observed for TmrAB [13]. When we compared map a's model to that of map e (which was the most different-rmsd 1.40 Å over 519 atom pairs within 2 Å, 2.64 Å overall 1182 atom pairs), the outwards bulging of the TM helices upon ivacaftor accommodation was more noticeable. In both cases, TM helices 1, 2, 7, and 8 move most whilst helices 6 and 12 move little (Figure 3b,c), and the bulge of the TMDs resulted in a wider separation of the NBDs. As previously mentioned, NBD1 also showed a downward rotation away from the TMDs (Figure 3). Assuming the additional density in map a is due to drug, then its presence is a ciated with a wider separation of the two halves of P-gp, similar to observations for co-crystal structures of murine P-glycoprotein with the marine pollutant and P-gp in itor BDE-100 [15,16,57]. Similarly, widening of the structure of the TmrAB heterodim peptide exporter was associated with substrate-binding [13]. We, therefore, exam whether other local changes in individual domains could be identified as being specif map a versus the other four maps. In this line of conjecture, we assumed that maps b were representative of multiple conformations of the apo-state. When map a's ato model was compared to the one most closely matching it: map d model (rmsd =1.17 Å 1018 atom pairs within 2 Å , 1.46 Å overall 1182 atom pairs), a local bulging outward the transmembrane helices were apparent (see Figure 3), similar to that observed TmrAB [13]. When we compared map a's model to that of map e (which was the m different-rmsd 1.40 Å over 519 atom pairs within 2 Å , 2.64 Å overall 1182 atom pa the outwards bulging of the TM helices upon ivacaftor accommodation was more ticeable. In both cases, TM helices 1, 2, 7, and 8 move most whilst helices 6 and 12 m little (Figure 3b,c), and the bulge of the TMDs resulted in a wider separation of the NB As previously mentioned, NBD1 also showed a downward rotation away from the TM (Figure 3). . (b,c) outward bulge of the TMDs in the atomic model from map a (pink ribbon) vs. models from map e (green) and map d (blue). The additional density associated with ivacaftor in map a is shown (red mesh). Panel b is a section viewed along the membrane plane; panel c is a section viewed from the extracellular surface. (d) NBD1 and NBD2 in experimental maps a and d viewed from between the NBDs (blue and purple semi-transparent surfaces). The real-space refined atomic models for P-gp fitted to the maps are in red and blue, respectively. The nucleotide-binding cavity is indicated with the red mesh and its volume is given in cubic Ångstrom. The net result of this movement of the NBDs in the atomic models was a downward (cytoplasmic) shift of the Walker A and B regions relative to intracellular loops 1 and 4 that connect NBD1 from the TMD, and in map a this is manifested as an opening up of the nucleotide-binding site in NBD1 (Figure 3d). Similar changes but with a smaller magnitude, were also observed for NBD2 in the maps and atomic models. Caution must be exercised when extrapolating from atomic models that have been fitted to worse-thanatomic resolution maps, but if these conformational shifts in the NBDs are valid, then they may be reflective of allosteric changes that lead to increased ATPase activity of P-gp upon binding of a transport substrate. Discussion Structural data for 3D conformations of P-gp were obtained with the Volta phase plate; and with this methodology the resolution of the 3D information obtained was around 6-4 Ångstrom. Whether these resolution limitations can be overcome with a larger dataset remains to be established. The use of sub-stoichiometric levels of ligand may be a suitable approach in future cryo-EM studies, provided that ligand-protein complexes can be distinguished from ligand-free protein on the basis of different conformational status and/or by a sufficiently large additional density due to the ligand. Clearly, atomic details of ligand binding mode will depend on close to atomic resolution data, but the overall location of the ligand and some modeling of likely binding modes will be possible at intermediate resolution, as described here and in other studies for various other proteins and ligands [30][31][32][33][34][35][36][37][38][39][40][41][42]. Drug and inhibitor binding locations in P-gp have previously been studied for both mouse and human versions of the protein [8,9,15,16,58] as well as for a human/mouse chimeric construct [7]. For mouse P-gp, published structural data from X-ray crystallography is available for cyclic peptide inhibitors and for a marine pollutant BDE-100 that is also thought to act as an inhibitor of the protein [15,16]. For human P-gp, cryo-EM data are available for well-characterized substrates such as taxol [8,9] and vincristine [8] and also for some inhibitors such as elacridar, zosuquidar, and tariquidar [8]. Ivacaftor has been shown in-vitro to stimulate P-gp ATPase activity and to reduce transport by P-gp of Hoechst 33342 [22]. In humans, ivacaftor administration caused increased blood digoxin levels [23], implying inhibition of P-gp. These studies suggest that ivacaftor can compete with Hoechst 33342 transport and digoxin transport by P-gp, hence ivacaftor could be transported by P-gp. Alternatively, the data could be consistent with ivacaftor acting as a non-competitive or uncompetitive inhibitor of the protein's transport (but not ATPase) function. The Hoechst 33342 binding site (H-site) has been modeled to be asymmetrically-located in P-gp [59,60], but this position is in the opposite lobe of the internal cavity compared to the modeled site for digoxin (D-site) [60], and the density associated with ivacaftor described here. Hence from the data and models, one could propose that ivacaftor may compete for both digoxin binding and transport. Whereas for Hoechst 33342, transport, but not binding, would be affected by ivacaftor presence. These suggestions remain to be tested experimentally. A comparison of murine P-gp models both with and without bound compounds is shown in Figure 4, panels a-c. Their conformations are similar to the murine P-gp model for map a that is described here, with root mean squared deviation (rmsd) between the main chain Cα atoms of the models of around 4 Å over 1179 comparisons (i.e., not including disordered linker and N-and C-terminii atoms). When these various atomic models are superimposed (Figure 4, upper panels), the positions of the inhibitors and drug (ivacaftor) are all found within the central aqueous cavity. However, the BDE-100 and cyclic peptide inhibitors appear to be located further towards the extracellular side of the membrane. within the central cavity. As discussed by Nosol et al. [8], whilst the P-gp inhibitors display two separate molecules in the cavity, the transported substrates (and ivacaftor) display only one molecule in the cavity. Density associated with ivacaftor in map a is consistent with a single molecule, but again, in this comparison, it would be located closer to the cytoplasmic side of the membrane than the other compounds, with the exception of one of the elacridar molecules (orange stick representation, Figure 4). Curved arrows indicate the 90° rotations involved. (d-f): Human and human/mouse chimera P-gp: no drug (6fn4, yellow ribbon); with zosuquidar (6fn1, green ribbon, green drugs, and black drugs-7a6f); tariquidar (7a6e, pink drugs); vincristine (7a69, cyan drug); taxol (6qex, blue drug); elacridar (7a6c, orange drugs). Wider separation of the murine NBDs necessitated alignment of the N-and C-terminal arms of the model (this paper, purple ribbon, red drug) with the human P-gp structures. Where major differences exist between the paths of transmembrane helices in human and murine models, they are numbered in both purple and green font. Taken together, the data for ivacaftor is equivocal: the single density observed would imply that ivacaftor shares P-gp substrate characteristics, whilst its overall loca- (d-f): Human and human/mouse chimera P-gp: no drug (6fn4, yellow ribbon); with zosuquidar (6fn1, green ribbon, green drugs, and black drugs-7a6f); tariquidar (7a6e, pink drugs); vincristine (7a69, cyan drug); taxol (6qex, blue drug); elacridar (7a6c, orange drugs). Wider separation of the murine NBDs necessitated alignment of the N-and C-terminal arms of the model (this paper, purple ribbon, red drug) with the human P-gp structures. Where major differences exist between the paths of transmembrane helices in human and murine models, they are numbered in both purple and green font. A comparison of the mouse/ivacaftor P-gp model with human and human/mouse chimera P-gp models in the presence and absence of other small molecules is shown in Figure 4, panels d-f. Separate alignment of the two 'wings' of the inward-facing conformation of the mouse/ivacaftor structure was required for this comparison because the human and chimera P-gp models all show a closer approach of the NBDs. In these comparisons, both substrates and inhibitors co-localize to the same approximate position within the central cavity. As discussed by Nosol et al. [8], whilst the P-gp inhibitors display two separate molecules in the cavity, the transported substrates (and ivacaftor) display only one molecule in the cavity. Density associated with ivacaftor in map a is consistent with a single molecule, but again, in this comparison, it would be located closer to the cytoplasmic side of the membrane than the other compounds, with the exception of one of the elacridar molecules (orange stick representation, Figure 4). Taken together, the data for ivacaftor is equivocal: the single density observed would imply that ivacaftor shares P-gp substrate characteristics, whilst its overall location in one lobe and over towards the cytoplasmic leaflet of the bilayer is suggestive of binding characteristics of an inhibitor according to the work of Nosol et al. [8]. It seems possible that the low concentration/stoichiometry of the drug employed in this study may result in a single bound ivacaftor molecule at a higher affinity site, even if it were an inhibitor with two potential sites. However, it should be noted that not all inhibitors appear to bind in the same position: The cyclic peptide inhibitors and BDE-100 studied with murine P-gp occupied a location different from the elacridar, tariquidar, and zosuquidar inhibitors [14,15]. In addition, of note is that in the human P-gp models, transmembrane helices 4 and 10, were kinked inwards and appear to partially close off the internal cavity on the cytoplasmic side. This behavior has not been observed thus far in the murine P-gp models (Figure 4). The closest approach of the two halves of the human and chimera P-gp atomic models is also noteworthy, although this may result from different solubilization methods employed (lipids in the nanodiscs for human P-gp; detergent in micelles for murine and chimera P-gp). Whether these differences between human and murine P-gp structures represent interspecies variation, or instead alternative conformational states in the transport cycle remains to be established. At first sight, it would seem somewhat counterintuitive that drug binding to P-gp would induce a further separation of NBDs, rather than bringing them towards the NBD dimerized and outward-facing state. However, other studies have also detected similar conformational shifts (e.g., [13,15,16], including P-gp structures co-crystallized with BDE-100. We would propose that the opening of the NBD1 nucleotidebinding site may be a precursor to further conformational shifts induced by nucleotide that then promote the formation of the outward-facing state. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/membranes11120923/s1, Figure S1: Murine P-gp affinity for ivacaftor measured by thermostability changes; Figure S2: CTF correction and initial selection of images step-by-step workflow; Figure S3: Cryo-EM images initially visualized with the cisTEM software suite; Figure S4: Initial classification of the automatically picked particles; Figure S5: 3D classification of particles; Figure S6: Assessment of map a resolution; Figure S7: Different assessments of the resolution of all five maps; Figure S8: Comparison of model-to-map fits; Figure S9: Asymmetry in P-gp. Examples of major asymmetrical features that distinguish between the N-and C-terminal halves of the protein.	2021-12-02T16:23:22.446Z	2021-11-25T00:00:00.000	{ "year": 2021, "sha1": "e0188e1207eb8e5d7b44dcc404ddd1b8564879b5", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2077-0375/11/12/923/pdf", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "2c09499044a88b822a59c65376d2255461896a95", "s2fieldsofstudy": [ "Medicine", "Chemistry" ], "extfieldsofstudy": [ "Medicine" ] }
259730229	pes2o/s2orc	v3-fos-license	Exploration of the Regulatory Pathways and Key Genes Involved in the Response to Saline–Alkali Stress in Betula platyphylla via RNA-Seq Analysis The pH of saline–alkali soil is high because of carbonate salts, and the deleterious effects of saline–alkali soil on the growth of plants are greater than those of saline soil. Few studies have examined the saline–alkali tolerance of Betula platyphylla at the molecular level. To clarify the regulatory mechanism underlying saline–alkali tolerance in B. platyphylla, RNA sequencing analysis of B. platyphylla seedlings treated with NaHCO3 was conducted. Differences in gene expression in the roots of B. platyphylla seedlings under saline–alkali stress (induced via NaHCO3) for 3 h and 6 h were characterized, and a total of 595 and 607 alkali stress-responsive genes were identified, respectively. Most differentially expressed genes were involved in stress, signal transduction, secondary metabolic process, regulation of jasmonic acid, and the abiotic stimulus signaling pathway. The single nucleotide polymorphism loci in the differentially expressed genes were associated with the alkaline-salt tolerance in birch germplasm. In addition, birch plants overexpressing WRKY70 and NAC9 were obtained using the A. tumefaciens-mediated transient transformation method, and these two genes were found to play key roles in saline–alkali tolerance. Additional study revealed that WRKY70 and NAC9 can increase resistance to saline–alkali stress by enhancing reactive oxygen species scavenging and inhibiting cell death in birch plants. The results of this study enhance our understanding of the saline–alkali stress tolerance of B. platyphylla at the molecular level, and provide several key genes that could be used in the breeding of birch plants in the future. Introduction The total area of saline-alkali land globally has reached 954 million hectares, and the area of saline-alkali land is increasing by 10% annually [1]. Saline-alkali soil is mainly caused by sodium carbonate (Na 2 CO 3 and NaHCO 3 ), which increases the pH and Na + concentration, decreases the water potential, and results in drought [2][3][4][5]. Thus, land saline alkalization has become an increasingly serious problem, as it can decrease the osmotic potential of soil, disrupt ion homeostasis, disturb physiological processes, inhibit the growth and development of plants, lead to declines in quality and yield, and even result in the death of plants [6,7]. Therefore, study of the mechanisms underlying the responses of plants to saline-alkali stress will aid the use of biotechnology to breed plants with improved saline-alkali tolerance. The root tissues of plants are the main organ responsible for the perception of several types of water-limiting stress, such as salinity and alkali stress [8,9]. Furthermore, the sensitivity of the roots to stress often limits the growth and development of the entire plant. Thus, the roots provide an excellent organ for clarifying the molecular mechanism underlying the saline-alkali stress tolerance of plants. The mechanisms regulating stress resistance in plants involve the expression of multiple genes and the interactions among them, rather than individual genes [10,11]; thus, achieving a complete understanding of abiotic stress-tolerance mechanisms requires study of gene expression patterns. RNA sequencing (RNA-seq) analysis can rapidly provide information on gene expression and identify novel genes that mediate responses to various physiological processes in plants. Betula platyphylla is a common pioneer tree in eastern Asia that plays a key role in maintaining ecosystem stability and forest regeneration. It is also widely used in architecture, furniture, and paper production [12][13][14]. Various aspects of the saline-alkali tolerance of plants have been analyzed in previous studies. However, plant saline-alkali tolerance is complex and controlled by a cluster of genes; it also varies among plant species and cultivars and involves various physiological, biochemical, and metabolic pathways. Study of the saline-alkali resistance mechanism of B. platyphylla could provide new insights into its saline-alkali tolerance and have important practical implications. Here, transcriptomes of B. platyphylla roots that had been exposed to saline-alkali stress for different lengths of time were analyzed using high-throughput RNA-seq technology. The transcriptional pathways and some differentially expressed genes (DEGs) were identified, and the roles of key genes in saline-alkali stress tolerance were analyzed. Our findings provide valuable information that will aid future efforts to enhance the resistance of plants to saline-alkali stress via genetic engineering. Quality Control of the RNA-Seq Data To obtain an overview of the transcriptome of B. platyphylla roots in response to salinealkali stress, nine cDNA libraries were constructed using Illumina sequencing technology. These cDNA libraries were from B. platyphylla roots that had been exposed to 0.2 M NaHCO 3 for 3 h (N3-1, N3-2, and N3-3) and 6 h (N6-1, N6-2, and N6-3); the control plants (ck-1, ck-2, and ck-3) were treated with fresh water for 6 h (three biological replicates for each treatment). A total of 66.45 Gb clean data were obtained, with each sample having at least 6.22 Gb. The GC content of each sample was approximately 46%, and the Q30 ranged from 92.8% to 93.95% (Table S1). The clean reads of each sample were mapped against the B. platyphylla reference genome, with alignment rates ranging from 92.28% to 93.08% (Table S2). Overall, these results suggest that the sequencing data were of high quality. Identification of DEGs The DEGs were identified by RNA-seq analysis. A total of 595 and 607 DEGs were identified following exposure to NaHCO 3 stress for 3 h and 6 h, respectively (Table 1). A total of 290 and 305 up-regulated and down-regulated DEGs, respectively, were identified in the 3 h NaHCO 3 treatment vs. the control comparison group; a total of 199 and 408 up-regulated and down-regulated DEGs, respectively, were identified in the 6 h NaHCO 3 treatment vs. the control comparison group. We made a Venn diagram to analyze the overlap in DEGs across plants treated with NaHCO 3 for different lengths of time; the results revealed that 188 co-expressed genes were differentially expressed in the 3 h and 6 h treatment groups (Figure 1). GO Enrichment Analysis and Functional Annotation of DEGs To clarify differences in gene expression related to biological pathways, Gene Ontology (GO) annotations of the DEGs in the three categories of biological process, cellular component, and molecular function, were obtained ( Figure 2). In the biological process category, cellular, metabolic, and single−organism processes, response to stimulus, and signaling were highly enriched. In the cellular component category, membrane, membrane part, cell, cell part, and organelle were highly enriched. In the molecular function category, binding, catalytic activity, nucleic acid binding transcription factor (TF) activity, and transporter activity were highly enriched. GO Enrichment Analysis and Functional Annotation of DEGs To clarify differences in gene expression related to biological pathways, Gene Ontology (GO) annotations of the DEGs in the three categories of biological process, cellular component, and molecular function, were obtained ( Figure 2). In the biological process category, cellular, metabolic, and single-organism processes, response to stimulus, and signaling were highly enriched. In the cellular component category, membrane, membrane part, cell, cell part, and organelle were highly enriched. In the molecular function category, binding, catalytic activity, nucleic acid binding transcription factor (TF) activity, and transporter activity were highly enriched. GO Enrichment Analysis and Functional Annotation of DEGs To clarify differences in gene expression related to biological pathways, Gene Ontology (GO) annotations of the DEGs in the three categories of biological process, cellular component, and molecular function, were obtained ( Figure 2). In the biological process category, cellular, metabolic, and single−organism processes, response to stimulus, and signaling were highly enriched. In the cellular component category, membrane, membrane part, cell, cell part, and organelle were highly enriched. In the molecular function category, binding, catalytic activity, nucleic acid binding transcription factor (TF) activity, and transporter activity were highly enriched. The clusters of orthologous groups (COG) classification statistics for DEGs revealed that signal transduction mechanisms, general function prediction only, carbohydrate transport and metabolism, and secondary metabolite biosynthesis, transport, and catabolism were highly enriched ( Figure 3). The clusters of orthologous groups (COG) classification statistics for DEGs revealed that signal transduction mechanisms, general function prediction only, carbohydrate transport and metabolism, and secondary metabolite biosynthesis, transport, and catabolism were highly enriched ( Figure 3). Hierarchical Clustering Analysis of DEGs DEGs encoding CYP82C2, cinnamyl−alcohol dehydrogenase (CAD) genes, and DREB, WRKY, NAC, ERF, and bHLH TFs, were involved in response to stress, signal transduction, secondary metabolic process, regulation of jasmonic acid, and the abiotic stimulus signaling pathway (Table S3). We performed a hierarchical clustering analysis of the expression patterns of genes related to the saline-alkali signal pathway. The expression patterns of most genes were similar in the 3 h and 6 h NaHCO3 treatments and the control ( Figure 4). According to the hierarchical clustering analysis, the genes could be roughly classified into five groups. The first group included genes with up−regulated expression in the 3 h NaHCO3 treatment but down−regulated expression in the 6 h NaHCO3 treatment. Genes with slightly up−regulated expression in the 3 h NaHCO3 treatment and highly up−regulated expression in the 6 h NaHCO3 treatment comprised the second group. The third group included genes with down−regulated expression in the 3 h Na-HCO3 treatment and up−regulated expression in the 6 h NaHCO3 treatment. Genes with down−regulated expression in the 3 h and 6 h NaHCO3 treatments comprised the fourth group. The fifth group included genes with up−regulated expression in the 3 h and 6 h NaHCO3 treatments. Hierarchical Clustering Analysis of DEGs DEGs encoding CYP82C2, cinnamyl-alcohol dehydrogenase (CAD) genes, and DREB, WRKY, NAC, ERF, and bHLH TFs, were involved in response to stress, signal transduction, secondary metabolic process, regulation of jasmonic acid, and the abiotic stimulus signaling pathway (Table S3). We performed a hierarchical clustering analysis of the expression patterns of genes related to the saline-alkali signal pathway. The expression patterns of most genes were similar in the 3 h and 6 h NaHCO 3 treatments and the control ( Figure 4). According to the hierarchical clustering analysis, the genes could be roughly classified into five groups. The first group included genes with up-regulated expression in the 3 h NaHCO 3 treatment but down-regulated expression in the 6 h NaHCO 3 treatment. Genes with slightly up-regulated expression in the 3 h NaHCO 3 treatment and highly up-regulated expression in the 6 h NaHCO 3 treatment comprised the second group. The third group included genes with down-regulated expression in the 3 h NaHCO 3 treatment and up-regulated expression in the 6 h NaHCO 3 treatment. Genes with down-regulated expression in the 3 h and 6 h NaHCO 3 treatments comprised the fourth group. The fifth group included genes with up-regulated expression in the 3 h and 6 h NaHCO 3 treatments. SNP Analysis of RNA-Seq Data A total of 578,863 single nucleotide polymorphism (SNP) loci were obtained from the RNA-seq data mapped against the reference genome. A-to-G transitions were the most frequent, accounting for 30.77% of the total. C-to-G transversions accounted for only 7.39% of the SNP mutation types ( Figure 5A). There were 47,833, 60,567, and 64,727 SNPs located in the introns, upstream regions, and downstream regions of genes. Additionally, 31,196 synonymous coding and 27,791 nonsynonymous coding SNPs were detected ( Figure 5B). SNP Analysis of RNA−Seq Data A total of 578,863 single nucleotide polymorphism (SNP) loci were obtained from the RNA−seq data mapped against the reference genome. A−to−G transitions were the most frequent, accounting for 30.77% of the total. C−to−G transversions accounted for only 7.39% of the SNP mutation types ( Figure 5A). There were 47,833, 60,567, and 64,727 SNPs located in the introns, upstream regions, and downstream regions of genes. Additionally, 31,196 synonymous coding and 27,791 nonsynonymous coding SNPs were detected (Figure 5B). We next analyzed the number of SNP loci, the ratio of transition types, the ratio of transversion types, and the ratio of heterozygous SNP loci identified from NaHCO3 treatments for 3 h and 6 h. The total number of SNP loci ranged from 262,684 to 275,847; the total number of SNP loci in genic regions ranged from 231,583 to 241,463; and the total number of SNP loci in the intergenic regions ranged from 31,101 to 34,383. The ratio of transition types was 61.3%; the ratio of transversion types was 38.7%; and the ratio of heterozygous SNP loci was approximately 61% (Table 2). SNP Analysis of RNA−Seq Data A total of 578,863 single nucleotide polymorphism (SNP) loci were obtained from the RNA−seq data mapped against the reference genome. A−to−G transitions were the most frequent, accounting for 30.77% of the total. C−to−G transversions accounted for only 7.39% of the SNP mutation types ( Figure 5A). There were 47,833, 60,567, and 64,727 SNPs located in the introns, upstream regions, and downstream regions of genes. Additionally, 31,196 synonymous coding and 27,791 nonsynonymous coding SNPs were detected (Figure 5B). We next analyzed the number of SNP loci, the ratio of transition types, the ratio of transversion types, and the ratio of heterozygous SNP loci identified from NaHCO3 treatments for 3 h and 6 h. The total number of SNP loci ranged from 262,684 to 275,847; the total number of SNP loci in genic regions ranged from 231,583 to 241,463; and the total number of SNP loci in the intergenic regions ranged from 31,101 to 34,383. The ratio of transition types was 61.3%; the ratio of transversion types was 38.7%; and the ratio of heterozygous SNP loci was approximately 61% (Table 2). We next analyzed the number of SNP loci, the ratio of transition types, the ratio of transversion types, and the ratio of heterozygous SNP loci identified from NaHCO 3 treatments for 3 h and 6 h. The total number of SNP loci ranged from 262,684 to 275,847; the total number of SNP loci in genic regions ranged from 231,583 to 241,463; and the total number of SNP loci in the intergenic regions ranged from 31,101 to 34,383. The ratio of transition types was 61.3%; the ratio of transversion types was 38.7%; and the ratio of heterozygous SNP loci was approximately 61% (Table 2). Confirmation of RNA-Seq Data Using qRT-PCR Analysis To further evaluate the validity of the RNA-seq data, quantitative real-time reversetranscription PCR (qRT-PCR) analysis was conducted. Twelve DEGs were randomly selected for qRT-PCR. Our results revealed that the relative expression levels of these genes were consistent with the fragments per kilobase of exon per million fragments mapped (FPKM) values ( Figure 6A), and their correlation coefficient (R 2 ) was 0.97, demonstrating that the expression of these genes based on qRT-PCR was strongly positively related to the results of the RNA-seq analysis ( Figure 6B). Thus, the differential expression analysis revealed that the RNA-seq data were reliable. Confirmation of RNA−Seq Data Using qRT−PCR Analysis To further evaluate the validity of the RNA−seq data, quantitative real−time reverse−transcription PCR (qRT−PCR) analysis was conducted. Twelve DEGs were randomly selected for qRT−PCR. Our results revealed that the relative expression levels of these genes were consistent with the fragments per kilobase of exon per million fragments mapped (FPKM) values ( Figure 6A), and their correlation coefficient (R 2 ) was 0.97, demonstrating that the expression of these genes based on qRT−PCR was strongly positively related to the results of the RNA−seq analysis ( Figure 6B). Thus, the differential expression analysis revealed that the RNA−seq data were reliable. Oxidative Stress and Cell Membrane Damage Were Alleviated in Plants Overexpressing WRKY70 and NAC9 The expression of the WRKY70 and NAC9 TF genes was significantly up−regulated under NaHCO3 treatment compared with the control. Therefore, these two genes were subjected to additional analyses. Birch plants overexpressing WRKY70 and NAC9 were obtained using transient Agrobacterium−mediated transformation. qRT−PCR was conducted to analyze the expression levels of WRKY70 and NAC9. The relative expression of WRKY70 and NAC9 was significantly higher in WRKY70− and NAC9−overexpressing plants than in pROKII−35S plants (Figure 7). The results indicated that this transient transformation system is efficient for the transformation of genes. In addition, the relative expression of WRKY70 and NAC9 was approximately 300 times higher in WRKY70− and NAC9−overexpressing plants than in pROKII−35S plants under saline-alkali stress, suggesting that the expression of WRKY70 and NAC9 can be strongly induced by stress. Oxidative Stress and Cell Membrane Damage Were Alleviated in Plants Overexpressing WRKY70 and NAC9 The expression of the WRKY70 and NAC9 TF genes was significantly up-regulated under NaHCO 3 treatment compared with the control. Therefore, these two genes were subjected to additional analyses. Birch plants overexpressing WRKY70 and NAC9 were obtained using transient Agrobacterium-mediated transformation. qRT-PCR was conducted to analyze the expression levels of WRKY70 and NAC9. The relative expression of WRKY70 and NAC9 was significantly higher in WRKY70and NAC9-overexpressing plants than in pROKII-35S plants (Figure 7). The results indicated that this transient transformation system is efficient for the transformation of genes. In addition, the relative expression of WRKY70 and NAC9 was approximately 300 times higher in WRKY70and NAC9-overexpressing plants than in pROKII-35S plants under saline-alkali stress, suggesting that the expression of WRKY70 and NAC9 can be strongly induced by stress. Furthermore, 3, 3 -diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining was used to detect H 2 O 2 and O 2− levels to determine whether saline-alkali tolerance was strengthened in WRKY70and NAC9-overexpressing plants. Leaves from WRKY70and NAC9-overexpressing plants and pROKII-35S plants were stained with DAB or NBT; the stained leaves from water-treated plants were used as controls. Under saline-alkali treatment, the H 2 O 2 and O 2− levels in the leaves of WRKY70and NAC9-overexpressing plants were greatly reduced compared with those in pROKII-35S plants (Figure 8). The content of H 2 O 2 and O 2− is negatively related to the reactive oxygen species (ROS) scavenging ability of plants. Therefore, our results suggested that the ROS scavenging ability of WRKY70and NAC9-overexpressing plants was enhanced. In addition, Evans blue staining was used to detect cell membrane damage. Less intense blue staining of WRKY70and NAC9-overexpressing plants was observed, compared with that of pROKII-35S plants under saline-alkali treatment (Figure 8), indicating that cell death was reduced. Physiological Characterization of WRKY70-and NAC9-Overexpressing Plants In this study, superoxide dismutase (SOD) and peroxidase (POD) activities, H 2 O 2 content, and electrolyte leakage were used to assess the resistance of WRKY70and NAC9overexpressing plants to saline-alkali stress, as well as that of the pROKII-35S transformants ( Figure 9). Water treatment was used as a control. SOD and POD play important roles in the removal of ROS in plants under stress. Our results indicated that SOD and POD activities in WRKY70and NAC9-overexpressing plants were significantly higher in WRKY70and NAC9-overexpressing plants than in pROKII-35S plants under saline-alkali stress ( Figure 9A,B). The H 2 O 2 content was substantially down-regulated in WRKY70and NAC9-overexpressing plants, relative to that in pROKII-35S plants ( Figure 9C). Cell death was evaluated using the electrolyte leakage rate. Electrolyte leakage was lower in WRKY70and NAC9-overexpressing plants than in pROKII-35S plants under saline-alkali stress ( Figure 9D). These results indicate that WRKY70 and NAC9 could enhance the ROS scavenging ability and inhibit cell death in plants. portant roles in the removal of ROS in plants under stress. Our results indicated that SOD and POD activities in WRKY70− and NAC9−overexpressing plants were significantly higher in WRKY70− and NAC9−overexpressing plants than in pROKII−35S plants under saline-alkali stress ( Figure 9A, B). The H2O2 content was substantially down−regulated in WRKY70− and NAC9−overexpressing plants, relative to that in pROKII−35S plants ( Figure 9C). Cell death was evaluated using the electrolyte leakage rate. Electrolyte leakage was lower in WRKY70− and NAC9−overexpressing plants than in pROKII−35S plants under saline-alkali stress ( Figure 9D). These results indicate that WRKY70 and NAC9 could enhance the ROS scavenging ability and inhibit cell death in plants. Expression Changes of Target Genes in WRKY70− and NAC9−Overexpressing Plants To further analyze whether WRKY70 and NAC9 could affect the expression of target genes, the relative expression levels of SOD and POD in WRKY70− and NAC9−overexpressing plants and pROKII−35S plants under saline-alkali stress were examined. Under NaHCO3 treatment, the relative expression levels of SOD and POD were higher in WRKY70− and NAC9−overexpressing plants, relative to the control, than in pROKII−35S plants ( Figure 10). The results indicated that WRKY70 and NAC9 can regulate the expression of SOD and POD. Expression Changes of Target Genes in WRKY70-and NAC9-Overexpressing Plants To further analyze whether WRKY70 and NAC9 could affect the expression of target genes, the relative expression levels of SOD and POD in WRKY70and NAC9-overexpressing plants and pROKII-35S plants under saline-alkali stress were examined. Under NaHCO 3 treatment, the relative expression levels of SOD and POD were higher in WRKY70and NAC9-overexpressing plants, relative to the control, than in pROKII-35S plants ( Figure 10). The results indicated that WRKY70 and NAC9 can regulate the expression of SOD and POD. Discussion The main aim of this study was to achieve an improved understanding of the molecular mechanism of saline-alkali tolerance and identify some key genes and regulatory pathways that play key roles in the response to saline-alkali stress in B. platyphylla. Thus, we performed high−throughput sequencing and comparative RNA−seq analysis of B. platyphylla roots under saline-alkali stress (0.2 M NaHCO3). Plants activate a series of complex regulatory processes to mediate adaptation to various types of stresses. In our study, some GO terms related to cellular response to hypoxia were identified; for example, CYP82C2 DEG was significantly up−regulated under Na-HCO3 treatments for 3 h and 6 h compared with the control. A previous study has shown Discussion The main aim of this study was to achieve an improved understanding of the molecular mechanism of saline-alkali tolerance and identify some key genes and regulatory pathways that play key roles in the response to saline-alkali stress in B. platyphylla. Thus, we performed high-throughput sequencing and comparative RNA-seq analysis of B. platyphylla roots under saline-alkali stress (0.2 M NaHCO 3 ). Plants activate a series of complex regulatory processes to mediate adaptation to various types of stresses. In our study, some GO terms related to cellular response to hypoxia were identified; for example, CYP82C2 DEG was significantly up-regulated under NaHCO 3 treatments for 3 h and 6 h compared with the control. A previous study has shown that CYP82, a member of the cytochrome P450 family, is involved in the resistance of plants to biotic and abiotic stress [15]. The results of our study indicate that CYP82C2 plays a positive regulatory role in the root system of birch plants experiencing alkali-stress-induced hypoxia damage. Generally, secondary metabolites play a role in the defense response to stress [16]. In this study, some DEGs that are potentially involved in secondary metabolic processes were identified. For example, three CADs involved in lignin biosynthesis were identified (CAD1, CAD3, and CAD5). The expression of these three genes was mainly down-regulated. A previous study has shown that the expression of CAD genes is generally down-regulated under salt stress [8]. Our results are consistent with the results of this previous study. TFs play key roles in regulatory cascades and are essential for the regulation of gene expression; they also play important roles in regulating the acclimation response of plants to various types of stress. We identified some TF families from the RNA-seq data of birch, such as DREB, WRKY, NAC, ERF, and bHLH TFs, involved in stress, secondary metabolic processes, and the jasmonic acid signaling pathway. A previous study has shown that the expression of a DREB gene under a stress-inducible promoter enhanced the drought and frost tolerance of transgenic barley and the frost tolerance of transgenic wheat seedlings [17]. Another study has shown that the overexpression of RcDREB2B from Rosa chinensis enhances sensitivity to high salt concentrations, abscisic acid, and polyethylene glycol at the germination and post-germination stages in Arabidopsis, suggesting that RcDREB2B negatively regulates the abiotic stress resistance of plants [18]. In our study, no differences in the expression of genes encoding three DREB TFs in the 3 h NaHCO 3 treatment group and control were observed; however, the expression of these genes was down-regulated in the 6 h NaHCO 3 treatment, relative to the control. Thus, the results of our study suggest that DREB TFs are involved in complex regulatory pathways. Previous studies have shown that WRKY TFs can negatively or positively regulate abiotic stress tolerance in plants [19][20][21]. In our study, the expression of WRKY41 and WRKY46 was upregulated, but the up-regulation of the expression of these genes was not pronounced under NaHCO 3 treatment for 3 h; however, their expression was significantly down-regulated in the 6 h NaHCO 3 treatment. The expression of WRKY70 was significantly up-regulated under NaHCO 3 treatment for 3 h, but no significant differences in the expression of this gene were observed in the 6 h NaHCO 3 treatment. Thus, the three WRKY TFs might play multiple roles in the response to saline-alkali stress in birch plants. Some studies have shown that NAC, ERF, and bHLH TFs can respond to various types of abiotic stress in plants [22][23][24][25][26]. In our study, the expression of NAC9, ERF019, and bHLH162 was upregulated in the 3 h NaHCO 3 treatment but down-regulated in the 6 h NaHCO 3 treatment, relative to the control. However, the expression of ERF012 was down-regulated in both the 3 h and 6 h NaHCO 3 treatments. These findings indicate that NAC, ERF, and bHLH TFs might play diverse roles in the stress responses of birch plants. In our study, the expression of WRKY70 and NAC9 was substantially up-regulated in the 3 h NaHCO 3 treatment, relative to the control. Therefore, birch plants overexpressing these two genes were used to study their stress resistance. Plants are often exposed to various adverse conditions that can lead to the accumulation of ROS [27]. Therefore, ROS scavenging is important for the resistance of plants to various types of stress. Two major ROS species, H 2 O 2 and O 2− , are important molecules in cells that are involved in oxidative injuries and stress signaling [28]. In our study, DAB and NBT staining revealed that ROS accumulation was lower in WRKY70and NAC9-overexpressing plants than in pROKII-35S plants under saline-alkali treatment (Figure 7), and this was consistent with the observed H 2 O 2 levels ( Figure 8C). SOD and POD played key roles in the removal of ROS. SOD and POD activities were significantly higher in WRKY70and NAC9-overexpressing plants than in pROKII-35S plants under saline-alkali stress ( Figure 8A,B). Given that SOD and POD activities in WRKY70and NAC9-overexpressing plants were altered in response to saline-alkali stress, we measured the expression of SOD and POD. Under NaHCO 3 treatment, the expression of all SOD and POD genes was higher in WRKY70and NAC9overexpressing plants than in pROKII-35S plants (Figure 9). These findings suggest that WRKY70 and NAC9 enhance SOD and POD activities by altering the expression of SOD and POD genes. Overall, these results indicate that WRKY70 and NAC9 could enhance SOD and POD activities by regulating the expression of SOD and POD to enhance ROS scavenging. The results of the Evans blue staining (Figure 7) were consistent with the electrolyte leakage rates ( Figure 8D). The WRKY70and NAC9-overexpressing plants had significantly less intense Evans blue stain, indicating that electrolyte leakage rates were lower in WRKY70and NAC9-overexpressing plants compared with pROKII-35S plants. These findings suggest that WRKY70 and NAC9 reduced cell death to enhance resistance to saline-alkali stress in plants. Plant Growth and Stress Treatments Birch seeds were sown in plastic pots with a mixture of perlite/vermiculite/soil (1:1:3) and subjected to a 16 h/8 h light/dark cycle, at an average temperature of 25 • C, and 60-70% relative humidity in a greenhouse. The pots were watered twice daily until seed germination, after which they were watered once daily. After two months of cultivation, healthy birch seedlings approximately 20 cm in height were treated with 0.2 M NaHCO 3 for 3 h and 6 h. Specifically, the 3 h treatment was performed backwards, with 6 h being the final treatment time point. The control plants were treated with fresh water for 6 h. The roots of birch seedlings were collected. Three independent biological replicates were conducted, and each replicate comprised six seedlings. All samples were quickly frozen in liquid nitrogen and stored at −80 • C. RNA Extraction, Library Construction, and RNA-Seq RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) per the manufacturer's instructions. The concentration and purity of RNA were measured using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA). The integrity of RNA was evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The cDNA library was constructed per the manufacturer's instructions of the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, E7530) and NEB Next Multiplex Oligos for Illumina (NEB, E7500). Briefly, the enriched mRNA was fragmented into RNA inserts that were approximately 200 nt, which were used to synthesize first-strand cDNA and second-strand cDNA. The double-stranded cDNA was end-repaired, A-tailed, and ligated to adaptors. The appropriate fragments were isolated using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and enriched via PCR amplification. Finally, the constructed cDNA library was sequenced on a flow cell using an Illumina HiSeq™ sequencing platform. RNA-Seq Analysis Using Reference Genome-Based Read Mapping Low-quality reads, such as reads containing adaptors, reads with greater than 5% unknown nucleotides, or reads with Q20 values less than 20% (percentage of sequences with sequencing error rates less than 1%), were removed by a Perl script. The clean reads that were filtered from the raw reads were mapped to the B. platyphylla genome using Hisat2 tools. Duplicate sequences were removed from the aligned sequences in BAM/SAM format. Gene expression levels were measured using FPKM values. Analysis of DEGs Analysis of DEGs in B. platyphylla plants under control conditions and after 3 h and 6 h of NaHCO 3 treatment was conducted using DESeq2 [29]. The p-value threshold in multiple tests for determining the significance of differences was calculated using the false discovery rate (FDR) control method. Here, only genes with an absolute value of log 2 ratio ≥ 2 and a FDR significance score < 0.01 were used for subsequent analysis. Gene Functional Annotation Genes were compared, via BLASTX, against various protein databases, including the National Center for Biotechnology Information (NCBI) non-redundant protein (Nr) database, Kyoto Encyclopedia of Genes and Genomes, COG, and Swiss-Prot database with an E-value cutoff of 10 −5 . In addition, searches of genes were conducted against the NCBI non-redundant nucleotide sequence database, using BLASTn, with an E-value cutoff of 10 −5 . Genes were retrieved according to the best BLAST hit (highest score), along with their protein functional annotations. Gene Ontology (GO) annotations were obtained using the Nr BLAST results and the Blast2GO program [30]. All the annotated genes were mapped to the GO terms in the database, and the number of genes associated with each term was determined. A Perl script was used to plot the GO functional classification of the unigenes with their GO terms to visualize the distribution of gene functions. The obtained annotations were enriched and refined using TopGo (R package). The gene sequences were also aligned to the COG database to predict and classify functions. SNP Analysis SNPs are genetic markers caused by single nucleotide variation in the genome. SNP calling Picard tools v1.41 and SAMtools v0.1.18 were used to sort and remove duplicated reads, and merge the BAM alignment results of each sample. GATK2 software was used to identify single-base mismatches between the sequenced samples and the reference genome to identify potential SNP loci. Raw vcf files were filtered using the GATK standard filter method and other parameters (cluster Window Size: 10; MQ0 >= 4 and (MQ0/(1.0×DP)) > 0.1; QUAL < 10; QUAL < 30.0 or QD < 5.0 or HRun > 5), and only SNPs with distances less than 5 were retained. Quantitative Real-Time PCR (qRT-PCR) Verification Total RNA was reverse-transcribed into cDNA using a PrimeScript RT Master Mix kit (Takara Bio Inc. Kusatsu, Shiga, Japan). A total of 12 previously identified DEGs were randomly selected for qRT-PCR verification. The genes encoding ubiquitin (GenBank accession number: FG065618) and tubulin (GenBank accession number: FG067376) were used as the internal controls; all primer sequences are listed in Table S4. PCR reactions were performed in a volume of 20 µL with 10 µL of TB Green Premix Ex Taq; 2 µL of 0.5 µg/µL cDNA; 1 µL of 10 µM Primer-F; and 1 µL of 10 µM Primer-R. The thermal cycling conditions were as follows: 95 • C for 30 s; 95 • C for 20 s, 55 • C for 30 s, and 72 • C for 30 s for 44 cycles; and 60 • C for 15 s. The relative expression levels of the genes were calculated using the delta-delta CT method [31] to verify the results of the RNA-seq analysis. Three independent experiments were conducted. Plasmid Construct Containing TF Genes and Plant Transformation TFs can control the expression of various target genes and therefore play important roles in stress responses in plants. In this study, some genes encoding TFs were differentially expressed according to the RNA-seq data. We used two up-regulated DEGs encoding the TFs WRKY70 (GenBank accession number: OR052244) and NAC9 (GenBank accession number: OR052245) for further analysis. The cDNA sequences of WRKY70 and NAC9 were obtained from the birch genome. The open reading frames of WRKY70 and NAC9 were cloned using In-Fusion ligase (Novo-protein, Suzhou, China) into the pROKII vector digested with Sma I (Takara Bio Inc. Kusatsu, Shiga, Japan) under control of the CaMV 35S promoter. The primers and amplicon sizes are shown in Table S5. The pROKII-35S::gene construct and pROKII-35S empty vector were transformed into Agrobacterium EHA105 competent cells, and then into one-month-old birch plants, via the A. tumefaciens-mediated transient transformation method following the method described in a previous study [32]. Relative Expression and Stress Tolerance Analysis of WRKY70 and NAC9-Overexpressing Plants Birch plants overexpressing WRKY70 and NAC9 were treated with 0.2 M NaHCO 3 for 6 h. The pROKII-35S empty vector transformants were also treated with NaHCO 3 . Water treatment was used as a control. Total RNA from each sample was reverse-transcribed into cDNA. The relative expression levels of WRKY70 and NAC9 in plants overexpressing WRKY70 and NAC9 and pROKII-35S birch plants were determined under control and 0.2 M NaHCO 3 conditions using qRT-PCR. The primer sequences are listed in Table S4. The relative expression levels of the genes were determined using the 2 −∆∆Ct [31]. Three independent experiments were conducted. The detached leaves of birch plants were infiltrated with DAB (1.0 mg/mL) and NBT (0.5 mg/mL) according to a previously described method [28]. Evans blue (1.0 mg/mL) staining was performed to detect cell death following previously described procedures [33]. SOD and POD activities, H 2 O 2 content, and electrolyte leakage were measured following previously described procedures [34,35]. Three independent biological replicates were conducted. Total RNA of birch plants overexpressing WRKY70 and NAC9 and pROKII-35S birch plants was extracted and treated with DNase I before being reverse-transcribed into cDNA using a PrimeScript RT reagent Kit (Lab). Real-time PCR was conducted on six SOD and eight POD genes. The primer sequences are listed in Table S6. Statistical Analysis All statistical analyses were conducted using SPSS 22.0 software (IBM, Bloomington, IL, USA). Analysis of variance was used to determine the significance of differences between groups; the threshold for statistical significance was p < 0.05. Conclusions In sum, 595 and 607 alkali stress-responsive DEGs were identified in the roots of B. platyphylla following exposure to saline-alkali stress (NaHCO 3 treatment) for 3 h and 6 h, respectively. These genes were involved in stress, signal transduction, secondary metabolic process, regulation of jasmonic acid, abiotic stimulus signaling pathway, and TFs. The SNP loci in the DEGs were associated with alkali tolerance in birch. In addition, the overexpression of WRKY70 and NAC9 increased alkali stress tolerance in plants, suggesting that WRKY70 and NAC9 TFs play key roles in mediating resistance to saline-alkali stress in birch plants. The results of this study enhance our understanding of the stress tolerance of B. platyphylla at the molecular level, and provide new insights that have implications for studies of the saline-alkali stress tolerance of other plants. The two key genes WRKY70 and NAC9 could also be used in plant molecular breeding programs to generate germplasm resources with high saline-alkali tolerance in the future. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12132435/s1. Table S1: RNA-seq data statistics. Table S2: The alignment statistics of the sequencing data against the reference genome. Table S3: Statistics of 100 DEGs for characterizing the responses to NaHCO 3 treatment. Table S4: The primer sequences used for qRT-PCR analyses. Table S5: Primers used for the construction of birch plants overexpressing WRKY70 and NAC9. Table S6: Primer sequences of SOD and POD used in qRT-PCR analyses.	2023-07-12T08:22:29.481Z	2023-06-24T00:00:00.000	{ "year": 2023, "sha1": "ca6ad3df802a3af11a79bde5ce751fecb7826b30", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2223-7747/12/13/2435/pdf?version=1687750429", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "0a19a000bd1905bb73d9038ed190cf73b247bc09", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Medicine" ] }
203830567	pes2o/s2orc	v3-fos-license	Overexpression of CASC19 indicates poor prognosis and facilitates proliferation, migration and invasion in colorectal cancer Background Recent studies reveal lncRNAs are associated with tumor progression or metastasis in colorectal cancer (CRC). However, the functional roles of lncRNAs in colorectal liver metastases (CRLM) are still poorly understood. In this study, we performed an integrative analysis of long noncoding RNAs (lncRNAs) of CRLM in Chinese population. Methods We first applied RNA-seq and Short Time-series Expression Miner (STEM) to identify differentially expressed lncRNAs in normal tissues, tumor tissues and matched liver metastasis tissues in discovery cohort including four patients with CRLM, and subsequently differentially expressed lncRNAs were checked in the validation cohort consisting of 21 patients with CRLM by qRT-PCR. Then we analyzed the association between lncRNAs and overall survival (OS) in the third cohort with 108 CRC patients. Finally, we investigated the function of lncRNAs in HCT-116 and LoVo cell lines. Results RNA-seq and STEM analysis identified four lncRNAs (HL-5, HL-20, HL-38, and HL-59) according with the expression trend of N (normal tissues) < T (tumor tissues) < M (matched liver metastasis tissue), which may be associated with CRLM. Univariate analyses showed that high CASC19 expression was associated with poorer prognosis (P=0.013). CASC19 knockdown inhibited cell migration more than 50% in LoVo cells and HCT-116 cells (P<0.001), and significantly decreased cell invasion (P<0.001), as determined with the Matrigel assays. In contrast, cell invasion and migration were increased when CASC19 was overexpressed in HCT-116 and LoVo cells (P<0.001). Conclusions Our results showed that liver metastasis tissues and primary tissues of CRC had different expression profiles of lncRNAs. We suggest that CASC19 is a promising biomarker for CRC patients. Introduction CRC is the fifth most frequent cause of cancer deaths in China, with the incidence increasing continuously (1). Approximately 50% of patients with colorectal cancer develop colorectal liver metastases (CRLM), which are one of major causes of deaths (2). Though there were a lot of alterations in oncogenes and tumor suppressor genes in CRLM (3,4), the exact molecular mechanisms involved in CRLM pathogenesis remain unknown. The long noncoding RNAs (lncRNAs) are a kind of RNA molecules over 200 nucleotides, which could be transcribed but have no protein-coding capacity (5). Previous studies found that lncRNAs have a crucial role in carcinogenesis and distant metastasis through regulating gene expression (6)(7)(8). Aberrant lncRNA expression has been reported in different kinds of cancers, including colorectal cancers (9)(10)(11)(12), suggesting that lncRNAs may be associated with tumorigenesis and tumor progression. In colorectal cancer, a number of lncRNAs were associated with tumor progression or metastasis, such as HOTAIR (13). Nevertheless, for the functional roles of lncRNAs in CRLM, there is still few integrative analysis. LncRNA expression has higher tissue-specificity than protein-coding gene expression (14), which suggests that lncRNAs are potential biomarkers for CRLM. Therefore, identifying lncRNAs associated with CRLM might provide new insights into the pathogenesis of CRLM as well as potential biomarker candidates. In this work, comprehensive analysis of the long noncoding transcriptome of CRLM and patient-matched specimens were performed to identify differentially expressed lncRNAs in normal tissues, tumor tissues and matched liver metastasis tissues. Furthermore, we used Short Time-series Expression Miner (STEM) and assessed the tissue specificity of the lncRNA expression and its relationship with CRLM. Finally, we examined in vitro effects of one long non-coding RNAs, termed CASC19, on tumorigenesis and tumor progression. Patients and samples This study included analysis of a total of 291 fresh tissue specimens from three cohorts of CRC. Cohort 1 (discovery cohort) included 4 patients with CRLM, which encompassed 12 samples of primary colorectal adenocarcinoma tumor tissues (T), normal tissues (N) and matched liver metastasis tissues (M). Cohort 2 (validation cohort) enrolled 21 patients with CRLM, which was used to confirm the discovery by cohort 1. And a total of 108 CRC patients were enrolled into cohort 3 to determine relationship between prognosis and lncRNAs identified in cohort 1 and cohort 2. Written informed consent from each patient with CRC was obtained at the FUSCC. Cell culture We purchased the human embryonic kidney cell line 293T and the human CRC cell lines (HCT-116, LoVo, SW480, SW620, RKO, HT-29) from the Type Culture Collection of the Chinese Academy of Sciences. The HCT-116 and HT-29 cells, LoVo cells and 293T cells were respectively cultured in McCoy's 5A, F12K medium, and Dulbecco's modified Eagle medium, and were maintained at 37 ℃ containing 5% CO 2 . The other CRC cell lines were cultured in RPMI 1,640 medium. All media were supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin (Gibco). Permanent stocks of purchased cells were prepared, and all cells were stored in liquid nitrogen. Cells were used for experiments within 6 months. Total RNA extraction and cDNA synthesis Total RNA was extracted from fresh specimens or cells by TRIzol reagent (Invitrogen, Life Technologies), and RNA was quantified by a NanoDrop spectrophotometer. Then RNA (500 ng) was reverse-transcribed by the PrimeScript™ RT Master Mix Kit (Takara Biotechnology). We amplified cDNA through PCR by SYBR Premix DimerEraser (Perfect Real Time; Takara Biotechnology), and realtime quantification PCR was performed using the Applied Biosystems 7900HT Fast Real-Time PCR System (Life Technologies). Real-time quantitative reverse transcriptase polymerase chain reaction qRT-PCR was performed using the 7900 Real-Time PCR System with the SDS 2.3 software sequence detection system (Applied Biosystems). The relative abundance of target transcripts was evaluated utilizing 5 ng of cDNA and SYBR Green (Takara), and the results were normalized to the expression levels of ACTB using the 2 −ΔCt method; ΔCt is the difference of Ct values between the lncRNA of interest and the normalizer. For each sample, qRT-PCR was performed in duplicate and the mean value was used to calculate the expression levels. Normalized values were further log transformed and standardized. Vector construction The lncRNA CASC19 was amplified by polymerase chain reaction (PCR), and then the PCR products were digested and ligated into the pLVTHM vector to construct a CASC19-overexpression vector. The sequences of the primers and siRNA are listed in Table S1. Migration and invasion assays The invasion assay was carried out using a 24-well Millicell chamber containing a Matrigel-coated membrane. The migration assay was carried out in a similar fashion but without the Matrigel coating. Cells (5×10 5 cells in serumfree medium) were seeded in the top chamber. The bottom wells were filled with complete medium. Cells on the upper membrane surface were wiped off using a cotton swab, and the lower membrane surface was fixed with methanol, stained with 0.1% crystal violet, and cells were counted in five random fields (original magnification, ×200). Cell proliferation assay We assessed cell proliferation by Cell Counting Kit (CCK)-8 (Donjindo) to estimate cell viability. Treated cells (i.e., overexpression and knocked down cell lines) were seeded into 96-well plates at an initial density of 1×10 3 cells/ well. After 24, 48, 72, and 96 h of cultivation, CCK-8 solution was added to each well and incubated for 2 h. The absorbance was measured by scanning with a microplate reader (MRX; Dynex Technologies, West Sussex, UK) at 450 nm. STEM analysis The specific tissue expression profiles were determined by STEM analysis. The values of lncRNA expression of tumor tissue were represented as log ratios relative to the expression of normal tissue. Then the approach selects a set of predetermined temporal model profiles and determines the statistical significance of the number of genes assigned to each profile compared to the number of genes expected based on chance. Statistical analysis We performed all statistical analyses using SPSS software. Error bars in figures represent the standard deviation. Expression levels of each lncRNA were compared by the paired Mann-Whitney test. The clinicopathological characteristics were compared between the two groups using the Fisher exact test or χ 2 test. Statistical significance was determined using the Student t-test. OS curves were analyzed using the Kaplan-Meier method, with P value determined by a log-rank test. Hazard ratios (HRs) and 95.0% confidence intervals (CIs) were calculated. A P value of <0.05 was regarded as statistically significant. The long noncoding transcriptome profile of CRLM To investigate the association between CRLM and lncRNAs expression, we applied RNA-seq technique to analyze cohort 1, which encompassed 12 samples of primary colorectal adenocarcinoma tumor tissues (T), normal tissue (N) and matched liver metastasis tissue (M) from 4 patients with CRLM. We organized the lncRNAs according to their expression profiles in 3 different types of tissues. Figure 1A showed the overall lncRNA expression profiles. Distinct groups of lncRNAs were expressed in 3 different types of tissues. The sequencing data of 3 tissues were normalized and the specific tissue gene expression profiles were identified by STEM analysis. Seven lncRNA profiles of total 16 model profiles showed significant difference ( Figure 1B). The change of lncRNA expression in T, N and M tissues was represented by the black lines in the profile boxes. The profile number on the top corner of each profile box presented each model profile and the number on the bottom represented the adjusted p-value of lncRNAs expression across 3 different types of tissues. We focus on continuous up-regulation patterns in 3 types of tissues such as profiles 12, 13 and 15. Finally, we identified 63 lncRNA according with the trend of N < T < M. Identification of novel CRLM related lncRNAs We evaluated the 63 lncRNAs expression by qRT-PCR analysis in cohort 2, which included 21 patients with CRLM. The results validated the expression trend of N < T < M in 9 candidate lncRNAs ( Figure 2 Up-regulation of CASC19 is associated with CRLM and poor prognosis Then we detected the expression of these 4 lncRNAs in 108 colorectal cancer tissues using qRT-PCR, and analyzed the prognosis value of these lncRNAs. The three lncRNAs (HL-5, HL-20 and HL-38) had no associated with overall survival. However, high expression of HL-59 (CASC19) was associated with poor prognosis (P=0.013, Figure 3). Furthermore, the patients were divided into high (n=32) and low (n=76) CASC19 expression groups according to the receiver operating characteristic curve of CASC19 levels. Univariate analyses showed patients with high CASC19 expression had a poorer prognosis compared to patients with low CASC19 expression (P=0.013, Table 1). The CASC19 aberrant high expression level was significantly correlated with distant metastasis (P=0.026) and midhighly differentiated histological grade (P=0.018) ( Table 2). However, there was no relationship between CASC19 expression and other factors such as sex (male vs. female), age (≤60 vs. >60), and lymph node metastasis (N0 vs. N1 or above). Knockdown and overexpression of CASC19 influence proliferation, migration and invasion in vitro HCT-116 and LoVo cell lines were used to analyze the function of CASC19. Figure 4A show that knockdown (with siRNA) and overexpression (with transfection of pLVTHM-CASC19) of CASC19 in these cell lines were successfully constructed (P<0.001). The growth curves determined by CCK-8 assays indicated that knockdown of endogenous CASC19 expression dramatically reduced cell proliferation in both HCT-116 and LoVo cells, whereas CASC19 overexpression enhanced the proliferative capacity in both cell lines (P<0.001, Figure 4B). Knockdown of CASC19 expression significantly inhibited the colony formation of HCT-116 and LoVo cells, whereas overexpression of CASC19 had the opposite effect (P<0.001, Figure 4C), which were shown in colony formation assays. Therefore these results collectively suggested that overexpression of CASC19 could enhance CRC cell growth. CASC19 knockdown inhibited cell migration more than in LoVo cells and HCT-116 cells (P<0.001), and significantly decreased cell invasion (P<0.001), as determined with the Matrigel assays. In contrast, cell invasion and migration were increased when CASC19 was overexpressed in HCT-116 and LoVo cells (P<0.001, Figure 4D). These results indicated that overexpression of CASC19 could facilitate CRC cell migration and invasion in vitro. Discussion A mass of lncRNAs encoded by human genes are dynamically expressed at different developmental stages, show highly specific expression patterns across different tissues, and play key roles in diverse biological processes (15). At present, a large number of lncRNAs have been identified and demonstrated to be involved in various types of cancers (16,17). In recent years, the usage of high-throughput technologies provides an easier way to profile the long noncoding transcriptome. As far as we know our research is the first study to investigate the differences in the long noncoding transcriptome profile of primary colorectal cancer tissues, adjacent normal tissues and matched colorectal cancer liver metastasis using RNA-seq technique. The study identified several CRLM-associated lncRNAs, such as CCAT1, GAS5 and PVT1, which have been reported to be involved in tumor progression and biological pathways and some lncRNAs have not been previously reported. Furthermore, we used Short Time-series Expression Miner (STEM) and focused on the lncRNAs which exert the expression trend of N < T < M and might act as promoting effect on CRLM. We identified 63 lncRNAs according with the trend of N < T < M, and after validation in cohort 2, 5 lncRNAs were showed to be associated with CRLM. Then we evaluated the prognosis of these five lncRNAs in 108 CRC cohort, and high expression of CASC19 was correlated with poor overall survival of CRC. In the present study, our results revealed that the expression of CASC19 increased in CRC tissues and matched liver metastasis tissues. CRC with higher CASC19 expression tended to be associated with high-middle differentiation state and distant metastasis. What's more, survival analysis showed that CASC19 overexpression was significantly associated with poorer overall survival of CRC patients, which suggested CASC19 could serve as a potential biomarker for advanced CRC patients. The CCK-8 assays and Matrigel assays showed that overexpression of CASC19 could enhance CRC cell growth, migration and invasion. Overall, our results indicated that CASC19 may act as tumor promoter in CRC. In conclusion, liver metastasis tissues and primary tissues of CRC showed different expression profiles of lncRNAs, and we identified five lncRNAs associated with CRLM. We suggest that CASC19 could serve as a promising biomarker associated with poor prognosis and liver metastasis in advanced CRC patients.	2019-09-17T02:50:23.518Z	2019-08-01T00:00:00.000	{ "year": 2019, "sha1": "ff99973b9b23db8d2637d73f1af1da5d614fc268", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.21037/tcr.2019.07.17", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "16eefab52eba913153b52e2003fc18594e2ab497", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
103149336	pes2o/s2orc	v3-fos-license	The effect of electric field on multiple exciton generation in lead chalcogenide nanocrystals Unique properties of lead chalcogenides have enabled multiple exciton generation (MEG) in their nanocrystals that can be beneficial in enhancing the efficiency of the third generation solar cells. Although the intrinsic electric field plays an imperative role in a solar cell, its effect on the multiple exciton generation (MEG) has been overlooked, so far. Using EOM-CCSD as a many-body approach, we show that any electric field can affect the absorptivity spectra of the lead chalcogenide nanocrystals (Pb4Te4, Pb4Se4, and Pb4S4). The same electric field, however, has insignificant effects on the MEG quantum probabilities and the thresholds in these nanocrystals. Furthermore, simulations show that Pb4Te4, among the aforementioned nanocrystals, has the lowest MEG threshold and the strongest absorptivity peak that is located in the multi-excitation window, irrespective of the field strength, making it the most suitable candidate for MEG applications. Simulations also demonstrate that an electric field affects the MEG characteristics in the Pb4Te4 nanocrystal, in general, less than it perturbs MEG characteristics in Pb4Se4 and Pb4S4 nanocrystals. Our results can have a great impact in designing optoelectronic devices whose performance can be significantly influenced by MEG. I. INTRODUCTION Multiple exciton generation (MEG) in a semiconductor with the energy gap EG is an alternative term used for the absorption of a single photon with energy E ≥ 2EG that leads to the generation of more than one exciton. Among semiconductor nanocrystals, lead chalcogenides (PbX, with X= S, Se, Te) have attracted ample attention, due to their interesting features such as small energy gaps, large bulk excitons Bohr's radii, low resistivities, large carrier mobilities, high dielectric constants, and high melting points. [1][2][3][4] These unique properties have led to the detection of MEG in the PbX nanocrystals. 5 MEG is always in competition with other relaxation processes such as Auger recombination or phonon emission. In this respect, strong quantum confinement, phonon bottleneck 6 and relaxation of crystal momentum conservation are the main factors that accelerate MEG in nanocrystals. This conjecture was confirmed experimentally for PbX, 5,7-10 CdSe, [11][12][13] InAs, [14][15][16] Si, 17,18 and Ge 19 nanocrystals. During the past decade, some research groups have focused on the application of the multi excitons generated in the PbX nanocrystals specifically for enhancing the efficiency of the third generation solar cells. 7,10,20,21 Moreover, solar cells share one common feature under thermal equilibrium. That is the presence of the intrinsic electric field, due to their electronic band structures. Meanwhile, the effects of E fields on the electronic and optical properties of semiconductor nanocrystals have been investigated intensively, in recent years. [22][23][24][25][26][27] However, none of the MEG related studies reported in the literature, so far, have considered the effect of electric fields on the MEG characteristics. Lack of such studies together with the unique properties of the PbX nanocrystals have motivated us to start this numerical investigation. Here, we report the results of our investigations about the electronic structures of the excited states in Pb4X4 nanocrystals, using the equation of motion coupled cluster single and double (EOM-CCSD) 28,29 as a high-level ab initio approach, as implemented in the GAMESS-US program suite. 30 Although the computational method used in this work is general, we have solely considered the eight-atom Pb4X4 nanocrystals with cubic geometry. In fact, the limitations in the available computational resources and the high computational cost of the EOM-CCSD method have forced us to consider these relatively small nanocrystals and use a pseudopotential for the core electrons. To understand the physics behind our results we have also solved the problem using a two-step perturbative approach for low electric fields and compared the results with those obtained by the exact numerical solutions. Any of the following three mechanisms can describe the MEG in a semiconductor nanocrystal: [31][32][33] (i) Incoherent coulomb scattering, analogous to the impact ionization mechanism in a bulk; 34,35 (ii) Coherent superposition of the single and the multiple excitons; 36-38 (iii) The direct mechanism that is based on a multi-exciton state, strongly coupled to a virtual single exciton. Hence, multiple excitons can be generated instantaneously by an absorbed photon via a perturbative process. 39,40 This mechanism is independent of the phonon coupling. Ab initio numerical methods [41][42][43][44] and the ultrafast time scale MEG experiments 18,20 have supported this explanation, and our approach is also based on this mechanism. II. BACKGROUND THEORY The details of the ab initio computational method for studying the MEG process in the absence of an electric field (E = 0), using the EOM-CCSD method are reported in Ref (B3) with Eq. (A1) reveals that the presence of a finite electric field results in some allowed optical transitions between any given state, k, and the states other than k can contribute in the MEG quantum probability of the k-state. These interactions could reduce down the transition from the single exciton to the multi exciton generation. Moreover, an electric field also affects the dipole moment between an initial state ( i ψ ) and a final state ( f ψ ), modifying the related oscillator strength and hence the corresponding optical absorptivity. Employing the first order perturbation approach, the approximated closed form formulas for dipole moment, the oscillator strength, and the optical absorptivity in the presence of an electric field can be written as in Eq. (B5)-(B7). III. NUMERICAL SIMULATIONS To optimize the geometry of each nanocrystal, we have used the gradient corrected DFT calculations with the B3PW91 functional. Moreover, we have employed the def2-SVPD basis set for atoms, performing the related calculations with the GAMESS-US package. The resulting maximum bond lengths, d, in the given cubic nanocrystals are shown in Table I. To calculate the eigenstates and the related oscillator strengths, we considered the C2V point group with N = 200 for each symmetry, including all valence orbitals in the active space and using the LANL2DZ basis sets with pseudopotentials representing the core electrons. A. The unperturbed case ( The open squares (□) in Fig. 1(a)-(c) represent the MEG quantum probabilities obtained for the unperturbed Pb4Te4, Pb4Se4, and Pb4S4 nanocrystals, using the similar numerical method that we have reported in Ref 32. The solid curves represent the best fit to the scattered data, using the cubic spline approach with the smoothing parameter of 0.99. The MEG thresholds estimated from these data are given in Table I Unlike Si, 32 Ge, 32 and CdSe, 39 all three Pb4X4 nanocrystals benefit from the similar electronic excitation behavior, having a very fast transition from the single excitation to multi excitation regime with small fluctuations in their spectra. A comparison between the estimated MEG thresholds shows that the MEG process in Pb4Te4 nanocrystal is the strongest of all, indicating that the "degree of quantum confinement" for this particular nanocrystal is the strongest among the three. We define the "degree of quantum confinement" as b a d γ  , as a figure of merit, where ab is the corresponding exciton Bohr's radius as shown in Table I for the given nanocrystals. 45 The corresponding "degrees of quantum confinement" are also given in the same table. As can be observed from the data given in the table, the larger the "degree of quantum confinement," the stronger the overlap between the carriers' wave functions, and hence the stronger the carriers' interactions and the smaller the MEG threshold. The open circles in Fig. 2 Moreover, dashes, dots-dashes, and the thick solid curves represent the exact numerical data for = E 2×10 −3 , 8×10 −3 , and 15×10 −3 au. Furthermore, we observe that the general behavior of MEG quantum probability for a given electric field differs from one nanocrystal to other. To be more specific, at any given field the MEG quantum probability in Pb4Te4 is affected the least and that in Pb4S4 is affected the most. In other words, the nanocrystal with the largest "degree of quantum confinement" is influenced the least by the electric field and vice versa. As can be seen form Fig. 4, the effect of a given electric field on the oscillator strength at a given excitation energy can be different from those related to other excitation energies. This effect depends on the degree of the state degeneracy in the absence of an electric field and the strength of the interactions between that state and the others. This can redistribute the density of states and hence the oscillator strengths. The coresponding absorptivity spectra will also be affected, acordingly, as shown in Fig. 5. Comparison of the thin black solid curve (the unperturbed absorptivity spectrum) with other curves (the perturbed absorptivity spectra), for a particular nanocrystal, shows that any of the given electric field reduces the spectrum main peak. This can be attributed to the decrease in the overlap between the wave functions of the paired electron-hole in an exciton, due to the presence of the electric field, and hence weakening the corresponding dipole moment and the related oscillator strength. However, the trend of this reduction may not follow the field intensity as it increases. This can be seen in Fig. 5 by comparing the tiny dots, dashes, and dotsdashes representing the spectra for = E 1×10 −5 , 5×10 −5 , 1×10 −4 au, and those representing the absorptivities for = E 5×10 −4 , 2×10 −3 , 8×10 −3 , and 15×10 −3 au, illustrated by thick dots, dashes, and dots-dashes, and solid curves. One may attribute this to breaking the existing degeneracies and creating new eigenstates that can redistribute the density of states and the oscillator strengths ( Fig. 4) that may have a constructive or destructive effect on the absorptivity, for specific energy levels. Alterations of the main peaks of the absorptivity spectra for different field intensities observed in Fig. 5(b) and 5(c) are the manifestation of the redistributions in the density of states and oscillator strengths for Pb4Se4 ( Fig. 4(b)) and Pb4S4 (Fig. 4(c)). Moreover, comparison between Fig. 3 and Fig. 5 reveals that the absorptivity, unlike the MEG quantum probability, is reduced significantly even for = E 1×10 −5 au. For example, the main peaks of the absorptivity spectra in the multi excitation windows of Pb4Te4, Pb4Se4, and Pb4S4 are reduced by 22%, 38%, and 50%, respectively. This example also verifies that while Pb4Te4 is affected the least by an electric field, Pb4S4 is affected the most. Finally, we have evaluated the perturbed MEG characteristics, using the perturbatition technique described in Appendix B, for low electric fields ( E =1×10 −5 , 5×10 −5 , and 1×10 −4 au). The MEG quantum probabilities obtained by this approximate technique (Eq. B(3)) did not show any significant differences from data represented by the thin black solid curves and the tiny pink dots shown in Fig. 3 (the exact numerical data for the same field intensities). Nevertheless, the oscillator strengths and the absorptivity spectra approximated by Eq. (B6) and (B7) exhibit some deviations from the exact values shown in Fig. 4 and 5. As an example, here, we illustrate the comparison between the absorptivity spectra approximated by Eq. (B7) represented by the solid curves in Fig. 6 and the exact data for the represented by the dots in the same figure. The comparison reveals that the absorptivity spectra approximated by Eq. B (7) for E =5×10 −4 au (red curves) match those of the exact numerical calculations (red dots), with the maximum tolerance of less than 9%, for all three nanocrystals. Moreover, the maximum tolerance in the approximated spectra for Pb4Te4 perturbed by E =5×10 -5 au is still below 9%. However, the maximum tolerances experienced by similar spectra obtained for the other two nanocrystals, perturbed by the same field intensity, both exceed an endurable value. The deviations between approximated and exact spectra for all three nanocrystals perturbed by E =1×10 -4 au also exceed an acceptable tolerance. Hence, the perturbation method can be confidently used for Pb4Te4 nanocrystal perturbed by electric fields of E ≤ 5×10 -5 au, and for Pb4Se4 and Pb4S4 nanocrystals perturbed byE < 5×10 -5 au. FIG. 6. Comparison of low field absorptivity spectra obtained by the exact (dots) and perturbation approximation (solid curves) methods. IV. CONCLUSION We have analyzed the MEG processes in Pb4Te4, Pb4Se4, and Pb4S4 nanocrystals, using the EOM-CCSD framework. In this analysis, we have calculated the MEG quantum probability and spectra for the absorptivities in the absence and presence of various electric fields in the range of 1×10 −5 −15×10 −3 au, using the exact numerical calculations. Simulations for = E 0, have shown that Pb4Te4, with the smallest MEG threshold and strongest absorptivity peak, is the most desirable candidate among the lead chalcogenides for MEG applications. Moreover, the main portion of the absorptivity spectrum for Pb4Te4 resides in the multi excitation window. Furthermore, we have shown that the effects of the electric fields up to E =5×10 −4 au on the MEG quantum probabilities and the related thresholds are insignificant, for all three nanocrystals. Nonetheless, the presence of an electric field as small as E =1×10 −5 au can have significant effect on the absorptivity spectra, for all three nanocrystals. In general, a finite electric field lowers the entire absorptivity spectrum for each nanocrystal, as compared with that for = E 0. However, this may not follow a specific trend as the electric field is increased monotonically. Because, different field intensities may perturb various degenerate eigenstates differently, redistributing the density of states and the oscillator strengths that may have constructive or destructive effects on the absorptivity, for some energy levels. Observations also reveal that among the three lead chalcogenide nanocrystals Pb4Te4 with the highest "degree of quantum confinement" is the least affected one by the electric fields. The significance of the results becomes more appreciated when the goal is to design a MEG based solar cells in which the role of the intrinsic electric fields is imperative. where υ′ ∆ represents the full-width half-maximum (FWHM) of the Gaussian functions, in units of υ′ . APPENDIX B: THE PERTURBED MEG CHARACTERISTICS ( The corrected MEG quantum probability is the dimensionless dipole moment corresponding to various n-states, and 0 , ,ˆˆ, n S n D n R r R R =+ + in which ,S n R and ,D n R are the single and double excitation operators in the n th state. Using Eq. (B1), one can easily obtain the modified MEG quantum probability analogues to Eq. (A1), 2 2 , , , , 0 , , , N N a ab n k n i n k n ij i a n ij ab n k N N N a ab n k n k n i n k n ij n i a n ij ab n M r M r M r M r M r As can be seen from Eq. (B2), for n = k the corresponding dimensionless dipole moment becomes unity and, hence, Eq. (B3) reduces to Eq. (A1) -i.e., the MEG quantum probability for the unperturbed case. In other words, the terms representing the contributions from all the allowed transitions between the k-state and the n-states other than k, manifest the effect of the perturbing electric field on the MEG quantum probability of the k th state. The corrected oscillator strengths and absorptivity spectrum Using the corrected eigen function (1) k ψ and its Hermitian adjoint, i	2019-04-09T13:04:50.645Z	2017-08-09T00:00:00.000	{ "year": 2017, "sha1": "85777a6055b8537abf74711f832087853c70e67a", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "2b8823dbbfb2ae8557e68e768c54363dee0d3232", "s2fieldsofstudy": [ "Materials Science", "Physics" ], "extfieldsofstudy": [ "Physics", "Materials Science" ] }

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