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15381746 | pes2o/s2orc | v3-fos-license | Age-related differences in treatment strategies and clinical outcomes in unselected cohort of patients with ST-segment elevation myocardial infarction transferred for primary angioplasty
Data concerning the benefits and risks of primary PCI in the elderly patients presenting with ST-segment elevation myocardial infarction (STEMI) are limited. Thus, the objective of the study was to assess age-dependent differences in the treatment and outcomes of STEMI patients transferred for primary PCI. Data were gathered on 1,650 consecutive STEMI patients from hospital networks in seven countries of Europe from November 2005 to January 2007 (the EUROTRANSFER Registry population). Patients <65, 65 to 74, 75 to 84, and ≥85 years of age comprised 49.3, 27.5, 20.2, and 3 % of the registry population, respectively. Elderly patients were higher risk individuals and have experienced longer delays to reperfusion than their younger counterparts and were more likely to be treated conservatively after coronary angiography. Despite similar frequency of TIMI 3 flow before PCI, elderly patients were less likely to achieve TIMI 3 flow and ST-segment resolution >50 % after PCI, and were more likely to have PCI complications. The rates of death at 30 days, as well as at 1 year were increased with age. In the Cox regression analysis model age was an independent predictor of 30-day mortality. A trend toward higher risk of major bleeding requiring transfusion was observed. Age was an important determinant of treatment strategies selection and clinical outcomes in the group of consecutive STEMI patients transferred for primary PCI. Further efforts should be made to reduce delays and to optimize treatment of STEMI, regardless of patients’ age.
Background
Elderly patients presenting with ST-segment elevation myocardial infarction (STEMI) are less likely to receive reperfusion therapies, both fibrynolysis and primary percutaneous coronary intervention (PCI) [1][2][3]. Common reasons for excluding older patients from reperfusion therapy are their delayed presentation and atypical symptoms. Also, up to 9 % of elderly patients have absolute contraindication to fibrynolytic therapy [4]. Nowadays, primary PCI is the preferred method of reperfusion for STEMI, also in elderly patients [5]. It has been shown to be more effective than fibrynolysis in reduction of ischemic events in patients C75 years old with STEMI with chest pain \6 h [6]. However, primary PCI carries a decreased success rate and an increased procedural risk in older patients when compared with younger ones. Elderly STEMI patients are also at the higher risk of death or other adverse ischemic and non-ischemic events as result of higher prevalence of comorbidities.
Elderly patients with STEMI are often excluded from randomized clinical trials, thus it is hard to generalize expected outcomes from randomized clinical trials to the real life setting. More reliable data on treatment and outcomes of elderly patients with STEMI can be extracted from multicenter registries. The objective of the present study was to assess whether there exist age-dependent differences in the clinical characteristics, treatment strategies and clinical outcomes in patients with STEMI transferred for primary PCI based on data from the European Registry on Patients with ST-Elevation MI Transferred for Mechanical Reperfusion with a Special Focus on Upstream Use of Abciximab (EUROTRANSFER) Registry [7][8][9].
Methods
The details of the EUROTRANSFER Registry (Clinical-Trials.gov number NCT00378391) protocol and main results have been previously published [7][8][9]. In this registry data concerning treatment and outcomes of 1,650 consecutive, transferred STEMI patients in 15 STEMI hospital networks from 7 European countries between November 2005 and January 2007 were collected. For the purpose of this analysis patients were divided into four age groups (\65, 65-74, 75-84 and C85 years of age). The study protocol and execution complied with the Declaration of Helsinki and has been approved by the Institutional Review Board.
All-cause death, reinfarction and urgent revascularization (PCI or coronary artery bypass grafting) and bleeding complications: puncture site hematoma, intracranial hemorrhage, major bleeding requiring transfusion were evaluated during 30-day follow-up [7]. Additionally 1-year mortality was assessed [8]. Data concerning Thrombolysis In Myocardial Infarction (TIMI) flow in the infarct-related artery before and after PCI, ST-segment resolution after PCI, and rate of PCI complications (no-reflow, distal embolization, side branch occlusion, artery perforation) were also provided.
Data were analyzed according to the established standards of descriptive statistics. Results were presented as percentages of patients or medians (inter-quartile range). Differences in dichotomous variables were analyzed using Chi-square test and the Fisher's exact test as appropriate. Continuous variables were compared by the Kruskal-Wallis test. The difference in death rates between groups during follow-up period was assessed by the Kaplan-Meier method using the log-rank test. In addition, multivariate Cox regression analysis was performed to find significant predictors of 30-day death. Risk of 30-day death was expressed as hazard ratios with 95% confidence intervals. All tests were two-tailed and a p value of \0.05 was considered statistically significant. All statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL).
Results
Data on 1,650 patients were entered into the EURO-TRANSFER Registry database. Patients \65, 65-74, 75-84, and C85 years of age comprised 49.3, 27.5, 20.2, and 3.0 % of the registry population, respectively. Characteristics of patient population according to age are shown in Table 1. The prevalence of female gender, diabetes mellitus, previous myocardial infarction, previous heart failure symptoms, previous stroke, current smoking and chronic kidney disease, as well as body mass index and diastolic blood pressure changed across age groups. Data concerning pharmacological and interventional treatment are summarized in Table 2. Elderly patients has experienced longer delays to individual stages of treatment than their younger counterparts ( Table 2) and they were less likely to be treated with diagnosis-to-balloon time \90 min (for age \65, 65-74, 75-84, and C85 percentage of patients with diagnosis-to-balloon time \90 min was as follows: 40.8, 34.1, 35.1, 34.7 %, p = 0.079). There was no difference in the frequency of unfractionated heparin and abciximab use across age groups, both in pre-cathlab and in the cathlab setting. However, a trend toward less frequent administration of clopidogrel in older patients was observed (Table 2).
In the coronary angiography prevalence of multivessel disease increased with age. A total of 1,537 patients (93.2 % of study population) underwent immediate PCI. Elderly patients were more likely to be treated conservatively after coronary angiography and were less likely to receive stents, especially drug-eluting stents during immediate PCI ( Table 2). TIMI grade 3 flow frequency before PCI was similar among age groups, but elderly patients were less likely to achieve optimal epicardial flow (TIMI grade 3 flow) after PCI, and were more likely to have PCI complications than their younger counterparts ( Fig. 1; Table 2). Similarly, rate of ST-segment resolution [50 % after PCI has shown age-dependency. As shown in Fig. 2a, the rates of death, death ? reinfarction and all major adverse cardiovascular events at 30 days were increased with age. In contrast, incidences of reinfarction and urgent revascularization at 30 days were independent of age. The Kaplan-Meier curves for survival according to age are shown in Fig. 3. In Cox regression analysis, independent predictors of 30-day death were: age, diabetes mellitus, previous stroke, heart rate on admission, systolic blood pressure on admission, cardiogenic shock (Killip IV) on admission, diagnosisto-balloon time, stent implantation during PCI, drug-eluting stent implantation during PCI, non-infarct-related artery PCI, left anterior descending artery as infarct-related artery, TIMI grade 3 flow after PCI, ST-segment resolution [50 % after PCI and major bleeding requiring transfusion (Table 3). As clearly shown in Fig. 2b there were no differences in the occurrence of puncture site hematoma and intracranial hemorrhage (which did not occur in either group) across age groups. A trend toward higher risk of major bleeding requiring transfusion and significantly higher incidence of all bleeding complications in elderly patients (especially C85 years of age) were observed.
Discussion
Our study suggests that age is still an important determinant of treatment strategies selection, even in well-organized networks of STEMI treatment. Elderly patients are treated less aggressively in terms of antiplatelet therapy, have experienced longer delays to successful reperfusion, and they are at higher risk of death during follow-up.
In our study, similar to previous studies, and as expected, higher mortality rate was observed in older patients. There are many reasons that contribute to this higher mortality. One of potential explanations of the short-and long-term clinical outcome worsening in elderly STEMI patients is higher prevalence of comorbidities. In line with previous studies, elderly patients were more likely to have diabetes mellitus, previous myocardial infarction, previous heart failure symptoms, previous stroke, and chronic kidney disease [1,[10][11][12][13][14]. Older patients experienced longer time-delays to admission and primary PCI, and presenting more frequently with acute heart failure symptoms. Importantly, ischemia time is a major determinant of survival in patients with STEMI. Another important risk factor for higher mortality in patients with acute coronary syndromes is the presence of renal function impairment [15,16]. Preexisting impairment of renal function, diabetes mellitus and advanced age are also associated with increased risk of contrast induced nephropathy development after coronary angiography and primary PCI, which may led to worsening of long-term prognosis [17,18]. Elderly patients are also at higher risk of non-cardiac death during follow-up related to cancer or lung diseases. Similarly, to previous reports in our study observed frequency of reinfarction and need of repeated revascularization was comparable across age groups [1,10].
It is well established that in elderly patients primary PCI success rate is lower, with higher risk of angiographic complications than in younger counterparts [12][13][14]. In our study, the frequency of optimal epicardial flow (TIMI grade 3 flow) after primary PCI decreases with age. In Fig. 3 One-year Kaplan-Meier survival curves stratified by age [10]. The complex coronary anatomy observed in elderly patients may be associated with a higher incidence of distal embolization, which is a important determinant of myocardial perfusion after primary PCI, as well as long-term clinical outcome [13,14,19]. In addition, De Luca et al. have found a relationship between increased age and impaired myocardial perfusion assessed by myocardial blush grade, and ST-segment resolution. Importantly, age and poor myocardial perfusion were independently associated with 1-year mortality [12]. The higher prevalence of multivessel disease and the fear of complications among elderly may account for more frequent selection of initial conservative approach, with postponed PCI or coronary artery bypass grafting. Presence of multi-vessel disease in STEMI patients influences the clinical outcomes of patients treated with primary PCI [20]. Also, as confirmed by our study patients with advanced age are less likely to be treated with drug-eluting stents in STEMI setting [21].
In the analyzed patients population there was a trend toward higher rate of major bleeding requiring transfusion in patients with advanced age [10]. Importantly, major bleeding occurrence is a strong predictor of short-and longterm mortality [22][23][24][25][26]. Also, major bleeding may be associated with higher incidence of ischemic events, for example myocardial infarction, unplanned ischemic revascularization, and stent thrombosis [22]. An increased likelihood of the vascular access site complications (hematomas or aneurysm) in elderly patients is a result of the presence of calcified, fragile and bleeding prone vessels in these patients. In addition, in elderly patients frequently reduced kidney function is leading to overdosing of antithrombotic drugs, and to the increased risk of bleeding. Access site bleeding complications could be decreased by broad usage of radial approach, which was used in \15 % of patients in the EUROTRANSFER registry [27]. Safety and efficacy of transradial catheterization in the elderly is similar to observed in younger patients. Importantly, it may improve the comfort of the patients, especially in the context of the age-related diseases that frequently affect elderly patients [28]. However, advanced age was identified as an independent predictor of selection femoral over radial access by the operator during primary PCI in our registry [27]. Also, a fear of bleeding may limit the use of antiplatelet agents, especially glycoprotein IIb-IIIa inhibitors in patients C75 years of age [29]. Importantly, previous studies have shown that patients who present with an acute coronary syndrome and do not receive guideline-recommended therapies, including glycoprotein IIb-IIIa inhibitors experienced higher short-and long-term mortality [30][31][32].
Limitations of the study
The present study has a number of limitations. First, the study group is relatively small, and the very eldery patient subset (C85 years of age) comprised only 3 % of the study population. Also, the study focused mainly on 30-day clinical outcomes. Secondly, patients were not screened for contraindications to use of each medication and appropriateness of used dosage was not assessed. It is very likely that in some of patients various therapies were not used due to an important clinical reason. The registry was conducted between November 2005 and January 2007 when new P2Y12 inhibitors (prasugrel, ticagrelor) were not available. Also, the frequency of bivalirudin monotherapy was rather low, as it was recommended as alternative to unfractionated heparin and glycoprotein IIb-IIIa inhibitors combination recently [5]. Thus, the study did not cover most contemporary pharmacological treatment patterns for STEMI. On the other hand, since year 2007 there was no significant change in the recommendations concerning the application of primary PCI in STEMI setting [5]. Finally, the interpretation of the TIMI flow grade measurements, as well as ST-resolution was limited by the fact that these represent not independent core-lab, but physician's assessments.
Conclusions
Age was an important determinant of treatment strategies selection and clinical outcomes in the group of consecutive STEMI patients transferred for primary PCI. Further efforts should be made to reduce delays and to optimize treatment of STEMI, regardless of patients' age. | 2016-05-12T22:15:10.714Z | 2012-03-29T00:00:00.000 | {
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73555695 | pes2o/s2orc | v3-fos-license | Capabilities of helmets for preventing head injuries induced by ballistic impacts
The limiting performance of ballistically loaded helmets designed to reduce head injuries is studied analytically. The projectile does not penetrate the helmet. This analysis evaluates the absolute minimum of the peak displacement of the helmet shell relative to the head, provided that criteria measuring the severity of head injuries lie within prescribed limits. Rather than optimize a specific design configuration, e.g. a viscoelastic foam liner, characteristics of a time-dependent force representing the helmet liner are calculated. The formulation reduces the limiting performance analysis to an optimal control problem.
Introduction
Helmets are widely used to prevent impact-induced head injuries in traffic, industry, construction work, sports, and other areas of human activity.Typical examples are helmets for motorcyclists, bicycle riders, soldiers, and ice hockey players and boxers.The design of a helmet, including the selection of materials, depends primarily on the characteristics of the impact disturbance of the helmet's outer shell with a fixed obstacle (for instance, with a roadway surface when a bicycle or motorcycle rider falls) or with a projectile (for example, with a bullet during a military operation or a puck when playing ice hockey).On the one hand, a good helmet must have shock isolation properties that correspond to head injuries lying within tolerable limits.On the other hand, the helmet must be fairly compact and light to be convenient for workers, soldiers or sportsmen to perform their normal functions when no impact has occurred.These requirements contradict each other, thereby giving rise to the problem of optimal design of helmets for head injury prevention.
To calculate the optimal design variables of a helmet, it is necessary to have a mathematical model that adequately describes the mechanical response of a helmeted head to an impact of a given type, as well as a set of functionals characterizing the severity of injuries in terms of this mathematical model.Then the determination of the optimal design variables is reduced to an optimal control problem or a mathematical programming problem.At present, a number of different models are available that enable one to estimate the nature and severity of head injuries depending on the parameters of the impact and shock isolation properties of helmets.For years the debate on the fundamental cause of brain injury has been between linear and angular acceleration.Now both of these are being questioned and other causes, such as strain rate, are being considered [1].The shock isolation properties depend on the mechanical characteristics of the liner that isolates the head from the helmet's shell and serves to cushion impacts.The head/brain injury models tend to be complicated, especially when nonlinear finite-element models are used.The simpler models are represented by a small number of concentrated masses or rigid bodies connected by springs and dampers in various combinations.In these models, the bodies describe the inertial properties of the skull and brain, whereas the springs and dampers represent their viscoelastic properties.Numerical parameters of the models are identified on the basis of experimental data.In these simple models, the severity of injuries is characterized by such quantities as the maximum absolute value of the acceleration of individual bodies of the model, maximum deformation of the springs, and the power or work of elastic and damping forces.The maximum values allowed for these characteristics are determined on the basis of biomechanical and medical data enabling one to establish relationships between the mechanical characteristics of the head's response to an impact and the severity of injuries.A number of mathematical models, which do not take angular motion into account, widely utilized for investigating the head's response to impact loads are presented in [2][3][4].
A helmet for head injury prevention is in essence a shock isolator and, hence, its design fits into the conventional design scheme for such systems.The first stage of this scheme involves a limiting performance analysis to evaluate the absolute minimum of the performance index of the system to be designed irrespective of the specific hardware implementation.For the limiting performance analysis, the isolation device, the shock-isolating liner, and in some cases, the helmet itself, are treated as generators of a control force that acts between the base (a body to which the impact load is directly applied) and the object to be protected.The desired estimate of the performance index is determined as its greatest lower bound over all possible laws of change of the control force.Thus, the limiting performance analysis of shock isolation systems is reduced to an optimal control problem.The current state of the art in the theory of optimal shock and impact isolation is presented in [5].A special section of this book is devoted to the limiting performance analysis of impact isolation systems for the prevention of spinal and head injuries.The book contains a lengthy bibliography on the optimal protection from impact and shock and related optimal control problems.
In the present paper, we study the limiting capabilities of helmets for the prevention of head injuries induced by impacting a helmet with a non-penetrating projectile.As a model of the head's response to an impact, we utilize the translational head injury model proposed by Stalnaker and colleagues [2][3][4].The performance index to be minimized is the peak value of the post-impact displacement of the outer shell of the helmet with respect to the head (the rattlespace of the helmet).The injury criteria subject to constraints are the peak power of the forces caused by the skull bone deformation and the peak acceleration of the brain.The action of the impact-isolating liner is modeled by the control force acting between the helmet shell and the skull.Earlier, a similar study was performed for a helmet to prevent injuries induced by an impact of a helmeted head against a fixed obstacle [6].
Mathematical model of a helmeted head interacting with a non-penetrating projectile
A sketch illustrating a helmeted head impacted by a projectile is shown in Fig. 1.The helmet consists of a rigid shell and a relatively soft liner that isolates the head from the impact-induced shock disturbance thereby reducing the severity of possible injury.To describe the response of a head to a shock we will use a two-mass translational head injury model suggested by Stalnaker, Low, and Lin [2].In the case of a high-speed projectile, there is little reason to believe that angular motion will play an important role.This model involves two masses (m 1 and m 2 ), a damper c 2 , as well as a combination of a spring k and a damper c 1 connected in series.See Fig. 2. The mass m 1 represents the mass of the skull bone interacting with the helmet liner.The mass m 2 represents the mass of the brain and the remaining portion of the head that may move with respect to the skull bone.The stiffness coefficient k and the damping factor c 1 model primarily the elastic and dissipative properties of the skull, whereas the damping factor c 2 models primarily the dissipative properties of the brain.This model accounts only for the translational motion of the head.Its parameters should be adjusted depending on the direction of the impact.For more details, see [2].
The mass m 4 in Fig. 2 represents that of the helmet shell, the force f is due to interaction of the projectile with the shell, and the force u characterizes the interaction of the helmet liner with the skull bone.The force u depends on the design of the helmet.Consider the model of the interaction of the projectile with the helmet shell.Let m p be the mass of the projectile and v p its velocity when hitting the helmet.We assume that the impact is perfectly inelastic, i.e., that the projectile adheres to the shell after the impact.In this case, using the law of conservation of momentum to calculate the velocity of the helmet shell (mass m 4 ) with the projectile embedded in it we obtain This relation is valid if the impulse of the force applied by the liner to the shell during the impact is much less than the momentum of the impacting projectile, m p v p .
An impact-induced force acting on an impacted body is frequently represented as the half-sine pulse where F 0 is the amplitude of the force and T 0 is the duration of the impact.The amplitude F 0 can be expressed in terms of the quantities m p , m 4 , v p , and T 0 .Let x and y be the coordinates of the impacted body (helmet shell) and the projectile, respectively, in a fixed inertial reference frame.The motion of these bodies is governed by the equations with the initial conditions Solving this initial value problem yields Since at the time instant T 0 the projectile adheres to the helmet shell, we have ẋ(T 0 ) = ẏ(T 0 ).Using this condition and Eq. ( 6) we obtain the desired relation If the mass of the helmet shell considerably exceeds that of the projectile, one may use the simplified relation The distance moved by the helmet shell during the impact is measured by If the projectile rebounds from the shell, the right-hand sides of Eqs ( 1), ( 7), ( 8) and ( 9) should be multiplied by 1 + µ, where µ is the restitution coefficient of the impact velocity.To obtain these relations, one should replace the condition ẋ(T 0 ) = ẏ(T 0 ) at the end of the impact interaction process by the condition ẋ(T 0 ) = ẏ(T 0 ) + µv p .
If x(T 0 ) is much smaller than a characteristic length of the system, for example, the thickness of the liner, one can neglect the duration of the impact and utilize the model of an instantaneous impact with where V is the post impact velocity of the helmet shell defined by Eq. ( 1) and δ(t) is Dirac's delta function.The equations of motion of the system have the form where x 1 , x 2 , and x 4 are the coordinates of the bodies representing the skull bone, the brain, and the helmet shell, respectively; x 3 is the coordinate of the point at which the spring k is connected to the damper c 1 .All these coordinates are measured with respect to a fixed inertial reference frame.The coordinate system is chosen such that when all of the coordinates are equal to zero (x i = 0, i = 1, 2, 3, 4), the brain, the skull bone, and the helmet are not stressed.We subject these equations to zero initial conditions The initial value for the velocity ẋ3 is not prescribed in Eq. ( 15), because it is uniquely defined by the initial values of x 1 , x 3 , and ẋ2 in accordance with Eq. ( 13).The initial conditions of Eq. ( 15) correspond to the case where the system is in a state of rest and is unstressed at the time of the impact.
Head injury criteria
To evaluate the severity of head injury in terms of the model described in the previous section, we will use the following two functionals: where T is the time interval on which the motion of the system is considered.
Criterion J 1 measures the peak value of the power developed in the spring k during the interval [0, T ].It represents the power developed by the forces due to elastic deformation of the skull.There is a relationship between the value of J 1 and the probability of skull fracture [3].
Criterion J 2 measures the peak acceleration of the mass m 2 during the interval [0, T ].It can be regarded as the peak acceleration of the head, since m 2 is about 90% of the total mass of the head.This criterion characterizes the severity of brain concussion [3].
To evaluate and optimize the design of the helmet, we supplement the head injury criteria with the criterion measuring the peak displacement of the helmet shell relative to the skull This criterion, the so-called rattlespace, characterizes the thickness of the liner of the helmet.
Limiting performance analysis. Problem formulation
The main idea and purpose of the limiting performance analysis of a shock isolation system is to investigate the theoretical possibility that all performance criteria lie within admissible ranges, thus guaranteeing operational integrity of an object or survivability of a person to be protected.In this analysis, the configuration and engineering implementation of the isolation system is not taken into account, and the control force can be treated as a function of time.Frequently, the limiting performance analysis is treated as an optimal control problem aiming at minimizing one of the performance criteria, provided that the other criteria are subjected to prescribed constraints.This approach is especially appropriate if the constraint to be imposed on one of the criteria is not established reliably or this criterion is critical for the design.Consider the following formulation of the limiting performance problem.Problem 1.For the system governed by Eqs (11)-( 14) with the initial conditions of Eq. (15), find the greatest lower bound J 0 3 for the criterion J 3 , provided that the other criteria are constrained by prescribed constants, i.e., where D i are specified positive numbers.
Transformation of the equations of motion
Some transformations will be introduced to simplify the equations of motion in order to facilitate the limiting performance analysis.First, proceed from the coordinates (x 1 , x 2 , x 3 , x 4 ) to the new coordinates (z, y 1 , y 2 , y 3 , y 4 ) defined by The variable z is the coordinate of the center of mass of the system, and y i are the coordinates of the bodies of this system measured from the center of mass.In terms of the new variables, the equations of motion take the form where The initial conditions for the system of Eqs ( 21)-( 24) corresponding to Eq. ( 15) are given by Note that the system of Eqs ( 21)-( 24) does not involve the variable y 4 .Use Eq. ( 20) to express this variable in terms of y 1 , y 2 , and y 3 as Introduce the notation to reduce Eqs.( 21)-(24) to the system with the initial conditions The performance criteria of Eqs ( 16)-(28) represented in terms of the new variables take the form The structure of Eqs ( 29)-( 33) and ( 35)-(37) implies that for the limiting performance analysis, in which one is interested primarily in the values of the performance criteria rather than in the behavior of the control force, it suffices to use only three equations (Eqs (31)-( 33)) considering w as a new (auxiliary) control variable.Once the control function w(t) is known, the system of Eqs (31)-(33) can be solved to find y 2 (t), ξ(t), and η(t).Then, if necessary, one can determine the control force u(t) from Eq. (30).
To further simplify the governing relations, introduce the dimensionless (primed) variables where In what follows, the primes labelling the dimensionless variables are omitted.The nondimensionalization reduces Eqs (31)-(33) to the form where The performance criteria become For Problem 1 formulated in terms of the dimensionless variables, we have D 1 = 1 and D 3 = 1 in Eq. (19).
Simplified optimal control problem
In addition to Problem 1, consider a simplified problem in which the performance index J 3 of Eq. ( 46) is replaced by Problem 2. For the system of Eqs (40)-(42) subjected to the initial conditions of Eq. (34), find the greatest lower bound of the performance index J3 under the following constraints With reference to Eq. ( 41), the constraint of Eq. ( 49) can be rewritten as Note that when solving Problem 2, Eq. ( 42) can not be taken into account.This is the case because the performance index and constraints of this problem involve only the variables y 2 , ξ and the control function w.For given control function w(t), the variables y 2 and ξ are completely and uniquely defined by Eqs (40) and (41) subject to the respective initial conditions.
The error of using J3 to approximate the criterion J 3 can be estimated as This estimate is based on an upper estimate of the variable η and the familiar inequalities From Eq. ( 52) it follows that and, hence, The solution of the system of Eqs ( 41) and ( 42) with zero initial conditions yields The last relation and the constraint of Eq. (48) imply the inequalities By substituting the notation of Eqs ( 46) and (47) into Eq.( 54) and using the inequality of Eq. ( 57) we obtain the estimate of Eq. ( 51).
Note that this estimate depends on the time T during which the motion is considered.The estimate is simplified and becomes independent of T in the case of α = β.In this case, Solve Problem 2 for the case of the instantaneous impact where the impact force is defined as with V given by Eq. ( 1).The minus sign on the right-hand side was taken for convenience and does not influence the final solution of the problem.In terms of the dimensionless variables, In this case, Eq. ( 40) with the initial conditions y 2 (0) = 0 and ẏ2 (0) = 0 is equivalent to the equation with the initial conditions
Lower estimate for the performance index in Problem 2
To obtain a lower estimate for the performance index we will solve Problem 2 on the time interval 0 t T where T is defined as the first instant at which the velocity of body m 2 vanishes, i.e, ẏ2 (T ) = 0.The time T is not prescribed beforehand but is to be determined during the solution.
The optimal control in this case takes on the maximum value allowed by the constraints of Eqs ( 48) and ( 50) at each instant.The control variable, being represented as a function of ξ, has the form To prove the optimality of this control, represent Eq. ( 41) in the form Let ξ w (t) be the solution of the differential equation of Eq. ( 64) subject to the initial condition ξ(0) = 0 for the admissible control w.Let w 0 (ξ) be the control defined by Eq. (63).By construction, w 0 (ξ) is the maximum value of the control variable admissible for the given ξ.Hence, for any admissible control w, the inequality f w0 (ξ) f w (ξ) holds.Therefore, in accordance with Chaplygin's theorem on differential inequalities [7,8], By substituting the left-hand side of Eq. ( 41) for w into Eq.( 61) we obtain ÿ 2 = − ξ − αξ.Integrate this relation over the interval [0, t] with the initial conditions of Eq. (34) for ξ and Eq.(62) for y 2 to obtain From Eqs (65) and (66) we conclude that ẏ(w0) where y (w0) 2 (t) and y (w) 2 (t) are the solutions of Eq. ( 61) for the control laws w 0 and w, respectively.Integration of the inequality of Eq. (67) from 0 to t with the initial conditions y To determine the minimal value of the criterion J3 one should define the functions ẏ2 (ξ) and y 2 (ξ) by integrating the system (obtained from the system of Eqs ( 61) and (41) by utilizing the independent variable ξ) with the control of Eq. ( 63) and the initial conditions Then solve the equation ẏ2 (ξ) = 0 to find ξ * .Finally, calculate J3 = y 2 (ξ * ).
The solution of Eq. ( 68) for α 1/4 is given by where For α 1/4, the solution has the form In this case, ξ * and J3 are expressed by the simple relations In the case of α < 1/4, these quantities have to be calculated numerically.
If ξ * α −1/2 , then the value J3 = y 2 (ξ * ) is attained.For example, this is the case for the control coinciding with that of Eq. ( 63) for 0 t T and being identically zero for t > T .It is apparent that such a control does not violate the constraint of Eq. ( 48).It will be shown that this control also does not violate the constraint of Eq. ( 50).On the interval 0 t T , this constraint holds in accordance with the construction of the control of Eq. (63).Integrate Eq. (41) on the interval T t < ∞ for w = 0 and ξ(T ) = ξ * to obtain ξ = ξ * e −α(t−T ) . (75) Then for the left-hand side of the inequality of Eq. (50), Therefore, the inequality of Eq. (50) holds for ξ * α −1/2 .If ξ * > α −1/2 , the constraint of Eq. ( 50) is violated at the time instant T after switching the control of Eq. ( 63) to w = 0.
Upper estimate for the performance index in Problem 2
If we apply the control of Eq. (63) to the system of Eqs ( 40) and (41) on the interval 0 t T and define w ≡ 0 for t > T, the criterion J3 will coincide with the lower bound calculated in the previous section.However, if ξ * > α −1/2 , the constraint of Eq. (50) will be violated.In fact, the left-hand side of Eq. (50) becomes αξ 2 for w = 0.When switching the control of Eq. ( 63) to zero at the instant T, we have αξ 2 = αξ 2 * > 1 immediately after the switch.
In this section, we will modify the control of Eq. (63) to assure that the constraint of Eq. ( 50) is satisfied during the infinite time interval.Denote the time history of the control of Eq. (63) by w(t).To define the function w(t) one should (i) substitute the control of Eq. (63) into Eq.( 41), (ii) integrate this equation with the initial condition ξ(0) = 0 to determine ξ = ξ(t) on the time interval 0 t T , and (iii) substitute the function ξ(t) into Eq.(63).Define the modified control w * (t) as follows: where The function w * (t) is defined by the condition that ξ ξ = −1 during the interval t 1 < t < T 1 (see Eq. ( 49)).Integration of the equation ξ ξ = −1 with the initial condition ξ(t 1 ) = ξ yields By substituting this function into the left-hand side of Eq. ( 41) we obtain Eq. ( 78).
The time instants t 1 and T 1 in Eq. ( 77) are to be defined by the conditions The control of Eqs (77), ( 78) and (80) satisfies the constraints of Eqs ( 48) and (50).Apparently, both of these constraints hold on the interval 0 t t 1 , since the control of Eq. ( 77) coincides with that of Eq. ( 63) on this interval.The constraint of Eq. ( 50) holds on the interval t 1 < t < T 1 , since the control of Eq. (77) on this interval is defined by the relation ξ ξ = −1.For t T 1 , the behavior of the function ξ(t) is governed by Eq. ( 41) with w = 0.
Integration of this equation subject to the initial condition ξ(T
Hence, the control of Eq. ( 77) satisfies the constraint of Eq. ( 50) on the entire nonnegative semiaxis, 0 t < ∞.
Define ẏ2 and y 2 to be functions of the variable ξ on the time interval t 1 < t < T 1 .Since ξ ξ = −1 on this interval, from Eq. (41) w = (αξ 2 − 1)/ξ.Substitute the last expression into Eq.( 68) to obtain Integration of the first equation gives where ẏ2 ( ξ) can be calculated in accordance with the relations of Eqs (70) or (73).Since the relations ξ = α −1/2 and ẏ2 = 0 hold at the time instant T 1 , Eq. (85) implies that One can utilize this relation to determine ξ.By substituting Eq. (86) into Eq.( 85) we finally obtain Substitution of the right-hand side of Eq. ( 87) into the second relation of Eq. ( 84) and integration of the resulting equation yields where y 2 ( ξ) is defined by Eq. (71) or Eq. ( 73).
The value of the criterion J3 for the control of Eq. ( 77) is equal to the value of the variable y 2 at the time instant T 1 , i.e., 4. Calculate the estimate for the absolute error, or the relative error, of the approximation of the minimum rattlespace of the helmet by the quantity Ĵ3 0 .Note that in the expression of Eq. ( 94) for ∆, the parameter T is the dimensionless characteristic time of the control process obtained by dividing the dimensional time by T * defined in Eq. ( 39).
Finally, we can represent the estimate of the minimum rattlespace as The relations of Eq. ( 111) obtained by executing steps 1 to 4 of the above algorithm are represented in terms of dimensionless units.To represent these relations in terms of the original dimensional units, multiply J 0 3 , Ĵ0 3 , and ).The relations of Eq. ( 111) define an interval in which the rattlespace minimum value certainly lies and provide information about the order of magnitude of this quantity.The length of this interval is determined by the quantity ∆ abs .If ∆ abs is sufficiently small as compared with Ĵ0 3 , the value Ĵ0 3 can be regarded as the final quantitative estimate of the minimum rattlespace, which completes the limiting performance analysis.
Numerical results
Two numerical examples are provided to illustrate typical values of the head injury criteria and the absolute minimum of the helmet padding thickness corresponding to the limiting performance characteristics.Consider two types of projectiles hitting a helmeted head -a bullet and an ice hockey puck.Of course, in the case of the bullet, it will be assumed that the helmet shell is strong enough to prevent the bullet from penetrating or substantially deforming the shell.We utilize the translational head injury model of [4] with the following parameters: We assume the mass of the helmet shell, m 4 , to be equal to 0.5 kg.The values D 1 and D 2 measuring the maximum tolerable injury in terms of the criteria J 1 and J 2 , respectively, are defined as For the bullet, define the mass and the impact velocity of the projectile as For the puck, typical values of these quantities are given by The dimensionless parameters α and β defined by Eq. ( 43) and occurring in Eqs (41) and (42) depend only on the parameters of the mechanical model of the response of the head to an impact (head injury model) and the maximum values allowed for the head injury criteria.These parameters are independent of the mass of the helmet, the mass of the projectile, and the impact velocity.For the numerical data of Eqs (112) and (113), α = 5.90 × 10 −2 , β = 5.85 × 10 −2 . (117) The dimensionless parameter λ depends on the characteristics of the head injury model and also on the mass of the helmet shell, but is independent of the mass and velocity of the projectile.In the case under consideration, this parameter is given by λ = 7.16 × 10 3 . (118) The dimensionless impact velocities v 0 of Eq. ( 60) for the bullet and the puck have the values The relative difference between the lower and upper bounds for the criterion J3 calculated in accordance with Sections 6 and 7 is 3 • 10 −5 .Within this error, the value of J3 is defined as According to Eq. ( 97), the relative difference of the optimal value of the criterion J 3 in Problem 2 from the optimal value of the criterion J3 in Problem 1 is estimated to be 40% for the bullet and 70% for the puck.To obtain these estimates, we defined the time T in Eq. ( 51) to be equal to the time of deceleration of the impact velocity v 0 to a complete stop under the action of the (dimensionless) control w = 1.In the dimensionless variables, T = v 0 , in accordance with Eqs (61) and (62).Of course, these estimates indicate a rather high possible error of approximation of the limiting performance characteristics of the helmet by solving Problem 2 instead of solving the original (more complicated) Problem 1.Nevertheless, in the cases under consideration, these estimates are useful, because they restrict the optimal value of the performance index J 3 of Problem 1 to a certain finite interval, while the optimal displacement of the helmet shell relative to the head is small (in comparison with the dimensions of the head and the helmet).These intervals are defined as 1.22mm Jbullet
Conclusions
An analytical technique has been formulated for the limiting performance analysis of a helmet intended to prevent head injuries due to being hit by a non-penetrating projectile.The goal of this analysis is to evaluate the absolute minimum of the peak displacement of the helmet shell relative to the head (the rattlespace of the helmet), provided that quantitative criteria measuring the severity of injuries lie within prescribed admissible limits.The response of the head to an impact was calculated in accordance with the translational head injury model by Rojanavanich and Stalnaker.This model leads to a seventh-order set of ordinary differential equations governing the behavior of the helmet -head system.Among the head injury criteria taken into account were the peak power of elastic forces due to deformation of the skull bone interacting with the helmet liner and the acceleration of the brain.This approach reduces the limiting performance analysis to solving an optimal control problem for a dynamical system governed by three first-order ordinary differential equations, subject to joint constraints on the phase and control variables.This substantially enables the analysis and makes the utilization of numerical methods necessary only for solving relatively simple transcendental equations.This approach was applied to evaluate the minimum rattlespace of helmets preventing the helmeted head from injuries due to being hit by a bullet and an ice hockey puck.The calculations show that a liner of a thickness of several millimeters could be sufficient.
helmet, m 4 v
p , m p m p : mass of projectile
Fig. 2 .
Fig. 2. Model of the head with helmet.
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52152200 | pes2o/s2orc | v3-fos-license | Comparison of single posterior debridement, bone grafting and instrumentation with single-stage anterior debridement, bone grafting and posterior instrumentation in the treatment of thoracic and thoracolumbar spinal tuberculosis
Background To compare the clinical efficacy of single posterior debridement, bone grafting and instrumentation with that of single-stage anterior debridement, bone grafting and posterior instrumentation for treatment of adult patients with thoracic and thoracolumbar spinal tuberculosis (TB). Methods We performed a retrospective analysis of 64 adult patients with thoracic and thoracolumbar spinal TB who underwent surgery between January 2011 and December 2014. Of the 64 patients, 34 patients were treated using a single posterior-only approach (posterior debridement, bone grafting and instrumentation; Group A). Thirty patients were treated with a combined anterior and posterior approach (single-stage anterior debridement, bone grafting and posterior instrumentation; Group B). Clinical manifestations, laboratory and imaging results were subjected to statistical analysis. Results The mean (±standard deviation) duration of follow-up was 16.8 ± 1.4 months (range, 10–34). Bony fusion was achieved in all the bone grafts with no loosening or breakage of internal fixation. In both of the groups, the visual analog scale (VAS) pain score, ESR and CRP at 6 weeks after operation and at the most recent follow-up were significantly lower than the preoperative level (p < 0.05). The operation time, intraoperative blood loss and length of hospital stay in group A were significantly less than those in group B (P < 0.05). As of most recent follow-up, no significant between-group difference was observed with respect to the American Spinal Injury Association classification status (p > 0.05). Furthermore, no significant between-group difference was observed with respect to preoperative kyphosis angle, and postoperative angle correction and angle correction rate (P > 0.05). One patient in group A relapsed 20 months after operation, and was successfully treated with debridement using the combined anterior and posterior approach. Conclusion Single posterior debridement, bone grafting and instrumentation for treatment of thoracic and thoracolumbar spinal TB can achieve similar curative effect as that with single-stage anterior debridement, bone grafting and posterior instrumentation, and is associated with additional advantages of shorter operation time, less bleeding and shorter length of hospital stay.
(Continued from previous page)
Conclusion: Single posterior debridement, bone grafting and instrumentation for treatment of thoracic and thoracolumbar spinal TB can achieve similar curative effect as that with single-stage anterior debridement, bone grafting and posterior instrumentation, and is associated with additional advantages of shorter operation time, less bleeding and shorter length of hospital stay.
Keywords: Thoracic and thoracolumbar, Spinal tuberculosis, Combined anterior and posterior, Single posterior, Debridement
Background
The incidence of spinal tuberculosis (TB) has shown a steady increase in developing countries and the condition often leads to severe kyphosis and permanent paralysis [1,2]. Chemotherapy is essential for the treatment of spinal TB; however, surgical treatment is often required to improve neurological function and kyphosis. Reasonable debridement, internal fixation and fusion can significantly relieve back pain, improve neurological function, and prevent or correct kyphosis in these patients [3].
The main purpose of surgical treatment is radical debridement, nerve decompression and reconstruction of spine to prevent or improve kyphosis. Currently, there is no clear consensus on the optimal surgical strategy for patients with thoracic and thoracolumbar spinal TB. The anterior approach provides direct access to the site of the lesion, which facilitates the removal of the lesion and reconstruction of the defect. However, the anterior approach does not provide adequate leverage for the correction of kyphosis; moreover, fixation performed through the anterior approach necessitates the ligation of the segmental arteries owing to the risk of massive bleeding [4,5]. The combined use of anterior and posterior approach in a single-stage surgery serves to overcome the limitations of the anterior-only approach and has been widely adopted with good results [6,7]. It should be noted that the use of a combined anterior and posterior approach increases the operation time, and is associated with greater trauma and higher risk of complications, not conducive to early rehabilitation of patients [3]. Good results have been reported with use of posterior approach [8,9]; however, it is associated with inadequate treatment of the lesion in front of the vertebral body.
Thoracic and thoracolumbar segments are the common locations of spinal TB. Due to the small volume of the spinal canal and the poor blood supply of the spinal cord, lower extremity weakness or other neurological deficits are liable to occur in patients with severe bone destruction or instability of the spine [10]. In this study, we explored the clinical outcomes of single posterior debridement, bone grafting and instrumentation and single-stage anterior debridement, bone grafting and posterior instrumentation for treatment of adult patients with thoracic and thoracolumbar spinal TB.
Patient population
We performed a retrospective analysis of 64 patients with active thoracic and thoracolumbar spinal TB (without active TB) treated between January 2011 and December 2014. The diagnosis of active spinal TB was based on clinical symptoms, laboratory investigations [high erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)], and radiographic examination [x-ray, computed tomography (CT), and magnetic resonance imaging (MRI)]. Moreover, pathological examination was performed to confirm the diagnosis. Other inclusion criteria were: (1) progressive nerve injury; (2) ineffective conservative treatment, and no significant decrease in inflammatory markers after anti-TB treatment; (3) kyphosis (kyphosis angle > 16°); and (4) back pain. The ethics review committee of the Shaanxi Provincial People's Hospital approved the study protocol. All the patients provided written informed consent for the use and publication of data for research purposes.
Preoperative preparation
Conventional anti-TB treatment was administered for 2-4 weeks before operation. HREZ anti-tuberculous regimen was adopted: isoniazid (300 mg, oral administration or intravenous drip, once a day), rifampin (450 mg, oral administration, once a day), pyrazinamide (1.5 g, oral administration, once a day), and ethambutol (750 mg, oral administration, once a day). Surgery was performed when the ESR had significantly decreased (≤ 40 mm/h), the temperature had returned to normal, and anemia and hypoproteinemia were resolved completely.
Operative technique
Patients in group A were placed in a prone position, and pedicle screws were inserted in the normal vertebral body adjacent to the upper and lower affected vertebrae. Internal fixation rod was temporarily installed on the less affected side, followed by the excision of the laminae to protect the spinal dura. From the more severe side of the lesion, the upper and lower segments of the diseased vertebra were removed and the intervertebral space was exposed. Then, the necrotic intervertebral disc, pathological vertebral body and paravertebral abscess were removed, followed by the removal of the lesion on the other side using the same method. After the removal of the lesion, a large amount of saline was used to wash the focal zone. After washing, a strip of bone graft was fixed between the residual vertebral bodies of the diseased vertebra. An autogenous iliac bone graft of appropriate size was closely embedded into the intervertebral bone grafting groove, and the pre-bent bar was installed, followed by the correction of kyphosis with moderate pressure. After confirmation of satisfactory internal fixation and correction of kyphosis deformity by C-arm fluoroscopy, a drainage tube was placed and the incision was closed layer by layer (Fig. 1).
In group B, the patient was placed in a supine position and the extra pleural or extra peritoneal anterolateral approach was used to avoid pleural or peritoneal injury. After the location of intervertebral space, the collapsed intervertebral disc and the vertebral body were removed along with the paraspinal and psoas abscess. After the complete removal of the lesion, an autologous iliac bone graft or titanium mesh filled with autologous bone was implanted into the bone graft to reconstruct the anterior column. Then, the patients were placed in a prone position, and pedicle screws were inserted in the normal vertebral body adjacent to the upper and lower affected vertebrae. The connecting rod was installed to stabilize the spine. Vertebral compression was determined according to the status of the compression of spinal cord from the posterior spine. Correction of kyphosis was achieved by moderate posterior compression. The incision was sutured after confirmation of internal fixation in good position with C-arm fluoroscopy (Fig. 2).
Postoperative care
The drainage tube was removed 24-72 h after the operation depending on the drainage volume. Prophylactic antibiotics were used for 3 days postoperatively. Anti-tuberculous therapy was continued after the operation. Pyrazinamide was withdrawn after 6 months of treatment, while the other anti-tuberculous drugs (HRE) were continued for 10-12 months. Non weight-bearing walking aided by braces was recommended 6-8 weeks after operation, while normal weight-bearing activity was allowed only after confirmation of intervertebral fusion on x-ray and CT examinations.
Evaluation standard
The American Spinal Injury Association (ASIA) scale was used to evaluate preoperative and postoperative spinal cord injury. Bone graft fusion, loss of correction angle and internal fixation failure were evaluated by imaging examination. Visual analog scale (VAS) scores were used to assess the severity of pain. Besides, ESR and CRP levels were assessed to evaluate the disease activity. Moon bone graft fusion method was used to evaluate the bone graft fusion [10]. In addition, preoperative and postoperative kyphosis angle was evaluated using the standard method for the measurement of kyphosis angle [11].
Statistical analysis
SPSS version 19.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used to process the data. Wilcoxon signed rank test was used to compare preoperative and postoperative ASIA classification. Paired t test was used to compare the preoperative and postoperative degree of kyphotic deformity, and ESR and CRP levels. Independent sample t test was used to assess the between-group differences with respect to various laboratory and physical parameters. The rank sum test was used for the analysis of discrepancy in normal distribution. P < 0.05 was considered indicative of significant difference.
Results
Out of the 64 patients, 34 patients [25 male and 9 female; mean age ± standard deviation (SD): 39.4 ± 12.0 years (range, 18-70)] were included in group A (treated with posterior-only approach for debridement, bone grafting and instrumentation), while 30 patients [22 male and 8 female; mean age: 40.6 ± 12.5 years (range, 19-71)] were included in group B (treated with combined anterior and posterior approach in a single surgery). All the patients underwent x-ray examination (CT examination in some patients) and showed bone graft fusion. None of the patients showed bone nonunion, false joint formation, internal fixation loosening, or fracture as of the most recent follow-up. The mean time of fusion in groups A and B were 8.6 ± 0.4 months (range, 6-12) and 8.4 ± 0.5 months (range, 5-12), respectively; the between-group difference was not statistically significant (P > 0.05). The operation time, volume of intraoperative blood loss, and the length of hospital stay were more favorable in group A as compared to that in group B (P < 0.05). Tables 1 and 2 list patient information, surgical details and postoperative characteristics.
The mean duration of follow-up in the entire study population was 16.8 ± 1.4 months (range, 10-34). In group A, superficial wound infection occurred in 1 patient, which healed after antibiotic treatment. In group B, 2 patients developed postoperative superficial skin infection, which was cured anti-infection treatment. Besides, 1 case of intercostal neuralgia was relieved after symptomatic treatment. Furthermore, 1 patient developed wound dehiscence 20 months after operation, which was confirmed to be TB relapse by imaging examination, and was treated with debridement using a combined anterior and posterior approach. None of the patients in group B experienced relapse of TB. No serious neurological complications occurred in any of the groups. Table 3 summarizes the changes in the VAS score, ESR and CRP levels 6 weeks after operation and at the most recent follow-up. The VAS score, ESR, and CRP level at 6 weeks after operation and at the most recent follow-up were significantly lower than the preoperative levels in both groups (P < 0.05). Furthermore, VAS score, ESR and CRP at 6 weeks after operation were significantly higher than those at the most recent follow-up (P < 0.05). Table 4 shows the changes in ASIA classification in the two groups. In group A, out of the 30 patients with preoperative neural deficit, 27 patients showed complete postoperative recovery. Out of the 28 patients with preoperative nerve dysfunction in group B, 25 patients recovered to normal postoperatively. As of the most recent follow-up, no significant between-group difference was observed with respect to improvement in ASIA classification status (P > 0.05). Table 5 presents data related to kyphosis correction and kyphosis loss. There was no significant between-group difference with respect to preoperative kyphosis angle (P > 0.05); besides, there was no significant between-group difference with respect to postoperative angle correction and angle correction rate (P > 0.05). Furthermore, the kyphosis angle loss and kyphosis loss rate did not differ between the two groups as of the most recent follow-up (P > 0.05). The kyphosis angle after operation and at the most recent follow-up were significantly lower than the respective preoperative kyphosis angle in both groups (P < 0.05).
Discussion
Spinal TB most commonly affects the thoracic vertebrae and the thoracolumbar spine. The latter represents the area of transition from a relatively fixed thoracic vertebra and a relatively mobile lumbar spine; therefore, the risk of back pain and paraplegia is greater once the disease occurs [12,13]. In clinical practice, patients with thoracolumbar TB often show severe bone destruction, spinal cord compression and/or kyphotic deformity. Therefore, conservative treatment alone is often fails to relieve spinal cord compression, improve nerve dysfunction and prevent spinal deformity, while surgery is often the preferred treatment modality [14]. The purpose of the surgical treatment of thoracic and thoracolumbar spinal TB is to remove the lesion thoroughly, relieve spinal cord compression and to reconstruct the spine.
Anti-TB drug therapy is an essential measure for the treatment of spinal TB and also the basis of surgical treatment. Without regular anti-TB treatment, surgery alone is extremely dangerous and ineffective. Effective surgical treatment can be performed only under effective anti-TB treatment. In this study, with the exception of 2 patients who showed progressive neurological impairment, all the patients were treated with anti-TB drugs for over 2 weeks prior to the operation. Furthermore, surgical treatment was performed only when ESR was < 40 mm/h, and hemoglobin level was at least 100 g/L.
Parthasarathy et al. [15] conducted a retrospective analysis of 235 cases of spinal TB treated with conservative and surgical treatment; the cure rates of spinal TB with isoniazid alone, and combination chemotherapy with rifampin were not worse than those achieved with surgery. However, the authors recommended active surgical treatment for patients who develop neurological deterioration or progressive kyphosis during the course of Time to abscess disappearance (postoperative months) 8.1 ± 0.5 (7)(8)(9)(10)(11)(12) 8.0 ± 0.6 (6)(7)(8)(9)(10)(11)(12) Time to bone fusion (postoperative months) 8.6 ± 0.4 (6)(7)(8)(9)(10)(11)(12) 8.4 ± 0.5 (5)(6)(7)(8)(9)(10)(11)(12) Hospitalization day (days)* 23.3 ± 4.5 (20)(21)(22)(23)(24)(25)(26)(27)(28)(29) 26.5 ± 3.5 (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) Data are presented as n (%) or mean ± standard deviation (range) * P < 0.05. Comparison between group A and group B anti-TB chemotherapy. In this setting, not only does surgical treatment aim to remove TB lesions, but it also aims to relieve spinal cord compression, correct kyphosis and reconstruct the spine. From this point of view, it is appropriate to apply internal fixation in thoracic and thoracolumbar spinal TB operations [16]. Single-stage anterior debridement, bone grafting and instrumentation have been considered as the gold standard for treatment of spinal TB [17]. However, this method often achieves inadequate fixation rigidity and orthopedic strength and is associated with a high risk of vascular injury due to poor access to the site of lesion [18,19]. Since the combined anterior and posterior approach overcomes the limitations of poor stability and correction associated with the single anterior approach, it has been used widely. However, the combined approach is associated with excessive intraoperative bleeding, prolonged operation time, increased length of hospital stage, and a relatively high risk of complications [6,7,20,21]. Wang et al. [20] evaluated 28 patients with thoracolumbar TB who underwent surgery using the combined anterior and posterior approach. They reported 90.4% correction of the kyphosis angle; however, compared to that with single posterior approach, the intraoperative bleeding, operation time and length of hospital stay were relatively greater. Moreover, they reported wound infection, sinus formation at the drainage tube and other surgical complications. Laheri et al. [21] reported 62.5% correction rate of kyphosis in their series of 38 patients with spinal TB who underwent surgery using the combined anterior and posterior approach. In the present study, all the patients in group B achieved 59.3% correction of the kyphosis angle, and the volume of intraoperative bleeding, operation time and length of stay were also greater than that in group A (single posterior approach). Besides, 3 patients in group B experienced surgical complications, as against 1 patient only in group A.
With the development of surgical techniques for spinal TB, single posterior debridement, bone grafting and instrumentation can achieve correction of kyphosis deformity and spinal stabilization. Zhang et al. reported good clinical outcomes in patients with thoracic vertebral TB [22]. Zhou et al. [3] reported good surgical results with single posterior approach for treatment of lumbar TB. In the present study, 34 patients in group A received single posterior debridement, bone grafting and instrumentation for the treatment of thoracic and thoracolumbar spinal TB. Postoperative ASIA classification showed significant improvement, accompanied by significant decrease in postoperative Cobb angle and ESR level (P < 0.05 vs. preoperative level), which suggests good efficacy of surgical treatment. No significant difference was observed with respect to the VAS score, ASIA grading, improvement in Cobb angle or ESR level when compared with those achieved with the combined anterior and posterior approach. However, due to poor exposure to the anterior structure of the spine, the single posterior approach is not suitable for patients with large paravertebral abscess. In this study, there was 1 patient with lumbar vertebral TB with paravertebral abscess in the single posterior approach group. Due to incomplete removal of the lesions, the large paravertebral abscess appeared again 20 months after operation in spite of the anti-tuberculous treatment, which exemplifies this shortcoming.
In this study, patients with spinal TB treated by posterior approach experienced obvious relief of back pain after operation. The intervertebral bone graft fusion was reliable, and there was less long-term kyphosis loss and low recurrence rate. However, it is necessary to evaluate the indications for use of this approach carefully, and due discretion should be exercised before use of posterior approach in patients with thoracic and thoracolumbar spinal TB with large paravertebral abscess. We believe that the rationale for use of the posterior approach is it allows for the removal of sclerotic bone around the lesion. Simultaneously, a small amount of residual TB-like lesion and pus can resolve with standard anti-TB chemotherapy after operation. Therefore, the importance of adequate anti-TB treatment after operation can hardly be overemphasized. Moreover, operations of "radical scavenging" are relative and not [23]. This study also has certain limitations, including the relatively small sample size and the duration of follow-up.
Conclusion
Single posterior debridement, bone grafting and instrumentation for treatment of thoracic and thoracolumbar spinal TB can achieve similar curative effect as that achieved with single-stage anterior debridement, bone grafting and posterior instrumentation, but with additional advantages of shorter operation time, less bleeding and shorter length of hospital stay. To sum up, single posterior approach is a favorable method for the treatment of thoracic and thoracolumbar spinal TB.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article. The raw data can be requested from the corresponding author on reasonable request.
Authors' contributions YZ, WL, JL1, LG, and JL2 participated in the recruitment, data collection and analysis. All authors contributed to the study design and drafting of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate Ethical approval from the Ethics Committee of the Shaanxi Provincial People's Hospital was obtained for this study. Each author certifies that all investigations were conducted in conformity with ethical principles. Written informed consent was obtained from all patients included in the study.
Consent for publication
All patients signed informed consent to publish their personal details in this article. One-way analysis of variance, compared pre-operative kyphosis angle between two groups, P > 0.05 Δ One-way analysis of variance, compared kyphosis angle with pre-operative in two groups, P A < 0.05, P B < 0.05 † One-way analysis of variance, compared angle correction between two groups, P > 0.05 ▲ One-way analysis of variance, compared correction rate between two groups, P > 0.05 □ One-way analysis of variance, compared angle lost between two groups, P > 0.05 # One-way analysis of variance, compared angle lost rate between two groups, P > 0.05 | 2018-09-05T14:05:13.411Z | 2018-09-03T00:00:00.000 | {
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14932121 | pes2o/s2orc | v3-fos-license | Urinary melatonin-sulfate/cortisol ratio and the presence of prostate cancer: A case-control study
The circadian-related hormones, melatonin and cortisol, have oncostatic and immunosuppressive properties. This study examined the relationship between these two biomarkers and the presence of prostate cancer. We measured their major metabolites in urine collected from 120 newly diagnosed prostate cancer patients and 240 age-matched controls from January 2011 to April 2014. Compared with patients with lower urinary melatonin-sulfate or melatonin-sulfate/cortisol (MT/C) ratio levels, those with above-median levels were significantly less likely to have prostate cancer (adjusted OR (aOR) = 0.59, 95% CI = 0.35–0.99; aOR = 0.46, 95% CI: 0.27–0.77) or advanced stage prostate cancer (aOR = 0.49, 95% CI = 0.26–0.89; aOR = 0.33, 95% CI = 0.17–0.62). The combined effect of both low MT/C ratios and PSA levels exceeding 10 ng/ml was an 8.82-fold greater likelihood of prostate cancer and a 32.06-fold greater likelihood of advanced stage prostate cancer, compared to those with both high MT/C ratios and PSA levels less than 10 ng/ml. In conclusion, patients with high melatonin-sulfate levels or a high MT/C ratio were less likely to have prostate cancer or advanced stage prostate. Besides, a finding of a low MT/C ratio combined with a PSA level exceeding 10 ng/ml showed the greatest potential in detecting prostate cancer and advanced stage prostate cancer.
The fact that patients with prostate cancer have lower melatonin levels than patients with BPH suggests that melatonin may protect against disease severity 23 . Melatonin has been found to have potential in the chemoprevention and treatment of prostate cancer in vitro studies 24 . Cortisol is another hormone important to circadian regulation 25,26 . One previous study revealed that cortisol secretion patterns may be impacted by shift work, which is known to cause circadian disruption 27 . This hormone has been found to influence cancer risk through its effects on immune function 28,29 . It is thus possible that carcinogenicity of circadian disruption can broadly affect many sites among both men and women via melatonin and cortisol levels. Therefore, we conducted this case-control study to examine the relationship between two urine biomarkers of circadian regulation hormones, melatonin and cortisol level, and the presence of prostate cancer and their impacts on the clinical stages of prostate cancer. Because PSA is also associated with the presence or relapse of prostate cancer, this antigen was also included to examine the interaction effect of circadian regulation hormones and PSA on prostate cancer.
Results
In total, we recruited 137 pathology-proved prostate cancer patients and 274 male controls (average: 70.53 years; range 45 to 85 years) between January 2011 and April 2014. After excluding cases with other cancers or cases without available individual matches, we were left with 120 cases and 240 controls to include in our final analysis. As can be seen in Table 1, summary of participant characteristics, the only significant difference between the cases and controls were PSA levels (p < 0.001). Most (75.0%) of those included in the study were 65 years old or older, had high school or college educations or less (76.7%), were married (87.7%), had no family history of prostate cancer (85.5%), did not smoke (70.1%), use alcohol (88.8%), chew betel nut (98.3%), or use vitamin D supplements (96.6%). There were no significant differences between the two groups with regard to these characteristics. Figure 1 shows the differences in the urinary biomarkers between cases and controls. Compared to the controls, the cases had a significantly lower levels of the urinary melatonin-sulfate (mean ± SD) ( After adjusting for other covariates, we found that subjects with high urinary melatonin-sulfate or MT/C ratios were significantly less likely to have prostate cancer compared to those with low urinary melatonin-sulfate or MT/C ratios (adjusted OR (aOR) = 0.59, 95% CI = 0.35-0.99 and aOR = 0.46, 95% CI = 0.27-0.77, respectively) ( addition, subjects with both high MT/C ratio and a pre-operative PSA level exceeding 10 ng/ml were 3.61 fold more likely (95% CI = 1.62-8.07) to have prostate cancer, when compared to those with both high MT/C ratio and a pre-operative PSA level less than 10 ng/ml. ( Table 2). The risk was even higher in the group with low MT/C ratios and pre-operative Table S1). Categorizing prostate cancer by clinical stage (localized and advanced), we found significant differences of urinary biomarkers when comparing advanced cancer and control groups. No multiplicative scale of interaction was obtained for the MT/C ratio and PSA level (Table 3). In addition, we found evidence of a combined effect of low MT/C ratio and the pre-operative PSA levels exceeding 10 ng/ml in the advanced cancer group compared with either control group or localized cancer group (Table 3). A similar trend pattern was also observed among the subjects when combining melatonin sulfate or cortisol and PSA level (Supplementary Table S2).
Discussion
In this case-control study, we found an inverse relation between first morning urinary melatonin-sulfate levels and the melatonin/cortisol (MT/C) ratio and the presence of prostate cancer overall and advanced stage prostate cancer.
Most prior studies have focused on evaluating the association between melatonin levels and breast cancer. Very few have focused on the association between melatonin and prostate cancer. One cross-sectional study by Bartsch et al. reported that men with prostate cancer had lower melatonin levels than men with BPH 23 . Another case-cohort study in an Icelandic population, conducted by Sigurdardottir et al., found that subjects with below-median morning pre-diagnostic 6-sulfatoxymelatonin (aMT6s) levels had a statistically significant 4-fold increased risk for advanced disease, compared to those with above-median levels (Hazard ratio = 4.04; 95% CI = 1.26-12.98) 30 . However, that study did not find a significant association between morning urinary aMT6s levels and prostate cancer risk overall. Our study contributes additional information about circadian hormones and the presence of prostate cancer in an Asian population.
The protective effect of melatonin on cancer risk may be related to its inhibition of cancer cell growth, its protection of cells from DNA damage, and its promotion of repair of DNA damage once it has occurred 1,31,32 . Recently, Blask et al. conducted a series of experiments using both steroid receptor-positive and -negative human breast cancer xenografts in rats and found an inverse relationship between melatonin level and tumor activity 33,34 . The same research group also reported similar results with prostate cancer xenografts 35 . Other studies have reported a reduction in growth of malignant prostate tumor cells achieved by the administration of both pharmacologic and physiologic doses of melatonin 24,36-38 , although their findings have not always been consistent 39,40 .
Our study also measured cortisol, another important circadian hormone secreted by the adrenal cortex. Cortisol has been found to help regulate both immunity and inflammation; a deficiency in this hormone may result in an unresponsive immune system and an overabundance of the hormone may suppress immune responses 41 . Moreover, chronic dysregulation of the circadian cortisol rhythm has been associated with higher levels of inflammation 28 , and such inflammation may play a critical role in carcinogenesis 42 . Mirick et al. found that circadian disruption can impact cortisol secretion patterns, which may in turn affect cancer risk 27 . Although this study did not find a significant association between one-spot morning urinary cortisol level and prostate cancer, we did find an inverse association between the MT/C ratio and the presence of prostate cancer and advanced stage prostate cancer. Previous studies have found MT/C ratio to be related to different types of depression and the severity of depression 43,44 . This is the first study to evaluate the association between the MT/C ratio and the presence of prostate cancer. It is difficult to decide clinically whether to perform an invasive prostate biopsy for subjects with abnormal PSA levels. Our finding that the combination of low MT/C ratios and PAS levels in excess of 10 ng/ml conferred the highest tendency that a person may have prostate cancer and advanced-stage disease prostate cancer suggests that we might consider the biomarker of MT/C ratio in urine to be an additional tool for judging whether the prostate biopsy is needed or not, when PSA levels are elevated. Further study is needed to investigate the clinical utility of the combining PSA and MT/C to detect the presence of prostate cancer and stage the disease.
A high proportion of controls in this study had PSA levels exceeding10 ng/ml. Increased PSA concentrations are found in the sera of patients with BPH or patients with prostate cancer, respectively 45 . An estimated 50% of men have histologic evidence of BPH by age 50, 75% by age 80, and 90% of men by age 85 years 46 . About 99% of prostate cancer cases occur in those over the age of 50 47 . This is of particular concern in older men, where BPH is more prevalent, as BPH increases gland volume which in turn increases the PSA level as well as the number of sampling errors associated with prostate biopsy 48,49 . For men with PSA 4-10 ng/mL, the detection rate of PCa in Caucasian men may be as high as 40% 50,51 but only 20% in Chinese men 52 . Although the inclusion criteria for our control group was either unremarkable digital rectal exams or remarkable digital rectal exams but histologically confirmed BPH, our control group may have included some men with undiagnosed prostate cancer. However, the inclusion of such patients as controls would only lead to underestimations of the true association.
The strengths of our study are that we were able to consider the most important prostate cancer risk factors, including clinical stage and PSA level in our analyses. Furthermore, we considered two important circadian biomarkers and found the M/C ratio is more relevant to prostate cancer. This study also has several limitations. One limitation is that it is a cross-sectional case-control study, so no clear causal relationship can be inferred. There is a likelihood of a reverse causality between circadian hormones and the presence of prostate cancer, if circadian disruption and/or sleep disruption following a cancer diagnosis results in the decline of melatonin levels. Another limitation is that we used one single measurement of one-spot morning urinary biomarkers, which may not represent long-term exposure level. Although we had broad information on various covariates and were able to control for possible confounders, we still lacked information on factors such as have sleep disorders or medication use, etc. Finally, the exposures of interest were collected using a questionnaire, which may lead to some recall bias.
Conclusion
Lower morning melatonin-sulfate levels or MT/C ratio were associated with the presence of prostate cancer. In addition, patients with both low MT/C ratios and PSA levels exceeding 10 ng/ml appeared to be far more likely to have prostate cancer and advanced stage disease. Because this is a cross-sectional case-control study, we could not establish a definite causal relationship between urinary MT/C ratio and risk of prostate cancer. Larger prospective cohort studies are needed to verify these findings. Data collection. Overview. Reference date for the cases was defined as date of pathology proof of prostate cancer. Controls were matched within the same month, the reference month. Subjects in both groups answered in-person interviews conducted by trained interviewers using standardized questionnaires collecting socio-demographic information (age, education, and marital status), family history of prostate cancer (PCa) and lifestyle (use of tobacco, alcohol, betel chewing, and vitamin D supplements). The medical charts of all cases were reviewed to collect data on serum prostate-specific antigen (PSA) level at diagnosis and disease stage. The medical charts of controls were reviewed to collect measured PSA results. All study procedures were reviewed and approved by the Institutional Review Board of the Kaohsiung Chung-Ho Memorial Hospital (KMUH-IRB-20110101). All the methods were carried out in accordance with the approved guidelines, and written informed consent was obtained from all subjects.
Urine sample collection. After their interviews, both case and control subjects provided the first-spot morning urine samples after waking up. The samples were divided into labeled cry-tubes of 4.5 mL volume each and then immediately stored at − 20 °C. Urine specimens from case subjects and matched control subjects were handled identically and assayed on the same day and in the same run. All samples were taken out of the freezer simultaneously and sent to the laboratory in the same parcel on dry ice. Laboratory personnel were blinded to the case-control status of all specimens. Analytic error was controlled for by including two standard samples in each assay. The morning urinary measurements showed good sensitivity and specificity in identifying individual differences in nocturnal plasma melatonin levels 53 . Statistical analysis. Urinary melatonin-sulfate/cortisol (MT/C) ratio was calculated by urinary melatonin-sulfate levels divided by urinary cortisol levels as a basis for measuring the combined impact of melatonin and cortisol levels simultaneously on the prostate cancer. Wilcoxon rank-sum test was used to compare the differences of urinary biomarkers, including melatonin-sulfate, cortisol, and MT/C ratio, between case and control groups. In addition, those urinary markers were dichotomized by medians, which were 43.23 ng/mg creatinine for melatonin, 24.06 ng/mg creatinine for cortisol, and 1.76 for MT/C ratio. Unconditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for the association between the urinary markers and the risk of prostate cancer. All analyses were adjusted for age at time of questionnaire completion (< 65 vs. ≥ 65 yrs), family history of PCa, personal habits (smoking, alcohol, and betel nut), and PSA level (< 10 vs. ≥ 10 ng/ml). We also categorized cases into localized prostate cancer (stage T1 or T2) and advanced cancer (extra-prostatic stage T3a or higher, N1/M1) 55 , and used polytomous logistic regression models to determine the risks of developing localized and advanced prostate cancer relative to the control group 56 . Multiplicative interaction was appraised by fitting polytomous logistic models containing categorical variables, as well as their cross-products. Wald Z-tests for cross-product terms were used to evaluate the significance of multiplicative interaction for each pair of comparison. All statistical operations were performed using the SAS 9.1 statistical package; all P-values were two-sided and considered significant if < 0.05. | 2018-04-03T01:37:44.650Z | 2016-07-08T00:00:00.000 | {
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6013877 | pes2o/s2orc | v3-fos-license | TGF-b2 Induction Regulates Invasiveness of Theileria-Transformed Leukocytes and Disease Susceptibility
Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.
Introduction
Cellular transformation is a complex, multi-step process and leukocyte transformation by Theileria is no exception, as parasite infection activates several different leukocyte-signalling pathways, the combination of which leads to full host cell transformation [1]. However, Theileria-induced leukocyte transformation is unusual in that it is rapid and appears to be entirely reversible with the host cell losing its transformed phenotype upon drug-induced parasite death [2]. Just like most cancer cells however, Theileria-induced pathogenesis (virulence) is associated with the invasive capacity of transformed leukocytes, which is lost upon attenuation of vaccine lines [3]. Attenuation of virulence has been ascribed to decreased matrix-metallo-proteinase-9 (MMP9) production and loss of AP-1 transcriptional activity [4]. Consistently, functional inactivation of AP-1 resulted in reduced tumour formation, when infected and transformed B cells were injected into Rag2gC mice [5].
Host leukocyte tropism differs with T. parva infecting all subpopulations of lymphocytes whereas T. annulata infects monocytes/macrophages, dendritic cells and B lymphocytes [1]. Despite this, the diseases they cause (called tropical theileriosis with T. annulata infection and East Coast fever with T. parva infection) are both severe, as susceptible animals usually die within three weeks of infection. The geographical distribution of their respective tick vector species determines areas where disease is widespread. Tropical theileriosis affects over 250 million animals and extends over the Mediterranean basin, the Middle East, India and the Far East, whereas East Coast fever is prevalent in eastern, central and southern Africa. It is noteworthy that in endemic areas indigenous breeds of cattle are more resistant to disease. For example, when Bos indicus Sahiwals are experimentally infected with T. annulata they exhibit fewer clinical symptoms and recover from a parasite dose that is fatal in the European Holstein-Friesian (HF) B. taurus breed [6][7]. Theileria-infected leukocytes are capable of producing IL-1 and IL-6 [8], as well as GM-CSF [9] and TNF [10]. Nonetheless, no differences in the level of expression of the pro-inflammatory cytokines TNF, IL-1b, or IL-6 were detected between disease-resistant Sahiwal-versus HF-infected macrophages [11]. Some additional inherent genetic trait of Sahiwal animals must therefore underlie their disease-resistance. Although transcriptome analysis of 3-5 times passaged Sahiwal and HF macrophages following infection with T. annulata revealed significant breed differences in both the resting and infected gene expression profiles, no clear candidate genetic trait was revealed [12].
Transforming growth factor beta (TGF-b) is a family of cytokines and both TGF-b1 and TGF-b2 can bind with high affinity to the TGF-b type II receptor (TGF-RII) leading to the recruitment of TGF-RI. The constitutive kinase activity of TGF-RII phosphorylates and activates TGF-RI, which in turns recruits and activates Smad2 and Smad3, which bind Smad4, and the whole complex translocates to the nucleus and induces the transcription of target genes [13]. The TGF-b signalling pathway can be negatively regulated [14] and an increasing number of non-Smad-mediated TGF-b signalling pathways have been described [15]. TGF-b can also regulate cytoskeleton dynamics via transcription-dependent and transcription-independent processes [16]. It is likely that all these different pathways contribute in different ways to the pleiotropic effects of TGF-b (see http:// www.cell.com/enhanced/taylor). TGF-b can exert opposite effects on cell growth: in most non-transformed cells TGF-b is usually growth inhibitory, but it can increase motility of certain mesenchymal cells and monocytes, but however, at some point in the transformation process TGF-b becomes pro-metastatic [17][18], for example in ovarian cancer [19]. We show here that TGFb plays a role in infected host leukocyte invasiveness. Importantly, the high level of TGF-b2 production by Theileria-infected HFtransformed macrophages renders them more invasive than those of disease-resistant Sahiwal animals. In addition, vaccination against tropical theileriosis uses live attenuated T. annulata-infected macrophages and attenuation leads to the loss of both TGF-b2 transcription and alteration in the expression of a set of TGF-btarget genes, and a drop in TGF-b-mediated invasion. Thus, Theileria-dependent TGF-b2 induction is a virulence trait that underscores susceptibility to tropical theileriosis.
Results
Induction of TGF-b2 by T. annulata is greater in infected macrophages isolated from disease-susceptible compared to disease-resistant bovine hosts As Theileria-transformed leukocytes are known to secrete a number of different cytokines we examined whether infection by T. annulata sporozites of the same parasite strain (Hissar) could induce TGF-b in macrophages 72h post-invasion, as described [12]. Prior to infection both Sahiwal and HF macrophages produced low levels of TGF-b transcript with slightly higher amounts of TGF-b1 (Fig. 1A). Interestingly, Theileria infection induces preferentially TGF-b2 in both Sahiwal and HF macrophages, and importantly, the induction after 72h is much greater in disease-susceptible HF cells.
We next examined the levels of TGF-b transcripts in a series of T. annulata-transformed cell lines derived from Sahiwal and HF animals. Again, TGF-b1 and TGF-b2 mRNA could be detected in all 10 transformed cell lines (Fig. 1B). Similar to freshly invaded cells the relative mRNA levels of TGF-b1 did not differ significantly across the T. annulata infected cell lines and there was no evidence of a breed-specific difference in TGF-b1 expression ( Fig. 1B grey bars, p = 0.710). In contrast, the relative TGF-b2 mRNA levels exhibit statistically significant differences ( Fig. 1B black bars, p,0.001), with TGF-b2 mRNA levels being higher in HF cell-lines. Additional qRT-PCR experiments revealed that the levels of expression of TGF-RI, TGF-RII and TGF-RIII were equivalent in HF versus Sahiwal T. annulatainfected cell lines (data not shown). Thus, disease susceptibility correlates to the level of TGF-b2 transcripts that are expressed 7-fold (p,0.001) more by T. annulata-transformed macrophages of HF origin.
The invasive capacity of Theileria-transformed HF macrophages is greater than Sahiwal-infected macrophages and is TGF-b-dependent In T. parva-transformed B cells a TGF-b-mediated signalling pathway is active and invasion is partially TGF-b-dependent (Fig.S1). We therefore compared the invasive capacity of T. annulata-transformed HF versus Sahiwal macrophages ( Figure 2). We found that disease-susceptible HF macrophages displayed 30% greater capacity (p,0.005) to traverse Matrigel than infected Sahiwal macrophages and that traversal is again TGF-bdependent ( Fig. 2A). The invasive capacity of the H7 cell line was reduced to levels equivalent to S3 cells upon treatment with the TGF-R inhibitor ( Fig. 2A) and conversely, when S3 cells were stimulated with conditioned medium from H7 cultures, S3 cells displayed increased invasiveness (Fig. 2B). Moreover, the reduced invasive capacity of disease-resistant S3 macrophages could be
Author Summary
Theileria annulata causes tropical theileriosis that is endemic in cattle in North Africa, the Middle East, India and China. T. parva causes East Coast fever that is prevalent in East and Southern Africa. In endemic countries indigenous cattle are more resistant to pathology, but produce little meat and milk and attempts to improve output by importing European and American breeds have failed due to a high susceptibility to these diseases that are often rapidly fatal. We examined T. annulata-transformed macrophages isolated from disease resistant Sahiwal compared to disease-susceptible Holstein-Friesian (HF) cattle, for their capacity to traverse synthetic extra-cellular matrix in vitro. The invasive capacity of all transformed macrophages was TGF-b dependent, but those of diseasesusceptible HF animals invaded better i.e. they were more aggressive. The greater invasive capacity of HF transformed macrophages matched their increased production of TGF-b2, since levels of TGF-b1, and all three TGF-b receptors, were the same as in transformed macrophages isolated from disease-resistant Sahiwal animals. TGF-b2 production therefore likely renders Theileria-transformed leukocytes more pathogenic and consistently, in a live attenuated line used to vaccinate against tropical theileriosis transcripts of TGF-b2 and those of a significant number of TGF-target genes drop and consequently, TGFb-mediated invasiveness decreases. restored to above virulent levels by addition of either TGF-b1, or TGF-b2 (Fig. 2C), demonstrating that the TGF-b signalling pathways are intact in these cells. Theileria infection therefore preferentially induces up-regulation of TGF-b2 and increased invasiveness of transformed leukocytes.
In vitro attenuation is associated with lower TGF-b2 expression and altered transcripts levels of TGF-bregulated genes T. annulata-infected cell lines can be attenuated for virulence by multiple in vitro passages to generate live vaccines that are used to protect against tropical theileriosis [3]. The molecular basis of attenuation is not known, but our above observations on preferential TGF-b2 induction and augmented host cell invasiveness suggest that attenuation might be lead to reduced TGF-b2 transcription and TGF-b2-mediated invasion. To directly test this prediction we examined the Ode vaccine line derived from an infected HF cow in India [20] and estimated the level of TGF-b2 transcripts and TGF-b-target gene transcription in virulent (early passage) and attenuated (late passage) infected macrophages ( Figure 3). As predicted, attenuation leads to a significant decrease in the amount of TGF-b2 message and surprisingly, a slight increase in TGF-b1 transcripts (Fig. 3A). This strongly suggests that upon attenuation the parasite's ability to induce host cell TGF-b2 has been impaired. Reduced levels of TGF-b2 should lead to an alteration in the transcription profiles of known TGF-b-target genes and to see if this is indeed the case, we performed microarray analyses and hierarchical clustering of transcript levels. The microarray representing 26,751 bovine genes included 1,158 targets of TGF-b (http://www.netpath.org/) and 76 of these genes were identified as differentially expressed upon attenuation. The heat-map, where low gene expression level is depicted as blue, intermediate as yellow and high expression as red, is present in Fig. 3B. Among the down-regulated TGF-target genes, five were chosen at random and their expression verified by qRT-PCR using mRNA from early and late passage Ode (Fig. 3C). In each case their transcription was reduced upon attenuation and could be restored by adding exogenous recombinant TGF-b2. Consistently, their expression was high in disease-susceptible infected HF macrophages that produce more TGF-b2 (see Fig. 1) and low in Sahiwal macrophages, but could be augmented by exogenous TGF-b2 stimulation (Fig.S2). Attenuation of virulence therefore, leads to ablation of TGF-b2 signalling and an alteration in the profile of expression of a set of TGF-target genes. Figure 2. The invasive capacity of Theileria-transformed HF macrophages is greater than Sahiwal-infected macrophages and is TGFb-dependent. A, The invasive capacity of H7 and S3 cells was determined by Matrigel chamber assays. Seven independent experiments were performed and each sample counted three times. The y-axis is the number of migrated cells for 10 independent fields. B, Conditioned medium from H7 cultured cells stimulates the invasive capability of S3 cells (*** p,0.0005). Invasion assay was performed on untreated S3 cells and S3 cells that had been incubated for 24h prior to the assay with culture supernatants from H7 cells grown overnight in low-serum (0.5% FCS) containing media. C, Addition of recombinant TGF-b1 and TGF-b2 leads to an increase in the invasive index of Theileria-infected S3 macrophages (** p,0.005). doi:10.1371/journal.ppat.1001197.g002 Figure 1. Induction of TGF-b2 by T. annulata is greater in infected macrophages isolated from disease-susceptible compared to disease-resistant bovine hosts. A, Macrophages isolated from Sahiwal and HF cows were infected in vitro with sporozoites of the Hissar strain of Theileria annulata. RNA was isolated immediately prior to infection (0h) and 72h post-infection and the relative amounts of TGF-b1 and TGF-b2 transcript measured by real-time RT-PCR. B, Total RNA was extracted from T. annulata-infected cell lines obtained from infection of Sahiwals (S1-5) and HF (H7-12) animals. TGF-b1 and TGF-b2 relative mRNA levels were determined by real-time RT-PCR. The relative value obtained for the cell line showing the lowest expression of each cytokine (H8 for TGF-b1 and S4 for TGF-b2) was used for comparison and arbitrary set at 1. Error bars represent standard deviations for triplicate samples. No significant difference between Sahiwal and Holstein-Friesian cells was found for TGF-b1 (p = 0.710), in contrast to TGF-b2 that was 7-fold upregulated (p,0.001) in all 5 H cells lines. doi:10.1371/journal.ppat.1001197.g001 Invasiveness is reduced upon in vitro attenuation of host cell virulence The observation that early passage Ode cells express higher levels of TGF-b2 message and have altered expression of 76 TGFb-target genes led us to compare early with late passage Ode and examine the contribution of TGF-b to their invasive capacity ( Figure 4). As previously described [4], attenuation of Ode leads to a significant drop in invasive capacity (***p,0.005) and receptor blockade by SB431542 gives an estimate (***p,0.005) of the contribution of TGF-b to early passage Ode virulence (Fig. 4A). The potential contribution of TGF-b to virulence has been ablated by attenuation, since the invasive capacity late passage Ode is insensitive to receptor blockade (Fig. 4A, right). When conditioned medium from early passage Ode is given to late passage Ode there is a marked (**p,0.05) regain in invasion (Fig. 4B). Virulent Ode therefore, secretes factors into the medium that contribute to invasiveness, one of which is clearly TGF-b2, and this capacity is lost upon attenuation. The partial inhibition of invasion by early passage Ode by SB431542 might also suggest that although virulent following 65/70 in vitro passages some attenuation of TGF-b2 induction might be occurring.
TGF-b promotes podosomal adhesion structures and ROCK-mediated cortical actin-rich membrane blebs
We next studied whether the TGF-b-mediated invasion programme might have a consequence on cellular adhesion and invasion structures such as lamellipodia, podosomes and membrane blebs. We first investigated by time-lapse video microscopy lamellipodia morphology of T. annulata-infected macrophages cultured without (control), or with SB431542 ( Fig.S3 and Movies S1 and S2) and found that the size of lamellipodia (shown boxed) decreased upon SB431542 treatment (Fig.S3A). Visualisation of the actin cytoskeleton with Texas red-labelled phalloidin showed that decreased lamellipodia size correlated with reduced actin dynamics ( Fig.S3B and C) and suggested that TGF-controls cortical actin dynamics in infected macrophages. We next compared the basal and central cortical actin cytoskeleton of S3 and H7 cells cultured on a gelatin/fibronectin matrix, which facilitates adhesion of these cells. In S3 cells, we observed only small podosomal adhesion structures that were rarely clustered and no actin-rich membrane blebs (Fig. 5A). In contrast, in H7 cells podosomal adhesion structures were markedly enlarged and clustered and the majority of cells displayed actin-rich membrane In vitro attenuation is associated with lower TGF-b2 expression and altered transcripts levels of TGF-b-regulated genes. A, Total RNA was extracted from early and late passage ODE cells. TGF-b1 and TGF-b2 relative mRNA levels were determined by real-time RT-PCR. B, Total RNA from early and late passage ODE cells (3 replicates each) was hybridised to a microarray representing known bovine genes, including 1,158 targets of TGF-b. 76 of these genes were identified as differentially expressed using the criteria of a false discovery rate ,5% (Rank Product analysis) and a fold change .2. Hierarchical clustering was performed on this gene set and the results are depicted as a heat-map. The gene symbol is shown on the right of the heat-map, where colour is used to represent gene expression level: blue (low), yellow (intermediate) and red (high). C, Total RNA was extracted from early and late passage ODE cells, as well as late passage ODE cells treated for 24h with rboTGF-b2 (5ng/ml). Relative mRNA levels for the indicated genes were determined by real-time RT-PCR and normalized to HPRT1 relative levels. doi:10.1371/journal.ppat.1001197.g003 blebs. Membrane blebbing was confirmed by live-cell imaging (Fig.S4A), which highlights the dynamics of bleb formation. Individual blebs expand within one to three seconds and persist for approximately 30-120 seconds, which is a time frame typically observed in bleb formation and retraction [21]. Reducing the serum concentration from 10% to 0.5% (starvation) resulted in a significant decrease in the number of membrane blebs ( Fig. 5B and C). Membrane blebbing in starved cells was rescued by the exogenous addition of TGF-b2. The TGF-b-induced membrane blebs were blocked by the Rho-kinase (ROCK) inhibitor H-1152 and in the presence of serum the formation of actin-rich membrane blebs was significantly reduced after treatment with the TGF-R inhibitor, but completely blocked in the presence of H-1152 (Fig. 5C). Thus, one consequence of increased TGF-b2 production is increased cortical actin dynamics, which likely gives rise to enhanced podosomal adhesion structures (invadosomes) and membrane blebs in macrophages derived from disease susceptible HF cattle.
We next investigated the functional significance of membrane blebs for cell motility in 3-D matrices. In the low rigidity fibrillar collagen or high-density Matrigel matrices, H7 macrophages acquired an amoeboid pattern of motility with characteristic polarized formation of membrane blebs (Fig. 5D and Fig.S4B and C). Membrane bleb formation required ROCK activity and inhibition of ROCK prevents local contractibility, polarized bleb formation and forward movement of the cell. Taken together, these data show that exposure of H7 macrophages to TGF-b2, results in ROCK-dependent membrane blebbing that drives motility in 3-D matrices.
Discussion
We have shown here that Theileria-induced leukocyte transformation results in the constitutive induction of a TGF-b autocrine loop that augments the invasive potential of infected leukocytes. We could find no evidence for a contribution of TGF-b signalling to host cell survival, or proliferation (data not shown), implying that leukocyte infection by Theileria essentially confers on TGF-b a pro-metastatic role. Smads and p53 are known to associate and collaborate in the induction of a subset of TGF-b target genes [22]. Recently, p53 has been described as being sequestered in the cytosol of Theileria-transformed leukocytes, as part of a parasiteinduced survival mechanism [23]. Although not addressed by Haller et al it is possible that cytosol located p53 might ablate nuclear translocation of Smads, thus counteracting the antiproliferative affect of TGF-b in Theileria-transformed leukocytes?
Importantly, comparison of disease-susceptible HF transformed macrophages to disease-resistant Sahiwal ones, showed that the degree to which Theileria (the same parasite strain) induces TGF-b2 influences the invasive potential of the infected and transformed host cell. The likelihood of developing a life-threatening cancerlike disease therefore appears to be due in part to the inherent genetic propensity of HF macrophages to produce high levels of TGF-b2 upon infection that might render the transformed macrophages more invasive. T. annulata-transformation of HF macrophages leads to the induction of higher amounts of TGF-b2 the levels of induced TGF-b1 mRNA being the same as in Sahiwal macrophages. As TGF-RI and -RII and -RIII [24] are expressed to the same extent in the two types of macrophages (data not shown) it suggests that only the amount of TGF-b2 is crucial. The predisposition of Theileria transformation to induce TGF-b2 over TGF-b1 in HF versus Sahiwal macrophages implies that there could be disease-associated sensitivity to infection linked to TGF-b2 over-production. Species-specific promoter differences, or some other unknown breed difference may explain the greater propensity of Theileria to induce TGF-b2 over TGF-b1 transcripts in HF compared to Sahiwal cattle. However, promoter sequence differences seem unlikely to underlie Theileria's ability to induce TGF-b2 over TGF-b1 transcripts, or explain the drop in TGF-b2 levels upon attenuation of the Ode vaccine line as here, both virulent and attenuated infected macrophages are of HF origin [20]. Loss of virulence of the Ode vaccine line upon long-term in vitro passage is clearly associated with a decrease in TGF-b2 transcripts and it would appear that the parasite's ability to specifically activate host cell transcription of TGF-b2 is impaired and one possibility is that attenuation is associated with altered epigenetic regulation of TGF-b2 promoter activity. Microarray analyses indicate that upon attenuation of virulence, not only do TGF-b2 levels drop, but also 76 TGF-b-target genes display altered transcription. It would appear then that preferential TGF-b2 induction following Theileria infection initiates a host cell genetic programme that contributes to more aggressive invasiveness of transformed HF macrophages. We believe the same TGF-b2initiated genetic programme also contributes to the invasiveness, albeit reduced, of disease-resistant Sahiwal macrophages, as Theileria infection also preferentially induces TGF-b2, just to a lesser extent.
We have used fluorescence and time-lapse video microscopy to examine the morphology of Theileria-infected Sahiwal and HF infected macrophages and the effect of TGF-b and Rho kinase (ROCK) on actin dynamics and lamellipodia formation. Theileria infected, disease-susceptible HF macrophages show increased actin dynamics in their lamellipodia and podosomal adhesion structures and a remarkable propensity to develop membrane blebs. Figure 5D shows the dynamic behaviour of infected cells embedded in 3-D matrices (see also Fig.S3 and movies S3 and S4), where either fibrillar collagen, or matrigel was used giving two 3-D matrices of low (collagen) and high (matrigel) rigidity. Membrane blebbing of motile H7 cells occurs in both matrices in a polarized fashion at the leading edge, clearly suggesting that membrane blebbing is required for infected cell motility in 3-D. Movie S3 shows bleb-driven membrane protrusions, which results in forward movement of the cell (see also kymographs of movie S3, Fig. 5D). Moreover, ROCK activity is required, because inhibition of ROCK with H-1152 impairs polarized bleb formation and forward movement in fibrillar collagen and matrigel. Spatio-temporal control of Rho-ROCK activity is also required for cell polarization and lamellipodia formation in 2-D [25]. Since TGF-b acts upstream of Rho-ROCK in infected cells, we would therefore predict that spatio-temporal activation of Rho-ROCK -controlled by TGF-b signalling -is required for lamellipodia formation as well. Combined, increased bleb and lamellipodia formation could give rise to more invadosomes on infected virulent macrophages in a similar manner to TGF-b-mediated increased adhesion of immortalised hepatocyte cell lines [26].
We showed that augmented invasiveness by TGF-b2 in diseasesusceptible HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho and its downstream target ROCK [27][28]. Given the important role played by ROCK in increased blebbing of infected host cells and the recent description that Rho/ROCK signals amoeboid-like motility [29], it is tempting to speculate that the TGF-b autocrine loop confers on Theileria-infected leukocytes an amoeboid-like motility that contributes to invasiveness and makes them more virulent. However, since prolonged TGF-b stimulation can result in decreased Rho activity due to the action of p190RhoGap [30], Theileria infected cells must have developed a mechanism to balance TGF-b-induced RhoGAP activities. It is possible that the parasite might also regulate the expression, activity or localization of Rho-family GEFs, as the Rho activator GEF-H1/Lfc has been shown to be a TGF-b1 target gene [31] and an analogous mechanism involving TGF-b2 might be exploited by Theileria parasites? Alternatively, the parasite could function by excluding negative regulators of Rho from specific subcellular compartments, analogous to the exclusion of the RhoGAP Myo9b from lamellipodia of macrophages [25].
Whatever the underlying mechanism of inducing TGF-b2 levels in Theileria-infected leukocytes, its induction and the genetic programme it initiates is clearly correlated with the invasive phenotype of transformed macrophages of disease-sensitive hosts. This implies that overall invasiveness of Theileria-transformed leukocytes is made up of amoeboid (TGF-b-& ROCK-dependent) and mixed amoeboid/proteolytic (MMP-dependent [4]) motility; reviewed in [32]. These Theileria-based observations also suggest that in some cases the propensity of human leukaemia patients to develop life-threatening cancer could be due to the inherent genetic predisposition of their tumours to produce high levels of TGF-b2, rather than TGF-b1, and the genetic programme this initiates on promoting an additional amoeboid-like invasive phenotype of their tumours.
Theileria-infected cell lines
TpMD409.B2 is a T. parva Muguga-infected B-cell clone (B2) and its B-cell characteristics have been previously confirmed [33]. The cell lines S1-S5 and H7-H10 have been described previously [6]. In vitro infection of uninfected S and H cells by Theileria sporozoites was done as described [12]. The characterisation of the Ode vaccine line has been reported [20] and in this study virulent/early Ode corresponds to passage 65-70 and attenuated to passage 318-322. It is possible that passages 65-70 have already become slightly attenuated. All cultures were maintained in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS) and 50 uM b-mercaptoethanol. Cell cultures were passaged 24h before harvesting to maintain the cells in the exponential growth phase.
Transfections, expression plasmids and reporter constructs
All transfections were carried out by electroporation as previously described [34]. The CAGA-luc (the Smad 3/4 binding elementluciferase construct) was transiently transfected into B2 cells with the inhibitory Smad7 plasmid (Flag-Smad7-pcdef3), or an empty vector (pcdef3). The major late minimal promoter (MLP -a minimal promoter consisting of the TATA box and the initiator sequence of the adenovirus major late promoter) was cloned into pGL3 (Promega) to generate the MLP-luc plasmid that was used as minimal promoter negative control [35][36]. Efficiencies of transfections were normalized to renilla activity by co-transfection of a pRL-TK renilla reporter plasmid (Promega #E6241). Luciferase assay was performed 24h after transfection using the Dual-Luciferase Reporter Assay System (Promega, #E1980) in a microplate luminometer (Centro LB 960, Berthold). Relative luminescence was represented as the ratio firefly/renilla luminescence.
Total RNA extraction
Total RNA was isolated from each of the T. annulata infected cell lines using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. The quality and quantity of the resulting RNA was determined using a Nanodrop spectrophotometer and gel electrophoresis and for microarray screens on an Agilent 2100 Bioanalyser (Agilent Technologies). mRNA was reverse transcribed to first-strand cDNA and the relative levels of each transcript were quantified by real-time PCR using SYBR Green detection. The detection of a single product was verified by dissociation curve analysis and relative quantities of mRNA calculated using the method described by [38]. GAPDH, HPRT1 and C13orf8 relative quantities were used to normalise mRNA levels. For list of primers used, see Table 1.
Microarray analyses
Host gene expression was investigated using a custom-designed microarray (Roche NimbleGen Inc., Madison, WI), which represented every bovine RNA reference sequence currently deposited in the NCBI database (n = 26,751). Each gene was represented by two identical sets of five 60-mer-oligonucleotide probes that were isothermal with respect to melting temperature. cDNA was generated from 10 ug total RNA using oligo(dT) primer and tagged with 39-Cy3 dye and labelled cDNA was hybridised to the array. Gene expression values were calculated from an RMA-normalised dataset [39] and differentially expressed genes were identified using Rank Product Analysis [40]. Genes were defined as differentially expressed using the criteria of a false discovery rate of less than 5% and a fold change of greater than two. Selected gene sets were subjected to hierarchical clustering based on Euclidean distance between expression values and the results were illustrated using a heat-map (ArrayStar3, DNASTAR Inc., Madison, WI).
Invasion assays of Theileria-transformed cells (B2, S3, H7 and Ode cells)
The invasive capacity of the bovine leukocytes was assessed using in vitro Matrigel migration chambers, as described [5]. After 26h of incubation at 37uC, filters were washed twice in PBS and the Matrigel was eliminated. In some cases, during this 12h period cells were also incubated with the TGF-b inhibitor SB431542 (10uM), or with recombinant TGF-b protein (10ng/ml). When added TGF-b was maintained in the top chamber, meaning that overall cells were incubated with TGF-b for 36h. Cells were then counted under the microscope (406 objective) to obtain a statistically significant mean number of cells per field (at least 10 fields per filter). The experiment was performed at least in triplicate.
Time-lapse imaging
Time-lapse imaging using video microscopy was performed with cells growing on glass bottom culture dishes (Willco Wells, the Netherlands) using a Nikon Eclipse TE2000-U inverted microscope equipped with a climate-controlled chamber. Data acquisition and image processing was performed using NIS software of Nikon Instruments. DIC images were acquired in intervals ranging from 0.5 msec to 1 min for 30 min and assembled in movies; acceleration of movies is approximately 600-fold. Kymographs were acquired along a one pixel wide line using NIS software. A 406 Plan Achromat Objective of Nikon was used for longworking distance image acquisition in Matrigel.
Lamellipodia morphology
Theileria-infected macrophages were seeded onto glass coverslips, or glass bottom culture dishes (Willco Wells, the Netherlands) and maintained in growth medium for 48h without or with 10uM SB431542 (SIGMA). SB431542 was replenished after 24h. Cells were either processed for live cell imaging or fixed in 3.5% formaldehyde, 15 min. Actin cytoskeletons of fixed and Triton X-100 permeabilized cells were visualized with Texas red-labelled phalloidin. Lamellipodia area and integrated fluorescence intensities in lamellipodia were determined using photoshop CS3 software.
Cell seeding onto gelatin/fibronectin matrix
Glass coverslips were coated with 0.1% poly-L-lysine for 15 min at room temperature and then fixed with 0.5% glutharaldehyde for 15min. After 3 washes with PBS, coverslips were inverted onto droplet containing 2mg/ml (0.2%) gelatin (MERCK) in H 2 O for 15min. After 3 washes with PBS, coverslips were inverted onto droplet containing 25ug/ml bovine plasma fibronectin (SIGMA) in PBS and then incubated 1h at room temperature. Coverslips were then transferred into 24 well plate washed once with PBS and kept in PBS until seeding of 50,000 cells per well. After 18h cells were fixed in 3.5% formaldehyde, 15min.
Embedding cells in 3-D matrices
Embedding in collagen: 3610 5 cells in 50ul medium were added to a mixture of 68ml sodium bicarbonate (7.5%, SIGMA), 240ul 106PBS and 2ml PureCol (3mg/ml, Inamed). The resulting gelatin solution with the concentration of 2.4mg gelatin/ml was distributed into live-cell imaging wells or dishes and transferred to 37uC for collagen polymerization.
Embedding in Matrigel: 1610 5 cells in medium were mixed on ice with 250ul growth factor reduced Matrigel (BD biosciences) and was distributed into live-cell imaging wells or dishes and transferred to 37uC for polymerization. Figure S1 A TGF-b autocrine loop leads to constitutive activation of Smad-mediated signalling and increased invasiveness of T. parva-transformed B2 cells. A, T. parva-transformed B2 cells were either incubated for 30min with rboTGF-b1 and rboTGF-b2 (5ng/ml), or treated for 24h with SB431542 (10uM). Nuclear extracts were analysed for phospho-Smad2 and Smad2 levels by Western Blot. PARP protein levels were used as a loading control. B, The CAGA-luc construct was transiently transfected into B2 cells and its constitutive activation is given as a relative luciferase activity (arbitrarily set at 100) that represents the ratio firefly/ renilla luminescence compared to the minimal MLP promoter. Where indicated, transfected cells were treated for 24h with SB431542 (10uM). CAGA-luc was also co-transfected with a | 2016-05-12T22:15:10.714Z | 2010-11-01T00:00:00.000 | {
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2507841 | pes2o/s2orc | v3-fos-license | Adaption to High Altitude: An Evaluation of the Storage Quality of Suspended Red Blood Cells Prepared from the Whole Blood of Tibetan Plateau Migrants
Hypoxia has been reported to cause the significant enhancement of hemoglobin (Hb) and hematocrit (Hct), which stabilizes at relatively high levels after an individual ascends to a high altitude. However, the quality of the suspended red blood cells (SRBCs) obtained from individuals at high altitudes such as Tibetan plateau migrants after storage has not been studied. In this study, we compared the storage quality of SRBCs prepared from Tibetan plateau and Deyang lowland populations by adding a normal volume of mannitol-adenine-phosphate (MAP), which is a common additive solution used in blood storage in Asian countries. The storage cell characteristics were examined on days1, 7, 14 and 35.We found higher Hct and Hb levels and viscosity in the high altitude samples. The metabolic rates, including those for electrolytes and lactate, were higher in plateau SRBCs than in lowland SRBCs; these findings were consistent with the higher osmotic fragility and hemolysis of plateau SRBCs throughout the entire storage period. In addition, the reduction rates of 2,3-diphosphoglycerate (2,3-DPG) and oxygen tension to attain 50% oxygen saturation of Hb (P50) in plateau SRBCs were higher than those in lowland SRBCs, and the oxygen delivering capacity in plateau SRBCs was weaker than that in lowland SRBCs. We concluded that the storage quality of plateau SRBCs was inferior to that of lowland SRBCs when using the same concentration of MAP. We suggested that the optimal formula, including the MAP concentration or even a new additive solution, to store the plateau SRBCs must be assessed and regulated.
Introduction
Environmental conditions such as high altitude affect blood cell morphology and phenotype [1][2][3]. Hypoxia in high altitude areas causes red blood cell (RBC), hemoglobin (Hb) and hematocrit (Hct) levels to significantly increase after individual persons ascend to high altitudes, and the enhancements stabilize at relatively high levels [4]. Han Chinese who migrated to the Qinghai-Tibetan Plateau had higher Hb concentrations than native Tibetans due to decreased plasma volume in the early arrival days and to increased numbers of red blood cells in the later days after arrival [5,6]. The increased Hb is potentially important for improving the oxygen-carrying capacity of RBCs. However, Hct values over 50% to 55% increase the risk in clinical transfusion through increased blood viscosity [7].
Donated whole blood (WB) is usually separated into various products for various clinical transfusion purposes. Suspended red blood cells (SRBCs) are the most commonly transfused blood product [8,9]. Our previous study [10] showed that blood collected from residents migrating to Tibet was separated into SRBCs with the normal volume of the additive solution (AS) and that the Hct level was over 65%. However, the biological change and the clinical transfusion suitability of storing Tibetan plateau migrants' SRBCs for up to 35 days have not been assessed. It has been shown that the SRBCs storage quality depends on the AS [11][12][13]. Mannitol-adenine-phosphate (MAP) is one of the most common AS in China and most Asian countries, and it enables the storage of SRBCs for up to 35 days following collection. Therefore, the aim of this study was to compare the quality between Tibetan plateau migrants' SRBCs and Deyang lowland residents' SRBCs stored using a normal volume of MAP.
In this study, we collected WB from the Tibet Autonomous Region blood center (elevation 3650 m) and Deyang Blood Center (elevation 400 m). SRBCs were prepared by removing plasma and adding MAP. The separated SRBCs units were stored under standard blood banking conditions at 4°C. The samples were examined aseptically on Days 1, 7, 14, and 35 poststorage. The following parameters were tested: extracellular pH, electrolytes, crystal osmotic pressure, glucose, lactate, adenosine triphosphate (ATP), 2, 3-diphosphoglycerate (2,3-DPG), hemolysis rate, osmotic fragility, and oxygen affinity.
Materials and Methods
Whole blood collection, RBC processing, and storage WB was collected according to standard procedures. We harvested the WB of 8 volunteer donors at the Tibet Autonomous Region blood center and of 8 lowland volunteer donors at Deyang Blood Center. All donors from Tibet were Han Chinese who migrated to the Tibetan plateau for more than one year. The 16 donors were male, and their ages ranged from 19-40. The information of weight and height of the donors was not supplied by the blood centers. They were regular donors, and this is the first time for them to donate. The study protocol was approved by the Institute of Blood Transfusion Ethics Committee. WB (400 mL ±10%) was collected into blood collection packs containing citrate-phosphate-dextrose-adenine-1 (CPDA-1) anticoagulant (56mL) (Nan Geer Biomedical Corporation, Sichuan, China). The formulation of the anticoagulant CPDA-1 was listed in Table 1. Each bag of WB was centrifuged at 3500 ×g for 15 minutes at 4°C. The plasma was removed, and 100 mL MAP (see Table 1 for the additive solution composition) was added to the packed RBCs to obtain the SRBCs. The SRBCs units were stored under standard blood banking conditions at 4°C and were analyzed on days1, 7, 14, and 35 of storage.
Routine RBC quality assessment
On each day of sample collection, blood examinations were performed on an automated hematology analyzer (BC-5800; Mindray, Shenzhen,China), and the RBC, Hb and Hct levels and mean corpuscular volume (MCV) were examined. Supernatant potassium (K + ) and sodium (Na + ) levels were measured on an electrolyte analyzer (HC-9885; Hangchuang Medical Instrument Co., Ltd, Shenzhen, China). Extracellular pH was measured with a pH meter (FE20; Mettler Toledo, Switzerland), crystal osmotic pressure was measured with an osmotic pressure instrument (SMC 30 C; Tianhe Medical Instrument Co., Ltd, Tianjin, China), and viscosity was assessed using a rheology instrument (990 BBT, South CNC Equipment Co., Ltd, Chongqing, China). RBC adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) were assessed using commercial ATP and 2,3-DPG kits according to the recommended protocols (Roche Diagnostics, Mannheim, Germany). Plasma glucose was measured with a commercial glucose kit (Rongsheng Biotech Co., Ltd, Shanghai, China). The lactate concentration was analyzed in RBC protein precipitant extracts using lactate kits (Jiancheng Bioengineering Institute, Nanjing, China) adapted for use with a spectrophotometer.
Hemolysis
Briefly, free Hb was assessed by measuring the absorbance of cell supernatants at 435 nm using a spectrophotometer (Ultrospec 6300 Pro; Amersham Biosciences, United States) and correcting for the absorbance of plasma, if necessary. Hemolysis rate was expressed as a percentage of the total Hb present in RBCs after correcting for the Hct.
Osmotic fragility
Osmotic fragility was assessed using a series of diluted sodium chloride solutions with concentrations ranging from 3.0 g/L to 9.0 g/L. Briefly, 30 μL of each RBC sample with 50% Hct was added to 3000 μL of each diluted sodium chloride solution and incubated at room temperature for 30 minutes, followed by centrifugation at 1000×g for 5 minutes. Supernatants were collected, and hemolysis was measured by a spectrophotometer at a wavelength of 540 nm. The percent hemolysis in each sodium chloride solution was plotted against the sodium chloride solution concentration to calculate the concentration that produced 50% hemolysis. Oxygen affinity The oxygen tension to attain 50% oxygen saturation of Hb (P50) was detected by a Hemox analyzer (TCS Scientific, USA). To obtain a suspension, 50 μL RBCs were added to the diluents. Oxygen was to the suspension until the oxygen partial pressure (PO 2 ) was greater than13.33 kPa (100 mmHg); oxygen was then stopped, and nitrogen was added until the PO 2 was less than 0.67 kPa (5 mmHg). The changes in PO 2 and oxy-hemoglobin were detected in the process, and oxygen dissociation curves (ODC) were drawn. Finally, P50 was measured according to the ODC.
Statistical analysis
The results are shown as the mean ±standard deviation (SD) of eight sample replicates, and every parameter has to be test for at least 3 times, unless otherwise stated. Independent sample t tests were used to compare the RBCs from various areas stored for the same lengths of time and the P values were corrected by Bonferroni correction. Repeated measures analysis of variance (ANOVA) was used to assess the influence of storage time on lowland or plateau SRBCs. Data were analyzed using computer software (SPSS Statistics17.0, IBM SPSS, United States). The levels of significance were indicated in the text.
Plateau SRBCs have higher RBC, Hb, and Hct levels and MCV than lowland SRBCs
At time of donation, we can't get all the baseline values of the parameters except the concentration of hemoglobin. After the donation, the blood has to do virus detection, and we can't have enough time and enough volume of blood to test all the parameters at time of donation. The hemoglobin of plateau and lowland blood was 165.62±10.15 and 137.43±7.38 g/L respectively. In China, leukodepletion was not asked for every blood center or hospital when preparing SRBCs, therefore, in this study, we didn't use leukodepletion both for plateau and lowland blood. We have tested the number of leukocyte in plateau and lowland blood at beginning of storage (3.89±1.23 vs 4.52±0.89 ×10 9 /L), and there was no significant difference between the two groups. On day 1 of storage, we found that Tibetan plateau SRBCs had significantly higher RBC, Hb, and Hct levels compared with Deyang lowland SRBCs (Table 2). We observed Hct enhancement in plateau SRBCs from 61.0% (day 1) to 64.3% (day 35), whereas the Hct value in lowland SRBCs had no significant relationship with the storage time. The MCV of plateau SRBCs also increased with the storage time, suggesting that the RBCs became more spherical ( Table 3). The viscosities of plateau SRBCs at low and high shear rates both increased with storage time, and these values were higher than those of lowland SRBCs from day 7 to day 35 (Fig 1).
Plateau SRBCs have higher crystal osmolality, higher rates of change of electrolytes and lactate, and lower pH compared with lowland SRBCs The pH of the plateau SRBCs and lowland SRBCs was 6.9 and 7.05, respectively, on day1, and decreased to 6.4 and 6.6, respectively, by day35. Plateau SRBCs had a significantly lower pH compared with that of lowland SRBCs from day 1 to day 35 of storage (Fig 2A). In contrast, crystal osmotic pressure increased with storage time in both plateau and lowland SRBCs ( Fig 2B). The glucose levels decreased with storage time in both plateau and lowland SRBCs, and no significant difference was found between the two groups ( Fig 2D). The production of lactate was higher in plateau SRBCs than lowland SRBCs on days 7 and 35 of storage (p< 0.05; Fig 2C). Plateau SRBCs had higher K + levels in the supernatant compared with lowland SRBCs on days 7 and day 35, whereas lowland SRBCs had higher Na + levels in the supernatant compared with plateau SRBCs (Fig 2E & 2F). We found that the RBC count in plateau SRBCs was much higher than that in lowland SRBCs and that the RBC metabolism parameters such as glucose, lactate, Na + and K + were correlated to the RBC number. To exclude the influence of RBC number on these parameters, we therefore propose the following formula to calculate the rate of RBC metabolism: where R represents the change rate of these parameters throughout the storage period, x represents the parameters, including glucose, lactate, Na + and K + ,V x1 represents the value of the parameters on day 1 of storage,V x35 represents the value of parameters on day 35 of storage, and N rbc represents the number of RBCs on day 1 of storage. The results (Table 4) revealed a significant difference between plateau and lowland RBCs during the entire storage period for the change rates of these parameters, with the exception of glucose, although the plateau glucose level was still higher than the lowland glucose level.
RBC morphology images
SEM studies showed a significant RBC shape transformation during blood storage. On day 1, discocytes were the dominant cell population in both lowland and plateau SRBCs (Fig 3A). On day 35, the number of normally shaped discocytes among plateau SRBCs was much less than among lowland SRBCs, and the number of abnormally shaped RBCs of the plateau group continued to increase with storage time (Fig 3B).
Plateau SRBCs have higher 2,3-DPG and P50 at the beginning of storage, and no significant difference exists for ATP between plateau and lowland SRBCs No significant difference in ATP levels was found between the two groups ( Fig 4A). 2,3-DPG levels in plateau SRBCs were higher than those in lowland SRBCs from day 1 to day7 (p < 0.01), whereas no significant difference was found on day 14 ( Fig 4B). When the SRBCs of the two groups were stored, there was a prompt rise in oxygen affinity, as shown in Fig 4C by a decrease in P50, the partial pressure of oxygen at which Hb is half Fig 1. The viscosities of stored suspended red blood cells (SRBCs) from plateau (circle) and lowland (square) populations were measured throughout the 35-day storage period. The viscosity at ahigh shear rate (A) (***t test p < 0.001 for plateau vs. lowland from day1 to day 35). The viscosity at low shear rate (B) (***t test p < 0.001 for plateau vs. lowland on day7, **t test p < 0.01 for plateau vs. lowland from day14 today 35).# ANOVA p < 0.05 for the viscosities of plateau SRBCs at high and low shear rates throughout the entire storage duration.
doi:10.1371/journal.pone.0144201.g001 saturated. Plateau SRBCs had significantly higher P50 values than lowland SRBCs on day 14, but no significant difference was found between the two groups at the end of the storage period.
Plateau SRBCs have higher osmotic fragility and hemolysis than lowland SRBCs
The plateau SRBCs were less resistant to osmotic stress than the lowland SRBCs. Whereas 50% of the plateau RBCs lysed at a NaCl concentration of 4.75 ± 0.25 g/L, the lowland RBCs were more resilient, lysing at a concentration of 3.81 ±0.15 g/L (Fig 5A). Although the hemolysis was within the COE acceptance limit of 0.8% throughout the storage of all RBC units (Fig 5B), the value for plateau SRBCs was higher than that for lowland SRBCs on day 1 and day 7, which was consistent with the results for osmotic fragility.
Discussion
In this study, we compared the quality of SRBCs from migrants to the Tibetan Plateau with those from lowland residents during a 35-day storage period. Our findings suggested that the storage quality of plateau SRBCs was worse than that of lowland SRBCs when using the same volume of MAP. Besides, the amount of citrate in the primary donation bag was identical in the two groups, and we didn't see any differences in clot formation during storage just from the observation by eyes, and the difference in blood coagulation between the two groups needs further study. At high altitudes, in response to the insufficient amount of oxygen, the bone marrow is stimulated by an erythropoietic factor to increase RBC production [14,15]. In addition to the Osmotic fragility (A) (***t test p < 0.001forplateau vs. lowland from day 1 to day35). Hemolysis (B) (**t test p < 0.01 for plateau vs. lowland on day1, ***t test p < 0.001 for plateau vs. lowland on day7). increase in RBCs, the Hb in plateau SRBCs is augmented. Our data showed that the average Hb in plateau SRBCs ranged from 185 to 218 g/L, which was increased compared with the lowland SRBCs levels, ranging from 164 to 183 g/L at sea level, similarly to the findings of previous studies [16][17][18]. Some investigators believe that increased RBC number is a necessary adaptation characteristic, whereas others suggest that better-adapted natives have lower Hct [19]. It was suggested that our findings of higher RBC and Hb levels in SRBCs from plateau migrants living at a high altitude for more than one year were indications of new adaptation features in these people.
Similarly, the MCV in plateau SRBCs also increased with storage time, and the MCV was higher in plateau SRBCs than in lowland SRBCs at the end of storage, indicating that plateau RBCs became swollen and spherical during storage. The gradual increase in MCV in plateau SRBCs over time might result from insufficient ATP-dependent Na-K pump activity at the RBC membrane, as these pumps cannot work properly under cold storage conditions [20]. The SEM findings of plateau RBCs were consistent with the MCV on day 35 of storage, and the number of abnormally shaped plateau RBCs was much higher than that of lowland RBCs. According to Bessis' and Brezina's classification and discrimination between reversible and irreversible membrane shape alterations over the storage duration [21,22], the number of reversible shapes such as echinocyte and stomatocyte shapes in plateau RBCs was much lower than that of lowland RBCs on day 35 of storage, and the percentage per500 cells was 68% vs. 75%, respectively. In contrast, the number of irreversible shapes such as spheroechinocyte and spherostomatocyte shapes was higher in plateau RBCs compared with lowland RBCs. Previous studies indicated that RBC shape changes are related to enhanced osmotic fragility and cation dysregulation, and are accompanied with increasing hemolysis and acidosis [23,24]. Our findings in this study were consistent with a previous study indicating that higher osmotic fragility, hemolysis, plasma K + accumulation and lower pH of plateau SRBCs may cause the abnormal shape changes in plateau RBCs in our storage conditions. Additionally, RBC shape was also influenced by other factors, including deformability and rheological properties [25]. The higher viscosities of plateau SRBCs at high and low shear rates may be caused by the higher Hct and Hb of plateau SRBCs and are also associated with the higher MCV and decreased RBC deformability [26]. Additionally, the viscosity of stored plateau SRBCs increased with storage time, indicating that the higher viscosity of plateau SRBC units at the end of their shelf-life increases the difficulty of clinical transfusion. Therefore, blood with an Hb concentration higher than 180 g/L was not collected by the Tibetan blood center.
A previous study found that that the initial pH value affected RBCs throughout the entire storage period [27]. Our study showed that from day 1 to day 35, the pH of plateau SRBCs was significantly lower than that of lowland SRBCs and was associated with continuous glucose metabolism, subsequently causing an accumulation of lactic acid and a stronger inhibition of glycolysis. Tighter packing of RBCs led to a faster rate of glucose depletion and more difficulty in in vivo recovery [28,29]. These findings suggested that the lower pH of plateau SRBCs was associated with higher Hct.
Plateau SRBCs had higher extracellular K + levels compared with those of lowland SRBCs during storage, whereas the Na + levels in plateau SRBCs supernatant were lower. Higher extracellular K + indicates that the intracellular K + loss rate was faster in plateau RBCs than in lowland RBCs. The loss of K + from RBCs causes dehydration, resulting in shape changes and rheological defects [30].The crystal osmotic pressure of plateau SRBCs was higher than that of lowland SRBCs and increased gradually during storage; the increased crystal osmotic pressure maybe associated with the increased K + leakage of plateau SRBCs. The results of the higher change rates of Na + , K + and lactate production in plateau SRBCs suggested that these parameters were caused not only by Hct and Hb but also by RBC properties, including osmotic fragility and hemolysis.
Osmotic fragility analysis indicated that lowland SRBCs had greater osmotic resilience compared with plateau SRBCs. A previous study suggested that the enhancement of osmotic fragility increased cell sphericity and cell volume, which was corroborated by a higher MCV in plateau SRBCs [31]. These findings were consistent with our hemolysis data, and the higher osmotic fragility of plateau SRBCs was in agreement with the higher spontaneous hemolysis rate after storage compared with that of lowland SRBCs. Therefore, the capacity to withstand osmotic stress was worse. We guess the deformability of plateau RBC would be worse and the RBC may not pass through capillaries smoothly when the plateau blood transfusion into recipients' body, thus, the oxygen of recipients' organ may not be enough. On the other hand, the half life of plateau RBC may be shorter than lowland RBC after it enters into the body. However, all the tests in the study are in vitro, and more transfusion outcomes need further study. Both values of hemolysis of lowland and plateau (0.066±0.068% and 0.18±0.13%, respectively) SRBCs on day 35 of storage are, however, well within the allowances of 0.8% in Europe and 1.0% in North America [32].
ATP concentrations in both lowland and plateau SRBCs gradually decreased throughout storage. As the ATP concentrations decrease, the Na/K membrane pumps begin to fail, and intracellular K + is leaked into the storage media [33].However, no significant difference in the ATP concentration between lowland and plateau SRBCs was found throughout the entire storage period, which was not consistent with the results of higher K + leakage in plateau SRBCs. Although the loss of ATP stiffens the RBC membrane due to calcium accumulation [11], we propose that the differences of K + , Na + and lactate concentrations in lowland and plateau SRBCs were not primarily caused by energy metabolism but rather by inherent differences in RBC membrane fragility at the beginning of storage [34].
At high altitudes, the affinity of Hb for oxygen is decreased to make Hb-bound oxygen more available to body tissues [35].In 1960s, Lenfant and Torrance studied the relationship between oxygen dissociation and RBC organic phosphate concentrations in subjects who moved from one altitude to another and found that 2,3-DPG and P50 both increased and that 2,3-DPG formed a complex with purified deoxyhemoglobin. This combination reduced the affinity of Hb for oxygen when subjects moved from lowland to plateau [18].In comparing the results of 2,3-DPG and P50 in plateau SRBCs to those of lowland SRBCs, the data reported here showed similar trends as a previous report, indicating that the plateau SRBCs had higher 2,3-DPG and P50 values at the beginning of storage. However, no significant differences in 2,3-DPG and P50 levels were found between lowland and plateau RBCs at end of storage, suggesting that the reduction rates of 2,3-DPG and P50 in plateau SRBCs were greater than those of lowland SRBCs and that the oxygen-delivering capacity of stored plateau SRBCs was weaker compared with that of lowland SRBCs.
Conclusions
In conclusion, we found an important difference in the storage quality of SRBCs from Tibetan Plateau migrants and Deyang lowland residents. Altitude may play a major role in these changes. In addition to the higher Hct and Hb levels and viscosity induced by the high altitude, the rates of electrolyte and lactate metabolism of plateau SRBCs were higher than those of lowland SRBCs during storage. These findings were consistent with the higher osmotic fragility and hemolysis of plateau SRBCs throughout the entire storage period. We concluded that the storage quality of plateau SRBCs was inferior to lowland SRBCs when using the same volume of the additive solution MAP. We recommend increasing the volume of AS for preparing plateau SRBCs, which can reduce the concentration of Hb and viscosity of blood for the convenience of clinical transfusion, or regulating the formula of the storage solution, for example, adding ascorbic acid to improve the membrane fragility of RBC, or finding a new suitable alternative solution for plateau SRBCs. | 2017-04-16T13:15:38.816Z | 2015-12-04T00:00:00.000 | {
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209509944 | pes2o/s2orc | v3-fos-license | Risk of ischemic stroke in patients with prostate cancer receiving androgen deprivation therapy in Taiwan
Background Androgen deprivation therapy (ADT) in the treatment of prostate cancer may be associated with an increased risk of thromboembolic disease. The aim of our study was to investigate the association of ADT in the treatment of prostate cancer with ischemic stroke risk. Methods We identified individuals older than 20 years of age who were newly diagnosed with prostate cancer between January 1, 2005, and December 31, 2012. Patients who experienced ischemic stroke or transient ischemic stroke before the index date were excluded. Patients who received at least one prescription for ADT within 6 months were defined as the ADT user group. Patients who did not receive at least one prescription for ADT within 6 months were defined as the ADT nonuser group. The patients were followed until the first occurrence of one of the primary outcome measures (ischemic stroke or death) or until December 31, 2013. The primary composite outcome was the time to any cause of death or ischemic stroke. Results There was no significant difference in the primary composite outcomes in the prostate cancer patients between the ADT user and nonuser groups. Prostate cancer patients who received ADT had a higher mortality rate than those who were not treated with ADT, and the adjusted hazard ratio was 1.907 (95% confidence interval: 1.278–2.844; P = 0.0016) after adjusting for age, comorbidities and comedication use. Conclusion ADT in the treatment of prostate cancer may not be associated with an increased risk of ischemic stroke. The differences in thromboembolic effects in cardiovascular disease and ischemic stroke secondary to ADT should be further discussed and evaluated prospectively.
Background
Androgen deprivation therapy (ADT) is the key treatment for advanced prostate cancer patients or patients with high serum prostate-specific antigen levels with intermediate or high-risk prostate cancer even without imaging findings or other evidence of disseminated disease [1,2].
Despite the frequently dramatic potential benefits after treatment, ADT has side effects that can greatly impact a patient's quality of life and may necessitate changing the treatment strategy. These side effects include sexual dysfunction [3], increased risk of bone fractures due to decreased bone mineral density [4], hot flashes [5], reduced muscle strength, decreased lean body mass, increased fat mass, decreased insulin sensitivity [6] and potential harm to the cardiovascular system [7].
There are existing conflicting reports regarding the effect of ADT on cardiovascular disease. Saigal et al. showed that ADT is associated with cardiovascular mortality and may decrease survival in patients with low-risk disease [8]. Tsai et al. reported that ADT significantly increased mortality due to cardiovascular disease in patients who underwent radical prostatectomy for localized prostate cancer [9]. Nanda et al. found that ADT increased the risk of allcause mortality in patients with a history of congestive heart failure or myocardial infarction, but this effect was not observed in patients without comorbidities or those with a single coronary artery risk factor [10].
However, a systematic review and meta-analysis found that ADT did not increase the risk of cardiovascular death and that ADT reduced prostate cancer-specific mortality and all-cause mortality [11]. A recent metaanalysis [12] suggested that ADT without estrogen increases the risk of thromboembolic events. A database study also showed that ADT significantly increased the risk of thromboembolic events [13]. A Swedish database study [14] also showed that the risk of thromboembolic events was increased in patients with prostate cancer. These studies focused on cardiovascular events. Teoh et al. [15] reported on 452 Chinese men with prostate cancer treated primarily with radical prostatectomy or radiotherapy and compared the risk of acute myocardial infarction (AMI) in patients who were given ADT with those who were not given any. The ADT group was associated with an increased risk of AMI (P = 0.004) when compared to the non-ADT group. The use of ADT (HR: 6.78, 95% confidence interval (CI): 1.31-35.05, P = 0.022) was a significant risk factor for AMI after multivariate Cox regression analysis. A retrospective study was then conducted by Teoh et al. [16] on 452 Chinese patients with prostate cancer. There were no statistically significant associations in the patients between administration of ADT and the development of ischemic stroke upon Kaplan-Meier analysis. However, upon multivariate Cox regression analysis, the use of ADT was a risk factor for ischemic stroke (HR: 3.32, 95% CI: 1.14-9.67, P = 0.028).
There are limited data about ischemic stroke and ADT in prostate cancer patients. The aim of our study was to investigate the safety of ADT as reflected by outcomes of ischemic stroke and mortality in a nationwide cohort study involving patients with prostate cancer using the Taiwan National Health Insurance Research Databases (NHIRD).
Database
A medical claims database, the NHIRD, was created for research and includes outpatient and inpatient claims data. The database includes information regarding each patient's age, gender, diseases, prescription drugs, and medical expenditures. This study was a population-based retrospective cohort study. Our data were retrieved from the Longitudinal Health Insurance Database 2005, which includes data from 1,000,000 beneficiaries collected from 1997 to 2013. In this observational and analytical study, the database was derived from the NHIRD, and the data were deidentified secondary data that were released for research purposes. The study was reviewed and approved by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital. According to both the NHIRD and hospital regulations, informed consent from the patients was waived for this retrospective cohort study because the information was deidentified secondary data. All procedures were in accordance with the principles of the Declaration of Helsinki.
Study population
We identified individuals older than 20 years of age who were newly diagnosed with prostate cancer (International Classification of Diseases, ninth revision, Clinical Modification [ICD-9-CM]: 185) between January 1, 2005, and December 31, 2012. Patients without certification for catastrophic illness were excluded. The index date was assigned as the date of diagnosis of prostate cancer.
We excluded patients who were diagnosed with other cancers before the index date and those who took any second-line ADT drugs (high dose bicalutamide, diethylstilbestrol, and medroxyprogesterone) within 180 days after the index date. Furthermore, patients who experienced an ischemic stroke or a transient ischemic stroke before the index date were excluded from our observational populations. We further excluded patients who had other cancer diagnoses, those who had received chemotherapy before the index date, those with a history of ischemic stroke or ADT exposure before the index date, and patients who had received second-line ADT.
Drug use
Patients with prostate cancer who received at least one prescription for ADT within 6 months after the index date were defined as the ADT user group. Patients who did not receive at least one prescription for ADT within 6 months after the index date were defined as the ADT nonuser group. Each patient was followed until the first occurrence of one of the primary outcome measures (ischemic stroke or death), until they started receiving ADT if they were in the ADT user group, or until December 31, 2013.
Comorbidities and medications
Patient comorbidities were identified according to diagnostic codes for each inpatient or outpatient diagnosis 1 year before the index date. Comorbidities included diabetes mellitus, hypertension, dyslipidemia, ischemic heart disease, congestive heart failure, chronic obstructive pulmonary disease (COPD), atrial fibrillation, thyroid disease, chronic kidney disease, and liver diseases. The demographic characteristics of the patients were also assessed at baseline.
Study endpoints
The composite primary endpoint was a combination of all-cause mortality or ischemic stroke. We also analyzed all-cause mortality and ischemic stroke as individual endpoints. For patients who visited the emergency department due to ischemic stroke, hospitalization due to ischemic stroke with a primary or secondary diagnosis code or hospitalization for carotid artery stent placement was defined as an ischemic stroke event.
Statistical analysis
We aimed to describe the demographics including the ages, comorbidities, and concomitant medication use of prostate cancer patients with and without ADT exposure. For the baseline characteristics, continuous variables are presented as the means and standard deviations and were compared by Student's t-tests. Discrete variables are presented as counts and percentages, and among patients with and without ADT exposure, these variables were compared by chi-square tests. Values of P < 0.05 were considered statistically significant in all statistical analyses.
We performed 1:1 propensity score (PS) matching on each sample. The variables including age groups and comorbidities were used to calculate the predicted probability from a logistic regression model. A Kaplan-Meier curve was used to estimate the differences between patients who did and did not use ADT after PS matching and was assessed by the log-rank test. A Cox proportional hazards model was used to assess the association of outcomes between patients with and without ADT exposure during the follow-up period. Models were adjusted for age group, comorbidities, and concomitant medication use. To assess the robustness of the outcomes, sensitivity analysis for ischemic stroke was performed in a competing risk model, and subgroup analysis was performed for ADT use (over one year and less than one year). All data management procedures and statistical analyses were performed using SAS software, version 9.4 (SAS Institute, Cary, NC, USA). The research was approved by the IRB of Kaohsiung Medical University Hospital (KMUHIRB-EXEMPT-20170011).
Results
We enrolled 1588 patients older than 20 years with prostate cancer. A total of 1292 patients met the inclusion and exclusion criteria. We performed 1:1 PS matching of these patients, and 272 patients were included for further analysis. Figure 1 shows a diagram of patient enrollment. Table 1 shows the patient characteristics before and after PS matching. The mean patient age was approximately 70 years, and more than one-tenth of the patients were older than 80 years. The five most common comorbidities in order were hypertension, dyslipidemia, COPD, ischemic heart disease and diabetes. The three most common medications used were NSAIDs, alpha blockers and CCBs. Table 2 shows the outcomes of ADT users and nonusers with prostate cancer after PS matching. The primary composite outcome was the time to any cause of death or to ischemic stroke. There was no significant difference in primary composite outcome among ADT users and nonusers with prostate cancer. In terms of any cause of mortality, prostate cancer patients who received ADT had a higher mortality rate than those who did not receive ADT, and the adjusted hazard ratio was 1.907 (95% CI: 1.278-2.844; P = 0.0016) after adjusting for age, diabetes mellitus, ischemic heart disease, congestive heart failure, COPD, hypertension, dyslipidemia, thyroid disease, atrial fibrillation, liver disease, renal disease and comedication, as shown in Table 1. The results showed that patients treated with ADT during follow-up did not have a significantly higher risk of ischemic stroke than those not treated with ADT (crude HR: 1.147, P = 0.3451; adjusted HR: 0.812, P = 0.3291).
In subgroup analysis, the patients who used ADT over one year did not have a significantly increased risk of ischemic stroke than the ADT nonuser patients (adjusted HR: 1.132, P = 0.5745); there was also no significant difference in ischemic stroke risk between patients who used ADT for less than a year and those who did not use ADT (adjusted HR: 1.217 (p = 0.2311)). Furthermore, we also repeated the analysis for ischemic stroke in a competing risk model (adjusted HR: 0.759, P = 0.2050), which also showed that ADT use did not significantly increase the risk of ischemic stroke. Figure 2 shows the probability of freedom from ischemic stroke after propensity score matching stratified by ADT users and nonusers. There was no significant difference in ischemic stroke between ADT users and nonusers. (P = 0.3805).
Discussion
In prostate cancer patients treated with ADT, the severity conferred by endocrine treatment was greater than that of the other treatments. These prostate cancer patients with more advanced disease may experience a poor prognosis and high mortality. Albertsen et al. [17] performed six phase 3 prospective randomized trials to show cardiovascular morbidity following initiation of gonadotropin-releasing hormone agonists compared with an antagonist. The risk of cardiac events within 1 year of initiating therapy was lower in patients received gonadotropin-releasing hormone antagonists compared with gonadotropinreleasing hormone agonists (HR: 0.44, 95% CI: 0.26-0.74, P = 0.002).
Teoh et al. [18] found that surgical castration was associated with an increased risk of cardiovascular thrombotic events when compared to gonadotropin-releasing hormone agonists. After multivariate Cox regression analysis, age, hyperlipidemia, and surgical castration were significant risk factors of cardiovascular thrombotic events. Chung et al. [19] also used the NHIRD and found that there was no significant difference in the risk of stroke in patients with prostate cancer who did and did not receive ADT in Taiwan, after adjusting for potential confounders. Some physiological and genetic differences have been observed in different ethnicities. The UGT2B gene and androgen receptor genetic polymorphisms, namely, the CAG repeat length polymorphism, may possibly lead to differences in the cardiovascular risk between Asians and Caucasians. Asians have longer CAG repeat lengths than Caucasians, and testosterone suppression with ADT may lead to higher cardiovascular risk in Asians than in Caucasians, although we need more evidence to support this hypothesis [20]. Prostate cancer patients who received ADT seemed to be at an increased risk of having thromboembolic events, including arterial embolism, pulmonary embolism or deep venous thrombosis. A recent meta-analysis including 10 studies found that ADT increased the risk of thromboembolic events with a risk ratio of 1.43 (95% CI: 1.15-1.77, P = 0.001) [12]. Another database study analyzed 58,466 patients treated with ADT [13] and found that ADT was associated with an increased risk of thromboembolic events (adjusted hazard ratio = 1.56; 95% CI: 1.50-1.61; P < 0.0001).
Malignant disease is associated with the risk of thromboembolic disease and has long been recognized in clinical practice. Previous studies have reported this risk of thromboembolism in patients with prostate cancer, but these studies did not focus on ischemic stroke [21][22][23]. Other malignant diseases, such as breast cancer, malignant gliomas and ovarian cancer, are also associated with a high risk of thromboembolic events affecting 1 to 28% of patients [22]. Graus et al. [24] conducted an autopsy study to assess the prevalence of pathological findings of cerebrovascular disease in cancer patients and found that 14.6% of patients had pathologic evidence of cerebrovascular disease, and 7.4% had clinical symptoms of cerebrovascular disease. The risk factors for cerebrovascular disease were obscured by pathophysiologic factors associated with malignancy, including direct effects of the malignancy, hypercoagulable state, susceptibility to infections, treatment medication and the process of treatment or diagnosis. Cestari et al. [25] used a registry database to evaluate the incidence and type of strokes in cancer patients and showed that the three most common primary cancers were lung cancer (30%), brain cancer (9%) and prostate cancer (9%). The most common cause of stroke was embolic stroke, occurring in 52 (54%) patients. Forty-four (46%) patients had nonembolic stroke. The mechanism of stroke was partially due to hypercoagulability, and atherosclerosis only explained 22% of strokes in cancer patients. Outcome s were primarily decided by the underlying cancer stage.
Bosco et al. [26] included 8 studies in a meta-analysis and found a positive association between ADT and the risk of cardiovascular disease. In their meta-analysis, most studies focused on myocardial infarction, and one study focused on ADT and ischemic stroke. Azoulay et al. [27] used the General Practice Research Database, a primary care database from the United Kingdom, to conduct a nested casecontrol analysis and found that ADT increased the risk of stroke or transient ischemic attacks in patients with prostate cancer. In contrast to their study, our study found that ADT was not associated with the risk of ischemic stroke in prostate cancer. There are some differences between their study and our study. First, although both retrospective studies used a database, our study design was a cohort study, while Azoulay et al. used nested case-control analysis. In the presence of competing risks, the use of a nested case-control design results in greater bias compared to the use of a cohort design whether treatment occurs at baseline or varies over time and there is a single outcome. The cohort design has greater precision and a lower mean squared error in all scenarios [28]. Second, we used PS matching and adjusted for more cardiovascular medications. Third, their definition of stroke included hemorrhagic stroke because the stroke subtype (ischemic vs hemorrhagic) cannot be differentiated from the General Practice Research Database in the United Kingdom. Fourth, they excluded patients with evidence of metastases at diagnosis. We enrolled patients with all stages of prostate cancer.
Our population-based cohort study has some advantages. First, this was a large population-based cohort of patients with prostate cancer who were followed for up to 9 years. Second, the National Health Insurance program in Taiwan is compulsory for all citizens and covers almost all services that can be provided by a healthcare system. Third, we enrolled all patients with prostate cancer who were treated with ADT. Fourth, in Taiwan, patients with malignant diseases have catastrophic illness certification and do not need to pay a copayment for outpatient or inpatient care. Their applications are formally reviewed by more than two physicians and have a low probability of misdiagnosis. The prescriptions in Taiwan are also written by specialists (urologists or oncologists); thus, patients have a low probability of being misclassified as ADT users or ADT nonusers.
There are some limitations to our study. The NHIRD lacks some confounders, such as body mass index, cancer stage and smoking status. Therefore, we were unable to adjust for these confounders. This study evaluated the association of ADT and ischemic stroke in all prostate cancer patients regardless of the stage of disease. Second, there may be some asymptomatic ischemic stroke patients who were not diagnosed. These patients did not receive comprehensive routine brain magnetic resonance imaging examinations; thus, small stroke lesions may have been omitted. It is also possible that a rapidly massive fatal ischemic stroke in a prostate cancer patient with advanced disease could have been interpreted as the fatal end-stage of prostate cancer and therefore was not classified as our interest outcome of ischemic stroke. However, we believe that only a small number of cases may have been misclassified in this condition. Babiker et al. [29] suggested that prostasomes, submicron secretory granules from the prostate gland, evoke blood coagulation in prostate cancer and result in thromboembolic disease. Other studies [30,31] have also shown a link between testosterone and enhancement of fibrinolytic inhibition via increased synthesis of the plasminogen activator inhibitor plasminogen activator inhibitor-1, but our study found that the risk of ischemic stroke did not significantly increase in prostate cancer patients who received ADT. We may need a prospective study with a large sample size to assess the association between ischemic stroke and ADT, and whether there is a different thromboembolic mechanism between myocardial infarction and ischemic stroke in prostate cancer patients receiving ADT needs to be further investigated.
Conclusions
In this nationwide population-based study, we compared the risk of ischemic stroke between patients with prostate cancer who were and were not treated with ADT. Our study showed that patients with prostate cancer who received ADT did not have an increased risk of ischemic stroke, but they did have an increased risk of mortality, which may result from confounding by indication. | 2019-12-31T14:45:13.595Z | 2019-12-01T00:00:00.000 | {
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4775888 | pes2o/s2orc | v3-fos-license | The plant sesquiterpene lactone parthenolide inhibits Wnt/β-catenin signaling by blocking synthesis of the transcriptional regulators TCF4/LEF1
The Wnt/β-catenin pathway is essential for embryonic development and homeostasis, but excessive activation of this pathway is frequently observed in various human diseases, including cancer. Current therapeutic drugs targeting the Wnt pathway often lack sufficient efficacy, and new compounds targeting this pathway are therefore greatly needed. Here we report that the plant-derived natural product parthenolide (PTL), a sesquiterpene lactone, inhibits Wnt signaling. We found that PTL dose-dependently inhibits Wnt3a- and CHIR99021-induced transcriptional activity assessed with the T-cell factor (TCF)/lymphoid enhancer factor (LEF) firefly luciferase (TOPFlash) assay in HEK293 cells. Further investigations revealed that PTL decreases the levels of the transcription factors TCF4/LEF1 without affecting β-catenin stability or subcellular distribution. Moreover, this effect of PTL on TCF4/LEF1 was related to protein synthesis rather than to proteasome-mediated degradation. Of note, siRNA-mediated knockdown of RPL10, a ribosome protein PTL binds, substantially decreased TCF4/LEF1 protein levels and also Wnt3a-induced TOPFlash activities, suggesting a potential mechanism by which PTL may repress Wnt/β-catenin signaling. In summary, PTL binds RPL10 and thereby potently inhibits the Wnt/β-catenin pathway.
Since the first Wnt protein was identified 35 years ago, the Wnt/-catenin pathway has been well known for its essential role in both embryo development and adult tissue homeostasis (1). In the absence of Wnt signals, cytoplasm -catenin is phosphorylated by the Ser/Thr kinases GSK3 and CK1. The phosphorylated -catenin is then recognized and ubiquitylated by -TRCP and subjected to proteasome-mediated degradation. Upon binding of the Wnt proteins with the membrane receptor Frizzled and LRP5/6, -catenin is stabilized and shuttled into the nucleus, where it binds to the transcriptional factor TCFs/ LEF1 4 and activates expression of various target genes. Excessive activation of Wnt/-catenin signaling is frequently observed in a wide range of tumors, such as colorectal cancer, liver cancer, melanoma, prostate cancer, and breast cancer (2), making this pathway an attractive target for cancer therapy. Many efforts have been made to identify and develop drugs that can modulate Wnt/-catenin signaling, especially by targeting -catenin and its downstream players (3). Methyl 3-{[(4methylphenyl)sulfonyl]amino}benzoate is a selective inhibitor of Wnt/-catenin signaling that has been found to bind to -catenin and induce its proteasome-mediated degradation (4). PNU-74654 and iCRT3 are two other small molecules that inhibit Wnt signaling by disturbing TCF4 --catenin interaction (5,6). ICG-001 has been found to compete with CREB-binding protein for binding -catenin, thereby inhibiting Wnt signaling (7). Our laboratory recently reported NC043 as a Wnt signaling inhibitor by targeting CARF (collaborator of ARF) to disturb TCF4 and -catenin interaction (8,9). PTL, a sesquiterpene lactone, was first purified from the shoots of the medicinal plant feverfew (Tanacetum parthenium) (10,11), which is a Chinese folk medical plant and has been used to treat fever, headache, and arthritis for many years (12). In recent years, it has attracted wide attention for its considerable pharmacological activities, including antimicrobial, anti-inflammatory, and especially anticancer effects (13). PTL is well known for its activity of inhibiting NF-B signaling and is commonly used as an NF-B inhibitor (14). However, the mechanisms behind the anti-cancer effects of PTL have not been completely understood. Some studies indicate that PTL inhibits tumor growth by increasing reactive oxygen species (ROS) (15,16). PTL was also found to target epigenetic factors, such as DNMT1 and HDAC1, to inhibit tumor growth (17,18). Of note, in 2005, PTL was identified as the first small molecule that selectively kills cancer stem cells while sparing normal stem cells in acute and chronic myelogenous leukemia stem cell models (19). After that, the selective killing of cancer stem cells by PTL was also observed in a number of other cancers, such as bone, breast, melanoma, mesenchyme, and prostate cancer (20 -25). On the other hand, Wnt/-catenin signaling is well recognized for its essential role in the maintenance and selfrenewal of embryonic and adult stem cells. However, it remains unknown whether PTL may act on Wnt signaling to inhibit tumor growth. In this work, through high-throughput screening, we identified that PTL potently inhibits Wnt3a-induced TOPFlash activity. Further studies indicated that PTL acts through reducing TCF4/LEF1 synthesis via targeting RPL10.
PTL inhibits Wnt/-catenin signaling in HEK293 cells
In our high-throughput screening effort for small-molecule compounds modulating Wnt signaling, we identified the small molecule PTL (Fig. 1A). Our results showed that PTL dosedependently inhibited Wnt3a conditioned medium (CM)induced TOPFlash activity in HEK293cells (Fig. 1B). Next we examined the effect of PTL on the expression of Wnt target genes. We treated HEK293 cells with PTL and then carried out real-time PCR. As expected, PTL inhibited expression of the endogenous Wnt target genes Axin2 and NKD1 in a dose-dependent manner (Fig. 1C). To further explore the structureactivity relationship of PTL, we synthesized a series of PTL derivatives and tested their effects on TOPFlash activity. We found that PTL-4 and PTL-5 exhibited similar inhibitory effects as PTL, whereas PTL-1, PTL-2, and PTL-3 lost the inhibitory effect on TOPFlash reporter activity (Fig. 1D). These results indicated that the ␣,-unsaturated lactone and the 1(10) double bond of PTL might be the functional groups that are responsible for PTL inhibitory activity against Wnt signaling. Because PTL-4 can undergo retro-Michael additions to regenerate the HEK293 cells were transfected with TOPFlash plasmids. After 18 h of transfection, the cells were treated with DMSO or PTL for 1 h, followed by Wnt3a CM or control (Ctr) CM plus the same dose of PTL for another 6 h and then lysed for luciferase assays. RLC, relative luciferase count. C, PTL inhibits Wnt target gene expression. Expression of the Wnt target genes NKD1 and Axin2 was determined by quantitative real-time PCR and normalized to GAPDH expression. D, distinct effects of PTL and its derivates on TOPFlash reporter activity. HEK293 cells were treated with DMSO or 20 M PTL, PTL-1, PTL-2, PTL-3, PTL-4, or PTL-5 in the presence of either the Ctr CM or Wnt3a CM. Data represent the mean Ϯ S.D. from one experiment. Each experiment was repeated at least three times. **, p Ͻ 0.01; ***, p Ͻ 0.001; significant relative to vehicle control; ns, no statistical significance.
Parthenolide inhibits Wnt/-catenin signaling
parent molecule that is PTL, it was not surprising to find that PTL-4 inhibits Wnt/-catenin signaling, as does PTL.
PTL does not affect -catenin stability or subcellular distribution
As mentioned before, -catenin is the central player of canonical Wnt signaling, and its accumulation and nuclear translocation are hallmarks for activation of the signaling pathway. Thus, we wanted to ask whether PTL affects Wnt signaling through regulating the accumulation of -catenin. To answer this question, we first tested the effect of PTL on TOPFlash activity induced by a glycogen synthase kinase (GSK) inhibitor, CHIR99021 (26). We transfected HEK293 cells with TOPFlash plasmids, and then we treated cells with 2 M CHIR, followed by PTL treatment for the TOPFlash activity test. As shown in Fig. 2A, PTL showed similar inhibitory effects on TOPFlash luciferase reporter activity regardless of CHIR treatment. This result indicates that PTL functions downstream of the "-catenin destruction complex". Next we wanted to investigate the activity of PTL in the case of constant -catenin activation. For this purpose, we used the colon cancer cell lines Caco2 and SW480, which harbor adenomatous polyposis coli (APC) mutations, as well as HCT116 and SW48, which harbor -catenin mutations. We transfected the plasmids of TOP-Flash/FOPFlash to those colon cancer cells, followed by treatment with PTL. As shown in Fig. 2B, PTL efficiently inhibited TOPFlash reporter activity in all four cell lines, indicating that PTL acts downstream of -catenin accumulation. To further examine the effects of PTL on -catenin activation, we treated HEK293 cells with two doses of PTL in the presence of either control conditioned medium or Wnt3a conditioned medium. We then separated the cytosol and nucleus of the cells and subjected them to Western blot analysis using -catenin/tubulin/SP1 antibodies. As shown in Fig. 2C, PTL had no effect on endogenous -catenin protein levels or its subcellular distribution. Consistent with this result, in an immunofluorescence does not respond to Wnt singling and was used as a control. C, PTL has no effect on -catenin (-cat) stability and subcellular distribution. HEK293 cells were incubated with DMSO or two doses of PTL. After 1 h, cells were treated with control or Wnt3a CM containing the same dose of PTL for an additional 2 h. The cells were then fractioned as described under "Experimental procedures," and the samples were analyzed by Western blot analysis. Tubulin was used as a cytosolic marker, and SP1 was used as a nuclear marker. The immunoblots were quantified by densitometry, and the values are given beneath each band. D, immunofluorescence staining shows that PTL does not influence -catenin protein levels and distribution. HEK293 cells were treated with 10 M of PTL for 1 h, and then cells were incubated with control or Wnt3a CM containing the same dose of PTL for an additional 2 h. The cells were then fixed as described under "Experimental procedures," and the images were collected and analyzed by Opera. Scale bars ϭ 50 m. Data represent the mean Ϯ S.D. from one experiment. Each experiment was repeated at least three times. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; significant relative to vehicle control.
Parthenolide inhibits Wnt/-catenin signaling
assay with a -catenin-specific antibody, -catenin levels and its subcellular distribution showed no differences between the PTL-treated and control groups (Fig. 2D). These results further confirm that PTL does not affect -catenin stability or subcellular distribution.
PTL decreases TCF4/LEF1 protein levels
Our results above suggest that PTL acts downstream of -catenin accumulation and nuclear localization. Thus, PTL very likely acts on the TCF/LEF1 transcriptional factors in the nucleus. To test this possibility, we first examined whether PTL affects TCF/LEF1 protein levels. The human TCF family has four members: TCF1, TCF3, TCF4 and LEF1. Before testing the effect of PTL on TCF family proteins, we measured the mRNA levels of TCF1, TCF3, TCF4, and LEF1 in HEK293 cells. We found that the mRNA levels of TCF4 and LEF1 are much higher (nearly 40-to 50-fold) than that of TCF1 and TCF3 (Fig. S2). Therefore we carried out our further studies on TCF4 and LEF1. HEK293 cells were cultured in control CM or Wnt3a CM with 5 or 10 M PTL for 6 h. As shown in Fig. 3, A and B, both TCF4 and LEF1 protein levels were reduced by PTL in a dosedependent manner regardless of Wnt stimulation. PTL also reduced TCF4/LEF1 levels in a time-dependent manner (Fig. 3, C and D). In view of the significant differences in mRNA levels of TCF family members, although the TCF1 and TCF3 protein levels in HEK293 cells were also decreased after PTL treatment, PTL inhibition of Wnt signaling was mainly due to the decrease of TCF4 and LEF1 (Fig. S3). However, there was no difference between the levels of TCF4/LEF1 mRNAs before and after 10 M PTL treatment in the control conditioned medium, indicating that PTL could not function by regulating the transcription of TCF4/LEF1. Consistent with this, upon Wnt treatment, TCF4 mRNA levels were also not affected by PTL treatment, supporting that PTL does not function through affecting gene transcription (Fig. S2). However, LEF1 mRNA levels were apparently reduced by PTL. This is due to the fact that LEF1 is a target gene of Wnt signaling. Because PTL treatment leads to inhibition of Wnt signaling, it was not surprising to find a decrease of LEF1 mRNA levels by PTL treatment. Thus, this decrease of LEF1 mRNA levels is most likely due to inhibition of Wnt signaling rather than a direct effect on LEF1 transcription by PTL.
PTL decreases TCF4/LEF1 levels by blocking protein synthesis
Because our data suggest that PTL does not act through affecting gene transcription, we asked how PTL reduces TCF4/ LEF1 protein levels. One possibility is that PTL may facilitate proteasome-mediated degradation of TCF4/LEF1 proteins.
Parthenolide inhibits Wnt/-catenin signaling
Therefore, we blocked proteasome-induced degradation by using MG132. We treated HEK293 cells with 20 M MG132 followed by PTL treatment. We found that blocking proteasome-induced degradation with MG132 does not affect PTL activity in decreasing TCF4/LEF1 protein levels (Fig. 4A), indicating that PTL does not act through promoting TCF4/LEF1 degradation. Next we tested whether blocking protein synthesis by using CHX affects the PTL activity of inhibiting Wnt signaling. As shown in Fig. 4B, when protein synthesis process is blocked by CHX, treatment with PTL could not further decrease TCF4/LEF1 levels (Fig. 4B). To further address whether PTL inhibits protein synthesis of TCF4 and LEF1, we carried out a nonradioactive metabolic labeling assay. As shown in Fig. 4, C and D, PTL significantly inhibits the protein synthesis of both TCF4 and LEF1. Thus, we conclude that PTL decreases TCF4 and LEF1 by blocking protein synthesis rather than affecting TCF4/LEF1 protein stability.
RPL10 knockdown decreases TCF4/LEF1 protein levels
RPL10, a ribosome protein, was recently reported as a target of DMAPT, which is a water-soluble analogue of PTL (27). Chen et al. uncovered that DMAPT targets and decreases RPL10, leading to reduced protein levels of p65 and IKK␥ (27). This mechanism is responsible at least partially for DMAPT inhibitory activity against NF-B (27). To test whether PTL could bind to RPL10, we first synthesized biotinylated PTL (Fig. 5A) and confirmed that biotin-PTL dose-dependently inhibits Wnt signaling, as does PTL (Fig. 5B). Next we overexpressed HA-tagged human RPL10 in HEK293T cells and carried out a pulldown assay using biotinylated PTL as the bait with or without addition of unlabeled PTL. As shown in Fig. 5C, biotin-PTL captured RPL10, and this binding could be competed off by free PTL, supporting PRL10 as the target of PTL. To examine the role of PRL10 in Wnt signaling, we deprived HEK293 cells of RPL10 using the method of RNAi. As shown in Fig. 5D, knockdown of RPL10 significantly reduced TOPFlash reporter activity. Moreover, RPL10 deprivation led to a marked decrease in TCF4 and LEF1 levels (Fig. 5E). Our results, combined with the findings of Chen et al., confirmed that PTL reduces TCF4/LEF1 protein levels by targeting the ribosome protein RPL10.
PTL inhibits proliferation of colon cancer cells
As mentioned before, overactivation of Wnt/-catenin signaling is closely related to tumor formation and progression. One anecdote is colon cancer. More than 80% of colon cancers harbor mutations in the scaffold protein APC and have overactivated Wnt signaling (28). Thus, we used human colon cancer cells to test the potential of PTL in cancer therapy. We found that PTL significantly reduced TCF4 levels in the colon cancer cell lines SW480, Caco2, and HT29 but has no apparent effects on -catenin levels in these cells (Fig. 6A). Consistently, PTL inhibited expression of the Wnt target gene NKD1 in these three colon cancer cell lines in a dose-dependent manner (Fig. 6B). A colony-forming assay also showed that PTL inhibited colon cancer cell proliferation at a level similar to its inhibitory activity toward Wnt signaling (Fig. 6C). These results demonstrate the potential of PTL in the treatment of colon cancers that are mostly driven by overactivated Wnt signaling.
Discussion
The Wnt/-catenin signaling pathway is one of the most important pathways in development and is involved in many aspects of tumorigenesis. Considerable efforts have been made to identify and develop effective inhibitors against the Wnt signaling pathway. However, successes in finding inhibitors of this pathway are very limited, and so far, no drugs specific for Wnt signaling have been approved for clinical applications. In this
TCF/LEF1 transcription factors are the downstream effectors of Wnt/-catenin signaling. Recent genome-wide studies in mammals and Drosophila suggest that all direct activation of -catenin target gene expression requires TCF/LEF1 as the final effectors (29,30). Moreover, the levels of TCF/LEF1 in colon cancer cells are substantially increased compared with normal tissues, and TCF4 expression is positively correlated with a poor prognosis in resectable esophageal squamous cell carcinoma (31,32). Therefore, targeting TCF/LEF1 would be a potentially effective way in reversing excessive activation of Wnt/-catenin signaling in these cancer cells. However, few of the compounds have so far been reported to target TCF/LEF1. Thus, our findings provide a valuable prototype for new therapeutic agents in treating tumors driven by overactivated Wnt signaling.
RPL10, a component of the 60S ribosome, functions in protein synthesis, and its mutations have been found in T-cell acute lymphoblastic leukemia patients (33). Here we found that TCF4/LEF1 levels were significantly reduced by deprivation of RPL10, but -catenin levels were not affected. An interesting question is whether the ribosome has selectivity in mediating protein translation. Recently, Shi et al. reported that heterogeneous ribosomes tend to translate distinct subpools of mRNAs genome-wide (34). Sendoel et al. reported that, during tumor initiation, the translational apparatus is redirected toward noncanonical upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs (35). Thus, it is possible that RPL10 may selectively regulate TCF4/LEF1 synthesis without affecting -catenin translation. Further studies are required to investigate this possibility and address the mechanisms behind how RPL10 regulates TCF/LEF synthesis. PTL has been reported to specially inhibit cancer stem cell growth without killing normal tissue cells in leukemia and solid tumors (13). Wnt/-catenin signaling is known to be essential for the maintenance of stem cells (1). Our work shows that PTL inhibits colon cancer cell growth. Whether this inhibition of colon cancer cell growth is caused by inhibition of Wnt/catenin signaling in cancer stem cells also warrants further study.
In summary, our study identifies PTL as a new inhibitor of Wnt/-catenin signaling by targeting RPL10 and blocking TCF4/LEF1 synthesis. Given that DMAPT, a more hydrophilic form of PTL, has been approved for clinical trial for hematologic malignancies, our findings provide proof of concept for the use of PTL as a potential drug for treating Wnt-driven tumors.
Isolation of PTL, synthesis of biotin-PTL, and PTL derivatives
For isolation of PTL, air-dried powdered leaves of Magnolia grandiflora (10.0 kg) were extracted with MeOH (20 liters ϫ 3) under reflux three times (4, 3, and 3 h, respectively) at 80°C. The combined MeOH extracts were concentrated in a vacuum to give a crude residue (600 g) that was suspended in water. The water layer was successively partitioned with ethyl acetate (EtOAc) (10 liters ϫ 3). The EtOAc portion (220 g) was repeatedly subjected to a silica gel column to get parthenolide (2.0 g). The purity of parthenolide was greater than 95% as determined by TLC and NMR spectra. Biotin-PTL and PTL derivatives were synthesized as described in Fig. S1.
Antibodies and reagents
Antibodies for -catenin (BD Transduction Laboratories), TCF4 (Millipore), LEF1 (Cell Signaling Technology), SP1 (Sigma), tubulin (Sigma), actin (Santa Cruz Biotechnology), and HA (Covance) were utilized in this study. Wnt3a CM and control medium were described previously (36). CHIR-99021 was purchased from Selleckchem. MG132 and CHX were purchased from Sigma-Aldrich. The full-length complementary DNA of human RPL10 was generated by PCR from HEK293 complementary DNA libraries, cloned into the mammalian expression vector pCMV tagged with HA, and verified by DNA sequencing. The siRNA sequences of RPL10 #1 and #2 were 5Ј-GGCCAAGUUAUCAUGUCCA-3Ј and 5Ј-CUGAUGC-CAAGAUUCGCAU-3Ј, respectively. The negative control siRNA sequence was 5Ј-UUCUCCGAACGUGUCACGU-3Ј. Binding of Wnt protein with its receptors induces cytoplasmic -catenin (-cat) to aggregate and translocate into the nucleus. In the nucleus, -catenin binds to TCF/LEF to induce Wnt target gene expression. When PTL binds to RPL10, TCF/LEF levels are reduced, leading to inhibition of Wnt/catenin signaling.
Parthenolide inhibits Wnt/-catenin signaling
cells were transfected by Lipofectamine 3000 (Invitrogen). For reporter gene assays, cells were seeded in 24-well plates. Each well of HEK293 cells was transfected with 250 ng of plasmids in total, including 30 ng of TOPFlash and 25 ng of GFP. The LacZ plasmid was added to equalize the total amount of transfected DNA. After 18 h, the cells were treated with Wnt3a conditioned medium or control medium for another 6 h and then lysed for luciferase assays. Each well of SW480, Caco2, HCT116, and SW48 cells was transfected with 250 ng of plasmids in total, including 50 ng of TOPFlash or FOPFlash, 50 ng of GFP, and 150 ng of LacZ. The cells were lysed for luciferase assays 24 h after transfection. The GFP expression levels were determined for normalization as described previously (37).
Drug treatment and Western blot analysis
Cells were treated with the indicated dose of drugs and harvested with 2ϫ loading buffer (0.3 M Tris-HCl, 10% SDS, 6% -mercaptoethanol, 50% glycerol, and 0.05% bromphenol blue). Then proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat dry milk for 1 h and then incubated with primary antibodies for 1 h at room temperature or overnight at 4°C. After being washed, membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibodies (Thermo Scientific) for 1 h. Results were visualized with Tanon 5200.
Cytosol and nucleus fractionation
HEK293 cells were grown in 6-well plates, harvested with a cell scraper into 1.5 ml of PBS, and spun at 700 ϫ g for 10 min. Then the cytosol and nucleus fractions were separated with a nuclear and cytoplasmic extraction kit (CW0199S, CWBIO) according to the manufacturer's instructions.
Immunofluorescence staining
HEK293 cells were seeded into a 96-well plate to make 6000 cells/well. 48 h later, the indicated dose of PTL was added to the wells for 1 h, followed by Wnt3a conditioned medium or control conditioned medium plus the same dose of PTL for an additional 2 h. Then cells were fixed with 4% paraformaldehyde (Alfa Aesar, 30525894) for 15 min and permeabilized with PBST (PBS and 0.1% Triton-X100) for 20 min at room temperature. The fixed samples were incubated with a primary antibody against -catenin overnight at 4°C, followed by Cy3-conjugated anti-mouse secondary antibody and 4Ј,6-diamidino-2-phenylindole. For each well, 10 random-field fluorescence images were captured by the Opera LX high-content confocal imaging system with a ϫ20 Air-LUCPLFLN objective (numerical aperture ϭ 0.45, PerkinElmer Life Sciences) (38).
Nonradioactive metabolic labeling assay
The nonradioactive metabolic labeling assay mainly includes four parts: nascent protein labeling, immunoprecipitation of the protein of interest (for example, TCF4), Click-iT reaction of the immunoprecipitated protein, and detection of the nascent protein of interest by immunoblotting (39). For nascent protein labeling, HEK293 cells at 80 -90% confluency were incubated in complete normal medium with the indicated dose of PTL or CHX for 1 h. Then HEK293 cells were washed once with warm PBS and incubated with methionine-free medium (Thermo Fisher Scientific) together with PTL or CHX at 37°C for 1 h to deplete methionine reserves. Cells were further incubated with methionine-free medium plus 40 M methionine analog L-homopropargylglycine (Thermo Fisher Scientific) with PTL or CHX for 4 h instead of the traditional [ 35 S]methionine. 4 h later, cells were harvested in lysis buffer (1% SDS in 50 mM Tris-HCl [pH 8.0]) with protease inhibitors. The immunoprecipitation experiment was performed using protein A/G Plus-agarose (Santa Cruz Biotechnology) and antibody against TCF4 (Millipore) or LEF1 (Cell Signaling Technology), following the recommended procedures. The immunoprecipitated TCF4 or LEF1 was further labeled with tetramethylrhodamine (TAMRA) azide (Thermo Fisher Scientific) via a copper-catalyzed reaction between azide and L-homopropargylglycine using the Click-iT reaction buffer kit (Thermo Fisher Scientific). The TAMRA-labeled protein was detected by standard immunoblotting using an antibody against TAMRA azide (Thermo Fisher Scientific). Of note, the molecular mass of detected protein should be a bit larger than expected, and the increased molecular mass should be the molecular mass of TAMRA azide (555 Da) multiplied by the number of methionine residues in the labeled protein, and this labeled protein is probably not recognized by the normal antibody because of the integration of TAMRA azide.
Biotin pulldown assay
HEK293T cells were transfected with HA-tagged RPL10 plasmids. After 24 h of transfection, the cells were lysed with 500 l of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Triton X-100, and 5 mM EDTA [pH 7.4]) containing proteinase inhibitors and centrifuged at 16,000 ϫ g for 15 min at 4°C. 20 l of the supernatant was used as the input by adding 20 l of 2ϫ loading buffer, and the remaining 480 l of supernatant was mixed with biotin-PTL (10 M) together with streptavidinagarose (Novex, Carlsbad, CA) at 4°C for 1 h. For competition experiments, PTL (20 M) was added to the mixture. The beads
Parthenolide inhibits Wnt/-catenin signaling
were washed three times and resuspended in 40 l of 2ϫ loading buffer.
Colony-forming assays
For colony formation assays, cells were seeded in 6-well plates (1500 cells/well for Caco2 cells, 2000 cells/well for HT29 cells, and 1000 cells/well for SW480 cells). The cells were treated with control (DMSO) or PTL with medium changes every 3 days until visible colonies formed. At the end of the experiment, cells were washed with PBS twice and fixed with cold methanol for 30 min. After fixation, cells were stained with 2% crystal violet (25% methanol) at room temperature for 1 h. After staining, the plates were rinsed with distilled water and dried.
Statistical analysis
An unpaired Student's t test was used to evaluate the difference between the two treatments. p Ͻ 0.05 was considered to be statistically significant, and a p Ͻ 0.01 was considered to be extremely statistically significant. Statistical analyses were performed using SYSTAT SigmaPlot 10.0 statistics software.
Author contributions-X. Z., C. Y., C. T., C. L., and F. N. conducted the experiments. X. S., R. Z., X. H., D. W., and L. L. analyzed the results. X. Z., C. Y., X. S., and L. L. wrote the manuscript with comments from all authors. L. L. and X. H. guided all aspects of this study. | 2018-04-03T05:12:19.443Z | 2018-02-09T00:00:00.000 | {
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15382604 | pes2o/s2orc | v3-fos-license | Three-Dimensional Anthropometric Database of Attractive Caucasian Women: Standards and Comparisons
Abstract The aim of this paper is to develop a database to determine a new biomorphometric standard of attractiveness. Sampling was carried out using noninvasive three-dimensional relief methods to measure the soft tissues of the face. These anthropometric measurements were analyzed to verify the existence of any canons with respect to shape, size, and measurement proportions which proved to be significant with regard to the aesthetics of the face. Finally, the anthropometric parameters obtained were compared with findings described in the international literature. The study sample was made up competitors in the Miss Italy 2010 and 2009 beauty contest. The three-dimensional (3D) scanning of soft tissue surfaces allowed 3D digital models of the faces and the spatial 3D coordinates of 25 anthropometric landmarks to be obtained and used to calculate linear and angular measurements. A paired Student t test for the analysis of the means allowed 3 key questions in the study of biomorphometric parameters of the face to be addressed through comparison with the data available in the literature. The question of statistical evidence for the samples analyzed being members of the populations samples reported in literature was also addressed. The critical analysis of the data helped to identify the anthropometric measurements of the upper, middle, and lower thirds of the face, variations in which have a major influence on the attractiveness of the face. These changes involve facial width, height, and depth. Changes in measurements of length, angles, and proportions found in the sample considered were also analyzed.
M ore and more patients feel the need to undergo surgical, orthodontic, and/or prosthetic treatments, not only for the restoration of good health and function of the cranio-maxillo-facial structures, but also to improve facial aesthetics and quality of life. Therefore for the maxillofacial surgeon, the orthodontist, the prosthetist it is essential to have diagnostic tools that enable them to make more detailed morphological analysis and to compare individual patient's data with updated date base, related to the patient's ethnicity. To respond to these needs, our scientific research work was started in 2010 and involved a team of doctors, orthodontists, and engineers to study the anthropometric measures of attractive young women of the same ethnic group to create a new update data base.
During the diagnostic and monitoring phase of orthodontic treatments 1 and maxillofacial surgery, 2 direct measurements of facial characteristics of the patient are performed. Historically, measurements of craniofacial structures are commonly used by most orthodontists and maxillofacial surgeons. First, specific landmarks are identified and derived from two-dimensional (2D) radiographs; 3 further craniofacial components, including measurements of distances, angles, and proportions are then calculated. 4,5 These measurements allowed us to produce diagnostic methods of analysis and anthropometric classification of craniofacial structures which will be referred to as cephalometric analysis.
Over the last few decades, extensive samples of 2D facial databases which include anthropometric characteristics have been recorded. These different populations have been analyzed on the basis of ethnicity, age, and gender, in both normal subjects 6 and subjects affected by pathological conditions. [7][8][9] The advent of three-dimensional (3D) radiographic technologies (CT and cone beam CT) has made it necessary to revise traditional methods of cephalometric analysis and adapt them to 3D information about reference points in space (landmarks), resulting in the need to create new dimensional databases. 10,11 Historically, the surfaces of the soft tissues of the face (facial surface anthropometry) have received less attention. The difficulties of identifying and measuring these landmarks and soft tissue deformability are major challenges. Despite these methodological difficulties, over the years there has been an increasing need to be able to measure and analyze these structures accurately and reliably, 12,13 as they are immediately caught and play an important role in the visual impact of the facial aesthetics of the patient. 14 Few studies [15][16][17][18][19] relating to these topics are reported in the literature prior to 2000.
The need for less invasive diagnostic procedures, 20 combined with the availability of X-ray-free systems with the advent of 3D technologies such as laser scanning, structured light, 21 stereophotogrammetry, 22 and multi-image photogrammetry, has provided researchers and specialists with new, powerful, computerized 3D methods to detect and analyze anthropometric facial features, with a largely positive impact on diagnostics and monitoring of treatments.
A growing number of researchers conduct anthropometric studies on samples of populations that reflect different anthropometric characteristics based on ethnic origin, 23 geographical place of origin, [24][25][26] age, 27 gender, 28 facial attractiveness, 29 or the presence of certain diseases, 7 to create new anthropometric databases of facial characteristics.
Groups of universities have developed these databases and made them readily available on the Internet. 30,31
Aims of the Study
In this research, specific equipment and a precise investigation protocol previously described by the authors 32,33 were applied to scan and analyze the faces of a sample of young Italian women finalists in a national beauty contest (Miss Italia 2010), referred to in this paper as ''attractive 2010,'' and to compare it with other similar samples found in the literature.
First step: analysis of the 2 main anthropometric facial measurements (height and width).
The first step was to consider the 2 main anthropometric facial measurements (facial height and width) and the relationship between them, to investigate whether a uniform distribution of the sample can be observed by grouping the individuals into classes based on the following variables: facial height (Tr-Sn-Me), facial width (T_l-T_r) and the ratio of these 2 measurements ((Tr-Sn-Me)/(T_l-T_r)).
A further aim of this study was to verify whether statistical evidence of a scale factor relationship between facial height and facial width, based upon the measurements taken within this sample, could be demonstrated.
Second step: comparison with the data available in the literature. With regard to anthropometric facial parameters, it was necessary to address 3 key questions: In studies found in the literature, do the samples analyzed (attractive women) and reference samples (''normal'' women) belong-based on statistical evidence-to the same population, or are the differences such as to suggest a population of ''attractive'' subjects that is different from the population of ''normal'' subjects? Do the Miss Italia 2010 sample (66 women, ''attractive 2010'') and the ''normal'' reference samples adopted by other researchers in the literature belong-based on statistical evidence-to the same population, or are the differences such as to suggest that the ''attractive 2010'' population is different from the populations of ''normal'' reference samples? Do the sample analyzed in the Miss Italia 2010 contest (''attractive 2010'') and the samples of attractive women analyzed by other researchers belong-based on statistical evidence-to the same population, or are the differences such as to suggest that the ''attractive 2010'' population is different from the populations of ''attractive'' women in other samples?
Taking of the Sample
A 5-camera photogrammetric facial scanner ( Fig. 1) was used, applying the method and the scanning protocol for facial samples described and referenced in (32,33).
The study sample (''attractive 2010'') consisted of 60 contestants and 4 finalists in the Miss Italia 2010 beauty contest, and the firstand second-placed contestants in the Miss Italia 2009 contest. Facial data was acquired using close range stereophotogrammetry following a specific, standardized protocol, described in detail in (33,34). Table 1 and shown in Figure 2 was completed by a direct method based on visual inspection and palpation, with the exception of points Ex_r, Ex_l, En_r, En_l, Ey_r, and Ey_l, which were marked afterward on photographs, and not directly on the face; for each point the precision of the measurement for coordinates x, y, and z and the precision of the vector length for the linear measurements were calculated.
Data and Measurement Processing
The 3D reconstruction of virtual faces enabled us to conduct our qualitative and quantitative analysis, to measure the surfaces and to To assess facial attractiveness, 13 linear and 9 angular measurements were considered, as shown in Table 2.
The paired Student t test analysis was used to answer the 3 key questions formulated above to approach the study of biomorphometric facial parameters, by means of comparisons with the data available in the literature (in the literature, samples were reported for each measurement: average, standard deviation, and sample size).
Tables 3-6 report the average, minimum and maximum, and standard deviation for each linear and angular measurement calculated for the sample analyzed here.
RESULTS
From the sample analyzed, 58 linear measurements, 22 angular measurements, 4 linear relationships between measurements and 1 percentage ratio between linear measurements were obtained, a total of 5610 data items, as shown in Tables 3-6.
The minimum linear measurement, 0.14 mm, was the ''lower lip to E-line distance'' measurement Li-(Prn-Pg); the maximum linear measurement, 140 mm, was the ''middle facial width'' measurement T_r-T_l. The tables for each linear measurement show the average, minimum and maximum measurement uncertainty (accuracy) values, the standard deviation, and the variation range.
Analysis of this very large amount of data enabled the main anthropometric parameters influencing facial attractiveness of the upper, middle, and lower thirds of the face to be identified. These parameters express variations in the measurements in the 3 spatial planes, and involve facial width, and height. In the sample, changes in measurements of length, angles, and proportions were also analyzed, to verify the existence of any canons with respect to shape, size, and measurement proportions which proved to be significant with regard to the aesthetics of the face.
With regard to the landmarks T_r and T_l, this problem may be caused principally by 2 factors: they may not be clearly visible due to the scanner being partially masked by the hair, and the anatomical points are classified and recognized only by a pair of cameras, which are located ipsilaterally in relation to the points.
For the landmarks Ex_r-Ex_l, En_r-En_l, Ey_r-Ey_l, the cause of uncertainty may be due to the fact that they were marked on the photographs and not directly on the face.
Analysis of the vertical linear measurements along the midline shows a much smaller uncertainty value (average 0.15, minimum 0.09, maximum 0.21 mm). Figure 3 shows the average, minimum, and maximum values measured on the sample, as well as the standard deviation, for linear measurements, and Figure 4 for angular measurement. The analysis of linear measurements obtained shows very different values between individuals, probably as a function of changes in morphology and facial type of the finalists analyzed.
Analysis of the Two Main Anthropometric Facial Measurements (Height and Width)
Some of the most significant facial measurements of the face were grouped to verify whether the data were normally distributed and whether there are any evident relationships between the measurements analyzed.
By grouping measurements of facial height (Tr-Sn-Me), width (T_l-T_r) and the ratio of these 2 measurements ((Tr-Sn-Me)/(T_l-T_r)), we may observe that the sample is neither normally distributed nor has the typical distribution of a Gaussian curve; only the distribution of facial height is closer to a normal distribution (Fig. 5).
The ''attractive 2010'' sample was then checked for evidence of a scale factor relationship between the faces, expressed through a relationship of proportionality between the measurements of facial height and width. Figure 6 shows the plots relating to the analysis of the distance of these measurements from the normal distribution. The P value (between 0 and 1) represents the value for which the null hypothesis is to be rejected at an alpha (a) level equal to 0.05. The AD value refers to the Anderson-Darling test, which compares the empirical cumulative distribution of the sample with the cumulative distribution which would be expected if the sample were distributed following a Gaussian curve.
To check for a correlation between facial height and width (Fig. 7), it is observed that the correlation coefficient of Pearson between Tr-Sn-Me and T_r-T_l is rather low, at 0.387 (with a P value of 0.001). The hypothesis of a statistically significant correlation between these 2 anthropometric facial parameters (according to which an increase in facial height in the ''attractive'' faces should correspond to a proportional increase also in facial width) was not confirmed: in fact, the distribution of measurements taken does not reflect a constant relationship of proportionality between the 2 parameters, as shown by the graph in Figure 7. Figure 8 reports the values of the main measurements of facial height and width. The data from the sample were ranked by increasing values with respect to the facial height/width ratio; they highlight the absence of a Gaussian distribution, as can be expected in a normal population. In the study of facial anthropometric parameters, we must verify the 3 key hypotheses formulated.
Hypothesis 1
It was necessary to determine whether the samples analyzed (''attractive'') and the reference samples (''normal'') of the studies found in the literature belong-based on statistical evidence-to the same population, or whether the differences are such as to suggest a population of ''attractive'' subjects that is different from the population of ''normal'' subjects. Using the paired Student t test for the analysis of the mean, assuming the difference to be NS (not significant), if alpha > 0.05 (probability > 95%), the differences with alpha ¼ 0.00 (probability 100%) will certainly be significant.
The scientific literature contains very few articles that report measurements and specific analyses on attractive women, compared with the normal population. Ferrario et al 35 published data on 10 ''beauties,'' which they compared with a reference sample of 40 ''normal'' women (standard). From an analysis of their data, it can be deduced that only a few measurements differ to such an extent as to constitute statistical evidence that they belong to samples of 2 different populations.
Sforza et al 37 published data on 24 ''beauties'' measured in 2006, which they compared with a reference sample of 71 ''normal'' girls. In this case, too, it was found that only some measurements differed to such an extent as to constitute statistical evidence that they belong to samples of 2 different populations.
Sforza et al 37 published data on 24 ''beauties'' measured in 2007, which they compared with a reference sample of 71 ''normal'' girls. As with the analysis of previous samples, only some measurements differed to such an extent as to constitute statistical evidence that they belong to samples of 2 different populations.
Sforza et al 36 published data on 23 ''attractive girls'' aged between 13 and 15, which they compared with a reference sample of 51 ''normal'' girls (standard). From an analysis of their data, it can be deduced that no measurement deviates to such an extent as to constitute statistical evidence for belonging to samples of 2 different populations. Table 7 shows the measurements which, in the analyses mentioned above, diverge to such an extent as to constitute statistical evidence of belonging to samples of 2 different populations.
Hypothesis 2
It was necessary to determine whether the ''attractive 2010'' sample and the reference sample (standard) adopted by other researchers in the works mentioned above belong-based on statistical evidence-to the same population, or whether they differ to such an extent as to suggest that the ''attractive 2010'' population can be considered different from the ''normal'' reference population. In this case, too, a paired Student t test was used; assuming the difference to be NS (not significant), if alpha >0.05 (probability >95%), the differences with alpha ¼ 0.00 (probability 100%) will certainly be significant.
The data for the ''normal'' sample published by Ferrario et al 35 in 1995 and data for the ''attractive 2010'' sample were thus compared. In this case, strong statistical evidence was found to In summary, through the analysis of comparative statistics on multiple samples, it was possible to identify the facial anthropometric parameters that provide statistical evidence of the samples belonging to 2 different populations (''attractive'' and ''normal'') and therefore define the anthropometric characteristics of attractiveness (Table 8) With regard to angular measurements, the major influences are those relating to:
Hypothesis 3
It was necessary to determine whether the ''attractive 2010'' sample and other ''attractive'' samples cited in this paper belongbased on statistical evidence-to the same population, or whether the differences are such as to suggest that the ''attractive 2010'' sample belongs to a population that can be considered different from the other ''attractive'' reference samples. Here, too, a paired Student t test was used; assuming the difference to be NS (not significant), if alpha >0.05 (probability >95%), the differences with alpha ¼ 0.00 (probability 100%) will certainly be significant.
In 1969, Peck and Peck 38 analyzed the characteristic angles of the profile of 52 actresses with a photographic method; it may be observed that the values of the data reported in (38) and those found in the ''attractive 2010'' sample are still perfectly comparable, despite the fact that over 40 years elapsed between the findings of the 2 samples and that the 2 samples relate to different ethnic groups.
The comparison shows that only the mandibular angle and the total vertical extent appear to be slightly higher in measurements on 3D models of the ''attractive 2010'' sample compared with the measurements made by Peck and Peck of landmarks on the skin for their ''attractive 1969'' sample. 38 The comparison between the anthropometric measurements of the ''attractive'' women in Sforza et al in 1995 35 and those found in the ''attractive 2010'' sample did not find statistical evidence of the samples belonging to 2 different populations. Similarly, the comparison between the anthropometric measurements of the ''attractive'' sample in Sforza et al 36 For angular measurements, the major influences are those relating to: upper facial convexity Ex_r-N-Ex_l; nasolabial angles Prn-Sn-Ls, T_l-N-T_r, T_l-Prn-T_r, angles T_l-Pg-T_r, (Sn-Ls)^(Sl-Pg), maxillofacial angle Mf (Pg-N-Ls). A further significant measurement is the ratio between the sizes (Sn-Pg)/(N-Sn) Â 100.
CONCLUSIONS
This research work highlights the importance of having an updated date base relative to the facial anthropometric measures of attractive young women of the same ethnic group, to set a proper diagnosis and appropriate therapy in female patients with outcomes of facial trauma or syndromic or suffering from malformations of the cranio-facial region.
By comparing the data obtained from the ''attractive 2010'' sample with data reported in the literature for ''normal'' and ''attractive'' samples, it was possible to identify the main anthropometric parameters which influence facial attractiveness at the levels of upper, middle, and lower facial third, and in reference to facial width, height, and depth, comparing linear, angular measurements and proportions. The measurements found in the sample analyzed do not have a normal distribution.
It was also verified whether or not the faces of attractive women show statistical evidence of a relationship between scale measurements: for example, the existence of proportionality between height and facial width measurements, according to which any increase in facial height should correspond to a proportional increase in facial width. This hypothesis was not, however, confirmed by the results of the measurements made: in fact, the distribution of any measurement does not reflect a constant relationship of proportionality between the 2 parameters.
Our study of this data also shows that there are some statistically significant differences between most of the measurements performed on the sample of ''attractive'' and ''normal'' samples in the literature: statistically significant differences were found for some measurements, and these measurements should be considered elements of evaluation for facial attractiveness.
From a comparison of the ''attractive 2010'' sample and the reference samples (standard) adopted by other researchers in the literature, it is not possible to deduce that these samples belong to the same population. In contrast, from a comparison of the ''attractive 2010'' sample and samples of attractive women analyzed by the researchers mentioned above, it can be deduced that they belong to the same population. Indeed, no significant differences were observed between most of the measurements of the 2 samples, and statistically significant differences were found only for some measurements. | 2018-04-03T02:19:29.009Z | 2016-07-25T00:00:00.000 | {
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234457329 | pes2o/s2orc | v3-fos-license | Site-Specific Conjugation of Cell Wall Polyrhamnose to Protein SpyAD Envisioning a Safe Universal Group A Streptococcal Vaccine
Abstract Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus (GAS) is a public health priority. The species defining group A cell wall carbohydrate (GAC, Lancefield antigen) can be engineered to remove its immunodominant N-acetylglucosamine (GlcNAc) side chain, implicated in provoking autoimmune cross-reactivity in rheumatic heart disease, leaving its polyrhamnose core (GACPR). Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization. Instead of conjugation to a standard carrier protein, we selected SpyAD, a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen. SpyAD was synthesized using the XpressTM cell-free protein expression system, incorporating a non-natural amino acid to which GACPR was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes. The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils. Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR, together with candidate vaccine antigens streptolysin O and C5a peptidase, protected against GAS challenge in a systemic infection model and localized skin infection model, without evidence of cross reactivity to human heart or brain tissue epitopes. This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.
Introduction
Group A Streptococcus (GAS, Streptococcus pyogenes) is one of the most important human bacterial pathogens, estimated to cause more than 600 million cases of pharyngitis ('strep throat') and 100 million cases of impetigo annually across the globe. 1 In particular, pharyngitis is highly prevalent in school-aged children and a major source of antibiotic prescriptions worldwide, driving selective pressure for antibiotic resistance throughout the human microflora. [2][3][4] In recent decades, GAS has been increasingly associated with severe invasive forms of infection, sometimes in previously healthy individuals, including sepsis, necrotizing fasciitis, and toxic shock syndrome. Finally, GAS is the trigger for important post-infectious immune-mediated diseases, in particular poststreptococcal glomerulonephritis, the most common cause of childhood glomerulonephritis worldwide, 5 and acute rheumatic fever (ARF) and rheumatic heart disease (RHD), a leading cause of cardiovascular mortality in the developing world. 6 At least 33 million people are currently affected by RHD, with approximately 275,000 deaths annually (60% age <70) and 9 million disability-adjusted life years lost. 7 Almost all RHD deaths occur in low-income and middle-income countries. 1 As a result of all its disease manifestations, GAS ranks among the top ten infectious causes of human mortality. 8 Despite the high disease burden and global demand, there is to date no safe and efficacious commercial vaccine against GAS. A number of phenotypic features of the pathogen pose particular challenges to vaccine development. First, in contrast to the diverse capsular polysaccharides of Streptococcus pneumoniae that are the basis of multivalent glycoconjugate vaccines in clinical use worldwide, 9,10 GAS possesses an invariant capsule of high molecular weight hyaluronic acid (HA). The capsule is thus structurally identical to the ubiquitous HA present in human connective tissues, and therefore immunologically inert. [11][12][13][14] Instead, GAS serology is classically assigned by its most abundant and highly immunogenic surface-anchored protein, the M protein, which poses a second vaccine challenge-M proteins are highly polymorphic. There are over 220 described sequence variants of the encoding emm gene, classified into 48 emm-clusters of closely related M proteins sharing functional and structural properties. 15 Heterologous M proteins do not typically elicit cross-protective immunity to M-types from other clusters. 16 Finally, an unique challenge to GAS vaccine development is its link to the serious post-infectious, immune-mediated ARF and RHD, whose precise molecular pathogenesis remains poorly understood. 7,17,18 A prevailing theory holds that GAS molecular mimicry of host cell epitopes mediates B and/or T cell crossreactions with human tissue antigens in the heart, brain, or other tissues. 19 Beginning in the late 1970's, the US Food and Drug Administration made the rare decision to suspend GAS vaccine development (21 Code of Federal Regulations 610. 19) for nearly three decades. This decision followed an immunization study in which three participants developed suspected or definite ARF. 20 In 2004, the US National Institutes of Allergy and Infectious Diseases convened experts to review epidemiologic, microbiological, and immunologic factors involved in preclinical and clinical development of a safe and effective GAS vaccine that facilitated lifting of the Food and Drug Administration ban. 21 Their summary report concluded that molecular mimicry represented a major obstacle to vaccine development, and that GAS antigens including M proteins and group A carbohydrate (GAC) possess epitopes linked to B and/or T cell reactivity to human tissue antigens. The panel recommended: "Because the precise role of molecular mimicry in the pathogenesis of ARF has not been established, every effort should be made to exclude tissue cross-reactive epitopes during vaccine development." 21 Recently, the World Health Organization, International Vaccine Institute, Wellcome Trust, Bill and Melinda Gates Foundation and other stakeholders convened in advisories on GAS vaccine development to address scientific challenges for this paramount global health need. 4,22 Ideal candidate antigens for inclusion in a GAS vaccine would be (a) highly immunogenic and elicit antibodies that promote opsonophagocytosis or inhibit virulence, (b) exhibit broad conservation across strains contributing to global disease epidemiology, and (c) be thoughtfully chosen to avoid autoimmune cross-reactivity with human tissue epitopes. 23,24 All GAS strains possess the species-defining Lancefield GAC, composed of a polyrhamnose backbone with an immunodominant Nacetylglucosamine (GlcNAc) side chain, 25,26 of which ∼25% is decorated with glycerolphosphate. 27 Representing 40%-50% of the GAS cell wall by weight, 25 GAC serves as the basis of current rapid antigen testing for GAS infection. 28 Earlier mouse immunization studies with protein-conjugated native or synthetic GAC vaccines show clear efficacy against multiple GAS M types. 29,30 Serum anti-GAC antibodies are likewise present in healthy individuals and peak around 17 years of age, strongly correlating with decreased GAS infection risk. 31 For the above reasons of immunogenicity and conservation, GAC has garnered considerable interest as a universal GAS vaccine antigen. However, this has also elicited concern, since experimental evidence for autoreactivity of antibodies that recognize the native GAC GlcNAc side chain against human tissues has been communicated by different research groups. For example, glycoproteins from human heart valves elicit antibodies that bind GAC in a manner blocked by GlcNAc (but not rhamnose or other glycans), 32 and persistence of anti-GlcNAc/GAC antibodies (up to 20 years) are a marker of poor prognosis of RHD valvular disease, whereas antibodies against streptolysin O (SLO) and the polyrhamnose core of GAC fade independently of valvular complications. 33 Also, anti-GAC GlcNAc antibodies that cross-react with heart or brain tissue are present in sera of ARF patients with cardiac or neurological complications. 34,35 An approach to modify the GAC to eliminate the potential cross-reactive GlcNAc epitope was achieved by Van Sorge et al. upon discovery of the 12-gene GAS gac gene cluster encoding the biosynthetic machinery for GAC production. 36 This work generated an isogenic mutant (DgacI) that expressed only the polyrhamnose backbone of GAC without the GlcNAc side chain. 36,37 This DgacI mutant was attenuated for virulence in mouse and rabbit infection models, and showed increased sensitivity to killing by human whole blood, neutrophils, and platelet-derived antimicrobials in serum. 36 A biotin conjugate of the modified GAC structure containing only the non-mammalian carbohydrate rhamnose (GAC PR ), generated high antibody titers in rabbits that promoted opsonophagocytic killing (OPK) of GAS strains of multiple M types by human neutrophils and protected against systemic GAS challenge in mice upon passive immunization. 36 No cross-reactivity of anti-GAC PR antisera was observed against human heart tissue lysates. 36 In the present work, we sought to generate a protein conjugate of the GAC PR for use in safe, universal subunit vaccines against this important pathogen, and to test the utility of this conjugate antigen in active immunization. As a novel approach, instead of conjugation to a standard carrier protein to engender T-cellmediated immunity, such as tetanus toxoid or CRM197, we selected SpyAD, a highly conserved GAS surface protein contains both B-cell and T-cell epitopes relevant to the bacterium and itself shows promise as a vaccine antigen. [38][39][40][41] The conjugates SpyAD-GAC PR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted OPK by human neutrophils. Active immunization of mice with a multivalent vaccine consisting of SpyAD-GAC PR in combination with candidate vaccine antigens SLO and C5a peptidase provided significant protection against GAS challenge in a systemic infection model and localized skin infection model, without evidence of cross reactivity to human heart epitopes.
Results
Expression and purification of GAS protein antigens GAS expresses several secreted and membrane anchored virulence factors that are important for disease pathogenesis in vivo. 42,43 For the present vaccine study, we selected three protein antigens that are strongly conserved with high genomic carriage rate across a globally representative and clinically diverse collection of 2083 GAS genomes, coupled to low amino acid sequence variation 24 and evidence of natural immunogenicity-SLO, C5a peptidase, and SpyAD. SLO is a secreted, poreforming, cholesterol-dependent cytolysin unique to GAS, and immunization of mice with inactivated SLO toxoid induces SLOneutralizing antibodies and protects against experimental infection with the pathogen. 44,45 C5a peptidase is a surfaceexpressed GAS peptidase that cleaves C5a complement to inactivate the chemoattractant, delaying recruitment of phagocytes to the site of GAS infection.C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. 46 C5a peptidase-specific antibody responses strongly correlate with anti-SLO titers in children with pharyngitis, 47 and the protein also elicited protective vaccine responses in murine models. 48,49 Finally, "S. pyogenes adhesion and division protein" (SpyAD), which plays dual roles in GAS adherence to host cells and bacterial cell division, 38 was identified as a potential GAS vaccine candidate in several high throughput screens of the GAS surface proteome, 39,50,51 and has been part of a 7-valent GAS vaccine that proved successful in murine immunization studies. 40 To express the above proteins for use as vaccine antigens, we applied Vaxcyte's proprietary XpressCF +TM cell-free protein synthesis (CFPS) expression platform, which is based on extraction of the E. coli cellular machinery required for transcription, translation, and energy production into a cell-free mixture capable of continuous oxidative phosphorylation. With this CFPS platform, we successfully expressed the immunogenic cores of SLO, C5a peptidase, and SpyAD. For native FL-SLO, we expressed both a N-terminal truncation fragment spanning aa 79-571 (designated SLO) as well as a N-and C-terminal truncation fragment comprising aa 79-470 [designated SLO (DC101)] ( Figure 1A). Truncated SLO were designed to optimize product yield in our CFPS (further improved with truncation of 101 amino acids off the C-terminus) while preserving key immunogenic epitopes and detoxifying the cytolysin by removing the tryptophan-rich loop, a domain that mediates insertion into cellular membranes. Additionally, we expressed C5a peptidase and SpyAD with precision mutations for enzymatic inactivation ( Figure 1A). Next, using amber stop codon suppression through addition of orthogonal tRNA and tRNA synthetase pair during CFPS, we successfully expressed a variant of SpyAD that contains 4 non-native amino acids (nnAA) in the form of p-azidomethyl phenylalanine (pAMF) in replacement of 4 native amino acids ( Figure 1A, marked by black triangles). The quantitative expression yield of each antigen was >200 mg/L as estimated by incorporation of 14 C-leucine into the translating polypeptide ( Figure 1B). SDS-PAGE analysis of synthesized proteins confirmed high purity fractions of each of the expected peptide antigens (>95% as shown in Figure 1C and 1D). Incorporation of pAMF into SpyAD was confirmed by conjugation to dibenzocyclooctyne-PEG4-tetramethylrhodamine (DBCO-TAMRA) dye and fluorescence readout. Unlike native SpyAD, the variant with 4 pAMF sites gets labeled with the dye to confirm pAMF incorporation ( Figure 1D).
Generation of SpyAD-GAC PR conjugate
Using the in-house isolation and purification protocol, highly pure preparations of mutant GAS cell wall carbohydrate containing only polyrhamnose (GAC PR ) were generated from a GAS mutant strain that lacked the GlcNAc sidechain of GAC and the surface hyaluronan capsule (DgacIDhasA). Using 1-Cyano-4dimethyl aminopyridinium tetrafluoroborate (CDAP) chemistry, GAC PR was dibenzocyclooctyne (DBCO)-derivatized for use in a conjugation reaction with SpyAD[4pAMF], shown schematically in Figure 2A. SEC-MALS analysis estimated an average molar mass of 7.2 kDa for GAC PR and 87.3 kDa for SpyAD[4pAMF], the latter in close agreement with the theoretical molar mass of a SpyAD monomer ( Figures 2B and 2C). The molar mass of the purified DBCO-derivatized GAC PR was similar to the molar mass of the native polysaccharide (data not shown). For conjugation, DBCO-derivatized GAC PR was incubated with SpyAD[4pAMF] at a 1:1 ratio to facilitate strain-promoted Cu 2+ -free click chemistry reaction to generate conjugates. After the conjugation reaction and dialysis to remove excess GAC PR , the resulting conjugates are analyzed using SEC-MALS. As shown in Figure 2D, the conjugates elute as 2 separate polydisperse distributions under Peak1 and Peak2 with average molar mass ranging from 2 MDa to 135 kDa respectively, yielding a combined average molar mass of 153.4 kDa for the conjugates, which is significantly higher than the average molar mass of either GAC PR (∼7.2 kDa) or SpyAD[4pAMF] (87.3 kDa) alone, thereby confirming successful conjugation into the final product, referred to as SpyAD-GAC PR from here onwards.
Evaluation of GAS vaccine antigens for immunogenicity
Immunization of New Zealand White rabbits was performed to generate antisera for functional evaluation of the candidate GAS vaccine antigens. Rabbits were immunized with 5 mg of individual protein antigen (SLO, C5a peptidase) or SpyAD-GAC PR conjugate adjuvanted with Adju-phos ® (Invivogen, San Diego, CA, USA). To determine immunoglobulin G (IgG) titers elicited by vaccination, terminal bleed (day 35) rabbit antisera were evaluated by enzyme-linked immunosorbent assay (ELISA). For all three protein antigens (SpyAD, C5a peptidase, and SLO), the group of rabbits immunized with the recombinant protein showed significantly increased (3-to 4-log 10 fold) antibody titers against the target antigen compared to either pooled serum from the rabbits before immunization ("pre-immune (pooled)") or the other immunized rabbit groups ( Figure 3A), confirming specificity of the IgG response to each respective antigen. The anti-SpyAD protein titer generated by the SpyAD-GAC PR conjugate was not inferior to that of the SpyAD recombinant protein alone, suggesting that key B cell epitopes were not impacted by pAMF sites. Flow cytometry was used to evaluate the binding affinity of rabbit-derived IgG to the surface of eight live wild-type GAS strains of different M protein serotypes (M1-6, M12, M28). For the great preponderance of strains, the respective immunized serum yielded an increase in binding of 100%-400% over the baseline IgG binding of the pre-immune serum ( Figure 3B). For six of the eight strains, the GAS surface binding of the antisera raised against the SpyAD-GAC PR conjugate roughly doubled the level of IgG binding seen with antisera raised against SpyAD alone. To examine the post-immunization antibody response to native GAC antigen, we generated a genetic knockout of SpyAD in M1 5448 strain (DSpyAD) and tested rabbit serum IgG binding to the cell surface of the live bacterium ( Figure 3C). Fluorescent signals detected similar IgG binding of SpyAD or SpyAD-GAC PR antisera to the surface of wild-type M1 GAS. As expected, the IgG fluorescent signal was lost for SpyAD antiserum against the DSpyAD, but a clear signal was still present for the SpyAD-GAC PR antiserum, demonstrating that it contains native GACbinding IgG.
Evaluation of rabbit vaccine antisera in GAS OPK, blocking SLO activity, and passive protection in murine challenge model The ability of the rabbit antisera raised against the GAS vaccine antigens (SLO, C5a peptidase, SpyAD, and SpyAD-GAC PR ) to promote human neutrophil OPK was evaluated using GAS strains of five different M protein serotypes (M1-M5). This assay was performed with 30 min pre-opsonization with the respective heat-inactivated antisera or pre-immune sera control, then 30 min exposure to freshly isolated human neutrophils at multiplicity of infection (MOI) = 0.1 bacteria per neutrophil in the presence of 2% baby rabbit complement (BRC). To test potential protection from a combination of antigens, we tested an antiserum mixture composed of one-third of each antiserum raised against SLO, C5a peptidase, and SpyAD-GAC PR to maintain a consistent total serum concentration (designated as "Combo" in Figure 4A). As a positive control for OPK, we used anti-M1 rabbit serum against M1 GAS and recovered a low percentage of colony forming units (CFU) due to effective killing ( Figure 4A, striped bar of first graph). All antisera induced statically significant increases by one-way ANOVA versus pre-immune sera in short-term OPK of GAS ( Figure 4A). Of note, our assay uses a 100 to 1000-fold greater GAS inoculum than published OPK assays employing the human promyelocytic leukemia HL-60 cell line at MOI = 0.001 52 or MOI = 0.0001, 53 with our goal being to more accurately recapitulate the immunization target of supporting primary human innate immune cell function. In our ex vivo human neutrophil OPK assay, antisera raised against the SpyAD protein and SpyAD-GAC PR performed similarly. Moreover, the antisera combination "Combo" performed similarly or better than the individual components, indicating no cross-interference in their OPK functions. As SLO is a secreted cytotoxin not anchored to the GAS surface, its contribution to enhanced neutrophil killing is accrued not from increased phagocytic uptake, but rather reducing SLO-mediated membrane damage and impairment of neutrophil antimicrobial functions. 45 We found that the anti-SLO rabbit immune serum, whether elicited by the SLO or SLO (DC101) antigens, significantly inhibited hemolytic activity of purified SLO against human red blood cells up to a dilution of 1:2048 ( Figure 4B). Furthermore, anti-SLO or anti-SLO (DC101) immune serum equally preserved neutrophil oxidative burst function (superoxide generation) against GAS supernatant (SLO)-mediated suppression ( Figure 4C).
The potential of vaccination using SpyAD-GAC PR alone or in combination with other GAS protein antigens, namely SLO or SLO (DC101) plus C5a peptidase, to provide protection against systemic GAS infection was evaluated in murine models of passive immunization. As a first proof-of-principle, rabbit antisera were transferred into adult CD-1 mice intravenously by retro-orbital injection before challenging each animal with 1 Â 10 7 CFU of the virulent serotype M1 GAS strain 89155 injected into the peritoneal cavity ( Figure 4D). Control mice receiving preimmune rabbit serum showed <10% survival within 24 h of infection whereas mice immunized with either the SpyAD-GAC PR antiserum or a combination of SLO + C5a peptidase antisera (volume divided in a 1:1 ratio) showed modest protection (20% and 26.7% survival through day 5, respectively), but most importantly the mice immunized with the multivalent SpyAD-GAC PR + SLO + C5a peptidase (volume divided in a 1:1:1 ratio) had significant protection against mortality with 53.3% survival through day 5 (P = 0.0048 vs pre-immune serum group, Figure 4D) In summary, due to the variation in IgG binding to GAS strains of different M types ( Figure 3B), likely due to the variation in antigen surface expression levels, and the superiority of the combination antisera in OPK assays ( Figure 4A) and passive immunization ( Figure 4D), we concluded that a multivalent vaccine formulation was required to broadly cover GAS strains and proceeded to active immunization experiments with antigen combinations.
Multivalent immunization generates antibody response with improved capacity to bind to native surface antigens and direct OPK Mouse sera were collected from nine immunized (and ten mock immunized) animals on day 42 following a three dose immunization (intramuscular injections of antigens on days 0, 14, and 28) with SpyAD-GAC PR + SLO (DC101) + C5a peptidase or mock immunization control, and the efficacy of these antisera tested for surface IgG binding to 20 GAS strains via flow cytometry. For all 20 GAS strains of differing emm types tested, the multivalent combination antisera from all mice bound clearly more IgG to the bacterial surface than the mock immunization animal sera ( Figure 5A). Murine IgG binding to methicillinresistant Staphylococcus aureus strain USA300 was comparable between antisera groups, confirming the specificity of bound murine IgG from the SpyAD-GAC PR + SLO (DC101) + C5a peptidase for GAS.
The mock and combination immunized mouse serum was subsequently tested for promoting ex vivo OPK killing of M1 89155 GAS by freshly isolated human neutrophils ( Figure 5B). When GAS were opsonized with post-immune SpyAD-GAC PR + SLO (DC101) + C5a peptidase mouse antisera, only ∼20% of the original inoculum of M1 GAS was recovered following neutrophil exposure, compared to ∼60% CFU recovery in Figure 3. Immunogenicity group A Streptococcus (GAS) vaccine antigens in rabbits. A: ELISA was performed on rabbit serum to quantify IgG titers raised against each protein antigen. Each point represents serum derived from one animal, with 2-3 rabbits per group. B: Eight GAS strains of different M-types were used to determine rabbit serum IgG binding to native antigens by flow cytometry. Histograms show representative fluorescent signals from IgG binding; red represents pre-immune sera and blue immune sera. Numbers in dark blue the top right corner of each histogram shows the mean percentage of increased signal of immunized rabbit serum over pre-immunized serum signal. C: Rabbit IgG binding to DSpyAD mutant GAS confirms the presence of IgG recognizing native GAC in the SpyAD-GAC PR antiserum.
control studies in which GAS were opsonized with mock immune mouse sera ( Figure 5B, P < 0.01).
Immunization with a multivalent glycoconjugate vaccine provides significant protection against systemic and intradermal GAS challenge Active immunization of mice was performed with intramuscular injections of antigens at days 0, 14, and 28 followed by intraperitoneal (i.p.) M1 GAS challenge on day 42 ( Figure 6A). In this model, the SpyAD-GAC PR + SLO + C5a peptidase triple combination vaccine yielded striking 100% protection against the lethal challenge (P = 0.0004), whereas SpyAD-GAC PR alone (20% protection) or two protein SLO + C5a peptidase formulation (40% protection) showed modest increases in mouse survival that did not achieve statistical significance ( Figure 6A).
Mice immunized with SpyAD-GAC PR + SLO (DC101) + C5a peptidase or mock (PBS and adjuvant alone) control were challenged using intradermal M1 GAS infection. The size of necrotic lesions generated by the resulting acute inflammatory response did not differ significantly between the two groups ( Figure 6B and 6C), but the recovered bacterial CFU/gram was reduced more than two-fold in the debrided tissue at the site of infection in the SpyAD-GAC PR + SLO (DC101) + C5a peptidase immunized group ( Figure 6D, P = 0.0012).
SpyAD-GAC PR immunization does not induce antibodies cross-reactive to human heart or brain tissue epitopes GAS vaccine development programs have the unique challenge of ensuring the formulation does elicit cross-reactive immune responses capable of recognizing self-antigens in heart tissue implicated in the pathogenesis of RHD. We performed western Figure 4. Evaluation of rabbit vaccine antisera in group A Streptococcus (GAS) opsonophagocytic killing, blocking streptolysin O (SLO) activity, and passive protection in murine challenge model. A: Opsonophagocytic killing of GAS of different M serotypes by human neutrophils in the presence of immune serum from immunized rabbits compared to serum from pre-immunized rabbit serum. B: Anti-SLO or anti-SLO (DC101) antisera block lysis of human red blood cells by purified SLO. C: Anti-SLO or anti-SLO (DC101) antisera enhance oxidative burst capacity of human neutrophils exposed to GAS supernatants containing SLO as quantified by 2,7-dichlorofluoroscein diacetate fluorescence. Statistical analyses were performed by one-way ANOVA compared to pre-immune serum; P < 0.05 ( * ), P < 0.01 ( * * ), P < 0.001 ( * * * ). D: CD-1 female mice receive an intravenous dose of rabbit antisera followed by intraperitoneal lethal challenge with 1 Â 10 7 M1 89155 strain GAS. Data shown are from two independent experiments with N = 15 (combined) mice per group; statistics are calculated from log rank Mantel Cox test for Kaplan-Meier plots. Methicillin-resistant Staphylococcus aureus USA300 served as a control to show specificity of IgG binding to GAS. B: M1 GAS bacteria was pre-opsonized with murine post-immune serum and tested in a human neutrophil opsonophagocytic killing (OPK) assay. Each bar shown is the result from an individual mouse serum, with error bars representing technical replicates. P < 0.01 ( * * ) for each mouse by one-way ANOVA versus mock immunized.
blot analysis of the SLO, C5a peptidase, and SpyAD-GAC PR rabbit immune sera on normal human heart lysates separated by polyacrylamide gel electrophoresis; antisera raised against the GAS M1 protein using the same rabbit immunization protocol served as a control. As shown in Figure 7, while anti-M1 antisera reacted to very high molecular weight components of the lysate, no cross-reactivity to the human heart tissue was seen for the SLO, C5a peptidase, and SpyAD-GAC PR antisera, although they recognized the respective cognate GAS protein (or protein carrier in the case of SpyAD-GAC PR ). This analysis is consistent with (a) the lack of sequence homology of SLO, C5a peptidase or SpyAD to human proteins, and (b) knowledge that GAC PR is comprised solely of repeating rhamnose, a sugar absent in humans, following the genetic deletion of its GlcNAc side chain, which represented a common mammalian sugar epitope. Though our data reveals a lack of cross-reactivity of rabbit serum antibodies to human tissues, this does not exclude the possibility of crossreactivity in humans nor does it alone guarantee vaccine safety. Figure 7. Assessment of antigen-specific antisera for cross-reactivity to human heart lysate. Antisera from rabbits immunized with M1 protein, full length streptolysin O (SLO), C5a peptidase or the SpyAD-GAC PR conjugates were used in western blot analysis against (A) human heart or (B) brain lysate. Unlike antisera raised against purified M1, antisera generated against each of the group A Streptococcus (GAS) vaccine antigens only react to the corresponding recombinant protein immunogen but do not possess detectable cross-reactivity to human heart lysate.
Discussion
In the present work, we deployed a novel conjugation technology to a universally conserved, highly abundant GAS cell wall carbohydrate molecule (GAC PR ) that has been engineered to eliminate a suspected cross-reactive epitope (GlcNAc). To functionalize SpyAD as a carrier, the protein was synthesized using the Xpress TM cell-free system, incorporating a non-natural amino acid (nnAA) to which GAC PR could be conjugated in a site-specific manner using click chemistry, generating high molecular mass SpyAD-GAC PR conjugates. The sites of nnAA incorporation were chosen to avoid disruption of important immunological epitopes, including both T-cell and B-cell moieties. The successful outcome expands the proven utility of this conjugation platform, which has previously been applied in a 32-valent preclinical pneumococcal conjugate vaccine program 54 and in studies of an experimental malaria conjugate vaccine. 55 The use of bio-orthogonal attachment chemistry incorporated into the non-natural amino acids theoretically allows for more efficient and potent antigen presentation to the immune system, simplified purification, and more well-defined structure of these semi-synthetic immunogens.
We also applied a cell-free in vitro transcription-translation system for GAS protein antigen production that has high potential for speed and linear scalability. An advantage of this cell-free system is that viability of a microbial expression strain is not required; removal of this constraint creates unique avenues for optimizing protein production, especially for antigens that might confer cytotoxicity in a cell-based system. Indeed, expression yields for the GAS protein antigens studied herein using the cell-free system were much higher than previously reported in the literature, in particular for the cytotoxin SLO (∼200-fold improvement). Additionally, the lack of a cellular membrane also allows for the exogenous addition of components to manipulate transcription, translation, and folding, and for sitespecific incorporation of non-native amino acids into the translating polypeptide. The protein expression process can be modulated and easily sampled over time with standard industrial microbial fermentation and process equipment.
Another innovative aspect of this work was our aim to devise a GAC PR protein conjugate that avoided using the common pneumococcal vaccine carrier protein antigen CRM197, 56 a nontoxic mutant of diphtheria toxin. 57 As the ultimate goal of a GAS vaccine development program would be to enter the pediatric immunization schedule, simultaneous vaccination with different glyco-antigens on the same carrier protein is potentially associated with dampened immune responses. First, pre-existing immunity to a carrier protein may diminish the ensuing immune response to a new antigen conjugated to the same carrier, that is, carrier-induced epitopic suppression. 58 This was seen in children immunized simultaneously with tetanus toxoid-conjugated pneumococcal vaccine and either (a) Haemophilus influenzae type B (Hib) polysaccharide conjugated likewise to tetanus toxoid or, (b) Hib conjugated to diphtheria toxin, and the anti-Hib immune response was demonstrably superior in the latter group. 59 Second, combining different vaccines with CRM197 as carrier can induce "bystander interference" extending even to unrelated antigens present in a multivalent subunit vaccine, speculated to derive from competition for limited molecular and cellular resources for antigen presentation within the lymph node. 58,60,61 In our study, GAC PR was conjugated to the surfaceanchored GAS protein (SpyAD), itself a highly conserved vaccine antigen candidate for the pathogen. [39][40][41] SpyAD retained its immunogenicity following rational targeted introduction of nonnatural amino acids for site-specific conjugation that left its critical human B and T cell epitopes exposed.
In the present proof-of-principle studies, the novel SpyAD-GAC PR conjugate was combined with two additional universally conserved GAS virulence factors, the surface anchored C5a peptidase and the potent secreted cytotoxin SLO, yielding a 3component, 4-valent combination vaccine rationally designed to avoid cross-reactivity with human heart muscle epitopes, here corroborated by western blot analysis. This vaccine elicited antibodies that bound the surface of intact GAS of different serotypes, promoted human neutrophil OPK, and showed protection in a murine model of systemic and localized skin M1 GAS infection.
Other multi-subunit formulations are currently under preclinical exploration for GAS vaccination. Among the protein antigens we selected, C5a peptidase and SpyAD were two of the seven proteins selected for inclusion in a multicomponent vaccine (Spy7) that showed efficacy in reducing GAS dissemination in a murine intramuscular infection model, 40 SpyAD and SLO were included in a five protein component vaccine that reduced murine GAS skin lesion development and accelerated lesion recovery, 41 and SLO and C5a peptidase were included in a formulation (Combo5) that produced a reduction in pharyngitis and tonsillitis in a GAS nonhuman primate mucosal infection model. 62 The use of GAS proteins, including SLO, SpyCEP or SpyAD, as both antigen and carrier protein to conjugate GAC, has also recently been reported, 63 but this approach (i) use non-specific conjugation methods that can disrupt the polysaccharide backbone through periodate mediated oxidation, and (ii) were applied to native GAC, containing the potentially cross-reactive GlcNAc side chain epitope. Our approach instead utilizes CDAP to only derivatize polysaccharide backbone hydroxyls, which allows a site-specific conjugation strategy that can preserve critical protective immune epitopes of the carrier protein when known.
In sum, our general approach may allow the signature, species defining GAC antigen to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.
Bacterial strains and generation of SpyAD knockout strain
The main GAS strain used in the paper was M1 (emm1) strain 89155, an invasive disease isolate of U.S. origin from the World Health Organization Collaborating Center for Reference and Research on Streptococci at the University of Minnesota showing the common small fragment chromosomal restriction enzyme analysis pattern 1c. 64 Additional U.S. Centers for Disease Control and Prevention reference strains used were 3752-05 (emm2), 4041-05 (emm3), 3979-05 (emm4), 4623-05 (emm5), 4045-05 (emm6), 3749-05 (emm9), 3979-05 (emm11), 4523-05 (emm12), 4626-05 (emm22), 4039-05 (emm28), 3756-05 (emm44), 3487-05 (emm49), 4044-05 (emm77), and 4264-05 (emm89) were kindly provided by B.W. Beall at the CDC Streptococcal Reference Laboratory. M1 5448 is an emm1 GAS isolate from a patient with severe invasive GAS disease. 65 All GAS strains were propagated in liquid Todd-Hewitt broth (THB) and either THB agar plates (THA) or tryptic soy agar plates with 5% sheep's blood (Hardy Diagnostics, Santa Maria, CA, USA, #A10) overnight at 37°C in ambient air without shaking. To generate the genetic knockout of SpyAD (5448DSpyAD) via plasmid integration into the chromosome, an intragenic fragment (300 bp) of spyAD (original locus tag Spy0269) was amplified from M1T1 GAS 5448 chromosome using forward primer Spy0629_For_EcoRI (5'-GAATTCAGCAGATCGTAATCGC-3') and reverse primer Spy0629_Rev_EcoRI (5'-GAATTCC-CACGTTTAATACC-3'). The PCR product was recovered by TA cloning into pCR2.1-TOPO (Invitrogen), and then subsequently cloned into the temperature-sensitive erythromycin (Erm) resistant plasmid pHY304. The resultant plasmid was transformed into wild-type M1 GAS strain 5448 by electroporation and transformants were plated on THA-Erm 2 mg/mL. Singlecrossover chromosomal insertions were identified by shifting to the non-permissive temperature (37°C) while maintaining Erm selection. Integrational knockouts were confirmed by PCR. Used as a control, methicillin-resistant S. aureus USA300 strain TCH1516 was originally isolated from an adolescent patient with severe sepsis syndrome at the Texas Children's Hospital in Houston. 66 GAC purification GAC PR was purified from GAS 5448DhasADgacI, a M1 serotype strain genetically engineered to lack both the HA capsule and the GlcNAc sidechain on GAC. A bacterial cell pellet from 6.25L growth culture was resuspended and sonicated in ice cold 48% aqueous hydrogen fluoride, stirred at 4°C for 48 h, then dialyzed against ice cold deionized H 2 O, centrifuged to remove cellular debris, and supernatants lyophilized. Cell wall material was treated with proteinase K overnight, dialyzed in deionized water to remove salts and proteins before supernatants were once again lyophilized. GAC PR was purified from these lyophilized supernatants by size-exclusion chromatography (SEC), with positive fractions pooled and re-lyophilized. GAC PR identification and purity were confirmed by gas chromatography/mass spectrometry of alditol acetate derivatives and linkage analysis performed on partially methylated alditol acetate derivatives as a service of the UC San Diego Glycotechnology Core. Controlled large-scale antigen expression utilized a DASbox mini bioreactor system for 10 h at 25°C with constant 650 rpm stirring, pH 7.2, dissolved oxygen 30%. After 10 h, reactions were harvested and spun down at 15,000 Â g at 4°C for 30 min, passed through a 0.45 mm filter, filtrate loaded on a 5 mL HisTrap excel column (Cytiva) equilibrated and extensively washed with Buffer A (50 mM Tris, 150 mM M NaCl, 10 mM imidazole) until absorbance returned to baseline. Proteins were eluted using a 50% Step gradient of Buffer B (50 mM Tris, 150 mM M NaCl, 500 mM Imidazole), and elution fractions pooled, concentrated, and incubated with excess purified his6-tagged TEV protease overnight while dialyzing against Buffer A. The dialyzed cleavage reaction was loaded back onto a pre-equilibrated HisTrap excel 5 mL column (Cytiva Life Sciences, Logan, UT, USA) and untagged proteins collected in the flow-through fractions. Thereafter, the flow-through was concentrated and loaded onto a Superdex 200 26/60 SEC column pre-equilibrated with Buffer S (50 mM Tris pH 8.0, 150 mM NaCl) and purity of eluted fractions assessed by SDS-PAGE gel analysis. Purified SpyAD [4pAMF] was incubated with excess DBCO-TAMRA dye for 1 h at room temperature to confirm pAMF sites by SDS-PAGE gel and fluorescence readout was recorded using a Syngene G-box gel imager.
Multi-angle light scattering (MALS) analysis
SEC MALS-UV-RI was performed with an Agilent HPLC 1100 degasser, temperature-controlled auto-sampler (4°C), column compartment (25°C) and UV-VIS diode array detector (Agilent, Santa Clara, CA) in line with a DAWN-HELEOS multi-angle laser light scattering detector and Optilab T-rEX differential refractive interferometer (Wyatt Technology, Santa Barbara, CA) coupled to three TOSOH columns in series: TSKgel Guard PWXL 6.0 mm ID  4.0 cm long, 12 mm particle; TOSOH TSKgel 6000 PWXL 7.8 mm ID x 30 cm long, 13 mm particle; and a TSKgel 3000 PWXL 7.8 mm ID  30 cm long, 7 mm particle. A mobile phase consisting of 0.2 mm filtered 1x PBS with 5% (v/v) acetonitrile was used at a 0.5 mL/min flow rate and 50-100 mg sample was injected for analysis. Agilent Open Lab software was used to control the HPLC, and Wyatt Astra 7 software was used for data collection and molecular weight analysis.
Dibenzocyclooctyne DBCO-derivatization of GAC PR and conjugation to SpyAD[4pAMF]
To a 6 mM solution of GAC PR in 100 mM Borate Buffer pH 8.5, three equivalents [to the polysaccharide repeating unit (PSRU)] of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP; from 100 mg/mL solution in acetonitrile) were added with vigorous stirring to facilitate cyanylation at reactive hydroxyl groups. 5 min after addition of CDAP, 2 molar equivalents of dibenzocyclooctyne-amine linker stock in dimethyl sulfoxide (DMSO) was added such that the final DMSO concentration was 5% (v/v). After DBCO-derivatization, 200 mM glycine was added to the reaction to quench unreacted cyanate esters. The DBCO-derivatized polysaccharide was purified via zeba spin desalting column and the purity of the recovered material was assessed by reverse phase. A single peak in HPLC when absorbance was monitored at 309 nm confirmed complete removal of excess DBCO linker and other reaction byproducts. Finally, the polysaccharide concentration was measured using anthrone assay and dibenzocyclooctyne concentration was measured using absorbance at 309 nm. These two values were combined to give an estimate of the percentage of polysaccharide derivatized with a dibenzocyclooctyne functional group. For conjugation, %DBCO derivatization of the GAC PR was kept between 5% and 10%. Thereafter, SpyAD[4pAMF] was mixed with DBCO-derivatized GAC PR at a 1:1 ratio (0.5 mg/ mL each) to facilitate conjugation via click chemistry. Postconjugation, the reaction mixture was dialyzed against a 50 kDa cutoff membrane to remove excess unreacted free polysaccharide. The recovered conjugates were analyzed by SEC MALS and the concentration was estimated using an anthrone assay.
Anthrone assay for total polysaccharide concentration A stock of 2 mg/mL of the anthrone reagent (Sigma-Aldrich, St. Louis, MO, USA, CAS#90-44-8) was prepared in cold sulfuric acid while a 1 mM stock of PSRU comprising 2x rhamnose was prepared in water as a standard. In triplicate wells, 100 mL of PSRU stock (serially diluted into reference standards) or the unknown samples (diluted 1:3) were plated (96-well plate) followed by addition of 200 mL/well of the anthrone reagent stock. All reactions were thoroughly mixed and sealed with a plate cover for incubation at 95°C for 10 min. The plate was briefly placed on ice to cool to ambient temperature before absorbance is measured at 620 nm using a UV/VIS plate reader.
To determine concentration of unknown samples, PSRU standard concentrations and absorbances were used to generate a least-square fit regression.
Generation of immunized rabbit serum
Age-matched New Zealand white rabbits were intramuscularly injected with 5 mg of protein antigens (SLO, C5a peptidase, M1) or SpyAD-GAC PR conjugate (equivalent of 5 mg of polysaccharide) in succinate buffer adjuvanted with Adju-phos ® (Invivogen). Immunizations (250 mL per injection per dose) were performed on day 0, 14, and 28 with terminal bleed for serum performed on day 35.
Antibody titer of rabbit serum by ELISA
Anti-protein antibody titers of rabbit antisera were determined by ELISA. 3 mg recombinant protein in PBS per well was incubated overnight in a high-binding flat bottom 96-well plate (Corning #3361) at room temperature, antigen-coated plates washed three times in PBS with 0.05% Tween-20 (PBST), blotted dry, and blocked for 2 h in PBS + 1% bovine serum albumin (R&D Systems cat#DY995). Plates were washed Â3 with PBST and blotted dry before addition of rabbit antisera, serially diluted in PBS with 1% BSA starting from 1:1000 dilution. Antisera were incubated for 2 h at room temperature, plates washed three times with PBST, blotted dry, then incubated with horseradish peroxidase (HRP)-conjugated goat-anti rabbit IgG secondary antibody (Southern Biotech cat#4050-05), diluted 1:4000 in PBS with 1% BSA, for 2 h at room temperature. Plates were washed three times with PBST and blotted dry before addition of TMB substrate (BD cat#555214) per manufacturer's instruction and incubated in the dark for 5 min. HRP reaction was stopped by the addition of 2N sulfuric acid (Sigma) before optical density was read at 450 nm. Antibody titer calculated as highest serum dilution where the signal exceeded the signal of blank wells plus three standard deviations, and all samples were run in at least duplicate for 2-3 rabbits per immunization group. For subsequent experiments, serum from the rabbits with the highest anti-protein antigen titers were used: 1:2,050,000 for SLO, 1:6,550,000 for C5a peptidase, 1:3,280,000 for SpyAD, and 1:6,550,000 for SpyAD-GAC PR . Statistics shown use one-way ANOVA with Dunnett multiple comparisons test to compare each immunized group with the dilution factor of the pooled pre-immune system.
IgG binding to GAS strains
All GAS strains were grown to mid-logarithmic growth phase (OD600 nm = 0.4) and washed in PBS before blocking incubation in 10% donkey serum for 1 h at room temperature. Murine or rabbit antisera was added to bacteria to 2% final volume and incubated for 1 h at room temperature to allow antibodies to bind to bacteria surfaces. Samples were washed with PBS and incubated with 1:200 donkey anti-rabbit IgG conjugated with AlexaFluor 488 fluorophore (Thermo Fisher, Waltham, MA, USA #21206), protected from light for 30 min at room temperature. Samples were washed in PBS once and run on a BD FACSCalibur. Signal intensity was analyzed using FlowJo software (Tree Star) and reported as a percentage of increased mean fluorescence intensity signal in individual rabbit antiserum compared to pooled pre-immunized rabbit sera.
Primary human neutrophil OPK assay
Neutrophils were isolated from blood drawn from healthy human donors with consent, as approved by UC San Diego institutional review board (Protocol #131002X). Neutrophils were pre-incubated with BRC (PelFreez cat#31061) and heatinactivated fetal bovine serum for 10 min. All GAS strains were grown to mid-logarithmic growth phase (OD600 nm = 0.4), washed in PBS, and incubated with heat-inactivated murine or rabbit antisera for 30 min at 37°C. For combination of rabbit serum, total anti-serum volume was kept the same but consisted of an even pool of serum from multiple rabbits. Neutrophils were added to bacteria at a MOI = 0.1 bacteria per neutrophil, briefly centrifuged to ensure contact, and allowed to incubate for 30 min at 37°C with 5% CO 2 . Final concentrations of components were 20% murine or rabbit serum, 2% fetal bovine serum, and 2% BRC, and the remaining volume comprised bacteria and neutrophils in PBS. At experiment termination, samples were serially diluted in PBS and plated onto THB agar plates for CFU enumeration. Sera from pre-immunized rabbits were pooled and used as control to measure non-specific, baseline bacterial killing by neutrophils for the rabbit antisera samples. At minimum, each serum or serum combination was tested in triplicate assays to ensure statistical confidence.
Primary human neutrophil oxidative burst assay
Primary human neutrophils were isolated as described for the OPK assay. 2 Â 10 6 /mL human neutrophils were loaded with 20 mM 2,7-dichlorofluorescein diacetate (Thermo Fisher, Waltham, MA, USA) in Hank balanced salt solution (Corning Cellgro, Glendale, AZ, USA) without Ca 2+ and Mg 2+ and incubated with rotation at 37°C for 20 min. Neutrophils were resuspended in Hank balanced salt solutionwith Ca 2+ and Mg 2+ to a density of 1 Â 10 6 cells/well before treatment with rabbit serum (1:64 final concentration) followed by supernatant from mid-logarithmic growth phase M1 89155 bacterial cultures (diluted 1:10). Lastly, 50 ng/mL phorbol myristate acetate was added to wells, and incubated at 37°C/5% CO 2 , and fluorescence intensity at 485 nm excitation/520 nm emission quantified on an Enspire plate reader (Perkin Elmer, Waltham, MA, USA) at the indicated time points.
In vivo mouse immunization studies
All mouse experiments were approved by the UC San Diego Institutional Animal Care and Use Committee (Protocol #S00227M) and conducted per accepted veterinary standards. For passive immunization experiments, 200 mL of rabbit antisera was intravenously delivered via retro-orbital injection in anesthetized wild-type female CD-1 mice (Charles River), 8-10 weeks of age. After 5 min, mice were challenged with 1 Â 10 7 CFU M1 strain 89155 GAS by i.p. injection and tracked for survival. For active immunization, wild-type female CD-1 mice (Charles River, Wilmington, MA, USA) were immunized every 14 days for a total of three doses starting at 5-7 weeks of age. Intramuscular immunizations delivered consisted of 100 mL total volume per mouse per dose, including 50 mL of Alhydrogel 2% aluminum hydroxide adjuvant (Invivogen), prepared per manufacturer's instructions. 14 days after final immunization, mice were infected with 1 Â 10 7 CFU M1 89155 GAS by i.p. injection and tracked for survival. Statistics of Kaplan-Meier survival curves calculated using log-rank Mantel-Cox test. 67 For intradermal infection, female CD-1 mice were immunized as previously mentioned. Mice were shaved and chemically depilated under isoflurane anesthesia and allowed to recover for a day prior to infection. Isoflurane-anesthetized mice were injected with 20 mL prepared M1 89155 culture (1 Â 10 6 CFU) intradermally using 26½ gauge needles on Hamilton 1000 mL glass syringe (cat# 81320) with PD600 repeating dispenser (cat# 83700) for reproducible precision. Developing lesions was tracked and photographed daily. On day 3 post-infection, mice were euthanized, lesions excised and homogenized using MagNaLyser (Roche, Indianapolis, Indiana, USA) and serially diluted in technical triplicate onto tryptic soy agar plates with 5% sheep's blood (Hardy Diagnostics A10) for CFU enumeration. Lesion sizes were quantified using FIJI. 68 Statistics of both lesion sizes and recovered CFU were calculated using unpaired t-test with Welch correction.
Western blot analysis for heart/brain lysate cross reactivity Varying amounts of normal adult human heart tissue lysate (Novus Biologicals, Littleton, CO, USA, cat# NB820-59217) or brain lysate (Novus Biologicals cat # NB820-59177) incubated with SDS-containing denaturing loading dye were separated by SDS-PAGE using 4-12% Bis-Tris gels before transfer onto a PVDF membrane using the manufacturer's protocol on iBlot (Thermo Fisher). The blot was blocked at ambient temperature for 1 h, followed by probing with rabbit antisera generated against each of the GAS antigens (diluted 1:1000). After three 30 min washes, HRP conjugated anti-rabbit (Jackson ImmunoResearch Laboratories, Inc., Cat # 211-035-109) secondary antibody (diluted 1:10,000) was added and chemiluminescence recorded on an Syngene G-Box F3 image scanner after incubation of the blot with the pico substrate (Thermo Fisher Scientific Cat # 34580). All blot blocking, washes, and antibody/serum dilutions were performed in TBS + 5% BSA. | 2021-05-13T00:03:35.668Z | 2020-12-29T00:00:00.000 | {
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8363015 | pes2o/s2orc | v3-fos-license | Effect of metalimnetic gradient on phytoplankton and zooplankton (Rotifera, Crustacea) communities in different trophic conditions
Theory predicts and recent study revealed that depth of the thermocline can strongly influence the nutrient availability and composition of plankton communities. We are focused on the effect of metalimnetic gradients on water chemistry and plankton communities in three stratified lakes with different trophic conditions. Vertical changes in water chemistry revealed significant increase of macroelement concentrations in the metalimnion of all studied lakes. However, there was no significant increase of nutrient concentrations in the thermocline of lakes with smoother metalimnetic gradient, whereas sharp and deep thermocline zone caused higher concentration of orthophosphates and dissolved inorganic nitrogen in the metalimnion. The maximum concentrations of phytoplankton were observed just below the thermocline and were caused mostly by the abundance of diatoms and cryptophytes. Vertical distribution of the crustacean zooplankton was similar to the distribution of phytoplankton. Especially, Daphnia cucullata was strongly related with the phytoplankton distribution and reached maximum densities in deep layers with high chlorophyll concentrations, and, conversely, smaller crustacean species and rotifers were not affected by the vertical distribution of phytoplankton.
Introduction
It is well known that metalimnetic gradients are key factors that influence nutrient cycling and structure of plankton communities in stratified lakes the during summer stagnation period (Gliwicz 1979;Cantin et al. 2011;Gauthier et al. 2014). During thermal stratification, nutrient depletion often occurs in the epilimnion of oligotrophic and mesotrophic lakes (Margalef 1983;Christensen et al. 1995;Padisak et al. 1998). Sharp vertical gradient of temperature in the metalimnion results in increased water density and viscosity, which decreases sinking speed of falling particles. These nutrient-rich waters of the metalimnion may play an important role in the functioning of phytoplankton in lakes of low trophy. Many previous studies revealed deep chlorophyll layers in thermally stratified lakes (e.g., Fee 1976; Barbiero and Tuchman 2004;Camacho 2006). The layers may be an important food source for zooplankton. The role of zooplankton is complex: they may enhance hypolimnetic algal growth by grazing in the epilimnion, which increases light penetration; but they may also feed on the hypolimnetic algal layer and reduce the biomass (Christensen et al. 1995). Finally, zooplankton could enriched the water with nutrients in a form available for phytoplankton. Phosphorus uptake by algae is directly affected by the rate of supply of available phosphorus forms, mostly by the orthophosphate phosphorus regenerated by the zooplankton rather than total phosphorus concentration (Ejsmont- Karabin and Spodniewska 1990).
The trophic state of a lake depends both on the intensity of nutrient loading from outside and on the rate of nutrient cycling within the lake ecosystem. Sharp metalimnetic gradients may cause the falling of particles which are trapped in the metalimnion. In lakes with sharp a thermocline, the symptoms of eutrophication may be more evident because of a decrease in water transparency in the epilimnion, whereas in lakes with a poorly defined thermocline, symptoms of eutrophication may be more visible in the hypolimnion due to an increase in the hypolimnetic oxygen consumption rate (Gliwicz 1979;Gliwicz and Kowalczewski 1981). Oxygen depletions could also prevent phosphorus loss from the epilimnion, because the orthophosphate phosphorus does not easily combined with metallic ions in reducing these conditions; thus, it is not carried down into the sediments (Gliwicz 1980).
The main goal of this study was to assess the effect of metalimnetic gradients on plankton communities in three stratified lakes in different trophic conditions. We predict that nutrient-rich waters of the metalimnion are preferred by phytoplankton and zooplankton communities. We analyzed the differences in concentration of orthophosphates, nitrogen forms (N-NH 4 + , N-NO 3 − , N-NO 2 − , DON, DN, PN, TN), and carbon forms (TOC, DOC, POC, IC, TC) in vertical profiles of the studied lakes. Furthermore, we expected that depth and shape of the thermocline could significantly influence water chemistry, plankton communities, and trophic state of lakes.
Study sites
The three studied lakes (Hańcza, Szurpiły, and Jaczno) are located in the Suwalki Landscape Park (SLP) in the north-eastern part of Poland. The area represents a typical landscape formed during the last Vistulian glaciation and nearly 10% of the SLP is covered with postglacial lakes. The studied lakes have diverse morphology. Lake Hańcza is a typical deep channel lake which has a maximum depth of 108.5 m and is the deepest lake in Poland and in the Central European Lowlands. Lake Szurpiły and Lake Jaczno are typical deep moraine lakes with varied coastlines (Table 1) formed by the melting of dead ice blocks. The concentrations of nutrients in waters of the SLP lakes are much lower than in the other lakes of the region. Maintaining a low trophic level in the studied lakes is possible because of large buffering properties and low phosphorus loads from the catchment (Jekatierynczuk-Rudczyk et al. 2014). The catchments of the lakes are mostly unforested with extensive agriculture and population density less than 30 persons per square km. The trophic status based on the biotic and abiotic parameters revealed differences between the studied lakes: Lake Hańcza is mesotrophic, Lake Szurpiły is meso-eutrophic (Jekatierynczuk-Rudczyk et al. 2014), and Lake Jaczno is eutrophic (Table 1). Detailed morphometric characteristics of the studied lakes were described by Borowiak et al. 2016. Hydrochemistry of the studied lakes was characterized by low orthophosphate content (Table 2). However, the lakes are clearly different as regards their electrical conductivity and concentration of oxygen in the hypolimnion zone ( Table 2). The lowest values of electrical conductivity were observed in Lake Hańcza and the highest in Lake Jaczno (Table 2).
Methods
The study was conducted in the peak of the summer stagnation (22-23 July 2015). The sampling stations were located close to the deepest point of Lake Hańcza Values in italics are the highest values in the lakes' profiles at p < 0.05 and Lake Szurpiły. Lake Jaczno was sampled in the central basin with maximum depth of 16 m. Water samples for chemical analyses and zooplankton samples were taken every meter from the surface to the hypolimnion zone (0-11 m) by the 5 L Limnos sampler. Additionally, one sample from the center of the hypolimnion was taken.
The field measurements included the Secchi disc visibility, conductivity and concentration of dissolved oxygen by the Hach Lange Sonde. Phytoplankton communities (green algae, cyanobacteria, diatoms, cryptophytes, total chlorophyll a concentration) and temperature were measured by the submersible spectrofluorometer (FluoroProbe, bbe-Moldaenke). Constant measurements of temperature every few centimeters of depth allow us to determinate precisely the thermocline (metalimnion) zone. The FluoroProbe spectrofluorometer provides in situ measurements of total chlorophyll a and also algae classes determination using differences among fluorescence excitation spectra. Changes in the resulting chl a emission allow for fluorometric estimation of algal classes based on differences in species and class-dependent peripheral antenna pigments (Beutler et al. 2002). The FluoroProbe identifies the four phytoplankton classes: green algae (Chlorophyta and Euglenophyta), cyanobacteria (phycocyanin-rich cyanobacteria), diatoms (Heterokontophyta, Haptophyta, and Dinophyta) and cryptophytes (Cryptophyta and the phycoerythrin-rich cyanobacteria).
Five-liter zooplankton samples were taken from each meter, then filtered through a 50-μm plankton net and fixed with 4% formalin. Rotifers and crustaceans were determined to species and counted in the whole samples. Additionally, 10-length measurements were made for each species. The animal length was used to estimate the dry weight of crustaceans by applying the equations after (Błędzki and Rybak 2016). The biomass of rotifers was established following Ejsmont- Karabin (1998).
Analyses of chemical parameters of water were conducted immediately after sample collection in the laboratory. . The concentrations of dissolved nitrogen (DN), total nitrogen (TN), total organic carbon (TOC), dissolved organic carbon (DOC), and inorganic carbon (IC) were analyzed by the high-temperature catalytic combustion in Shimadzu TOC-L Series analyzers. Particular nitrogen (PN) was calculated from the differences between TN and DN. Particulate organic carbon (POC) was calculated from the differences between TOC and DOC. Total carbon (TC) was calculated as the sum of TOC and IC (Cudowski et al. 2015).
The differences between analyzed variables were tested with the non-parametric Kruskal-Wallis test (p < 0.05). The agglomerative hierarchical classification (AHC) based on the Bray-Curtis similarity matrix was used to visualize the differences in water chemistry in vertical profiles of the studied lakes. The relations between the abundance of dominant zooplankton species to the vertical environmental variables (hydrochemistry and phytoplankton) in the studied lakes were visualized by the Canonical Correspondence Analysis (CCA). CCA is very useful tool for ecologists to relate the abundance of species to environmental variables (ter Braak 1986). Statistical analyses were performed with XLSTAT 2013 (Addinsoft).
Hydrochemical gradients
The differences were observed in vertical gradients of water temperature in the studied lakes. Lake Hańcza was characterized by sharp temperature gradient and the greatest depth of the thermocline zone (Fig. 1a). Smoother temperature gradients were observed in Lake Szurpiły and Lake Jaczno (Fig. 1a). Oxygen concentrations were high in the epilimnion zones of all studied lakes; however, the maximum concentrations of oxygen were observed in the upper part of the metalimnion (Fig. 1a). Lake Hańcza has well-oxygenated hypolimnion with saturation above 100%, oxygen saturation of the hypolimnion in Lake Szurpiły ranged from 30 to 40%, while in Lake Jaczno was below 1%. The lakes clearly differed by electrical conductivity (EC). The lowest values of EC were observed in Lake Hańcza and the highest in Lake Jaczno (Table 2). Vertical changes of EC revealed significant increase in the metalimnion zone. The increase of conductivity in the metalimnion of Lake Hańcza was at about 5%, while in Lake Szurpiły about 9.5%, and in Lake Jaczno approximately 11.5% (Fig. 1b). Generally , Cl − ) were found in the metalimnion and hypolimnion zones ( Table 2). The studied lakes were characterized by low orthophosphate content. Lake Hańcza had the highest concentration of orthophosphate in the metalimnion, while in Lake Szurpiły the concentrations of orthophosphate were higher in the upper water layers than in the hypolimnion zone. There were no significant differences in vertical concentrations of orthophosphate in Lake Jaczno ( Table 2). The vertical differences of organic and inorganic carbon concentrations were found in all lakes. Inorganic carbon (IC) had higher concentrations in the hypolimnion zones, while total organic carbon (TOC) had higher concentrations in the epilimnion and metalimnion ( Table 2). The analyzed lakes differed in the concentrations of nitrogen forms in the vertical profiles. Only ammonium ions were found in higher concentrations in the upper water layers of all lakes (Table 2). Higher concentrations of dissolved inorganic nitrogen were found in the deep water zone of Lake Szurpiły and Jaczno, while in Lake Hańcza the highest DIN concentration was found in the epilimnion.
The agglomerative hierarchical classification of similarity of hydrochemical parameters in vertical profile distinctly divided the studied lakes (Fig. 2). Very high similarity of hydrochemical parameters was noted in the epilimnion zones of the studied lakes, whereas these parameters in the deep water zone of Lake Hańcza and Szurpiły were very similar to the thermocline zone (Fig. 2).
Vertical distribution of phytoplankton
Total chlorophyll a concentration in the studied lakes ranged between 0.88 and 17.62 μg l −1 . The maximum concentrations of phytoplankton were observed in the upper part of the hypolimnion and in thermocline zone (Fig. 1c). In Lake Szurpiły and Jaczno, there were observed two significant increases of phytoplankton caused by different groups of algae (Fig. 2). The first increase of phytoplankton concentration in Lake Szurpiły was recorded in the lower epilimnion zone and was caused by diatoms and green algae. The second increase of phytoplankton in the upper hypolimnion zone of Lake Szurpiły was caused by diatoms and cryptophytes (Fig. 2). In Lake Jaczno, first significant increase of phytoplankton was observed in the metalimnion zone and was caused by diatoms and green algae, while the maximum concentration of phytoplankton in the upper hypolimnion was caused mostly by cryptophytes (Fig. 2). Often different groups of algae reached maximum density at different depths. Maximum concentrations of diatoms were found in the lower thermocline zone, while cryptophytes were reaching the Fig. 2 The Bray-Curtis similarity matrix of hydrochemical parameters in vertical profiles of Lake Hańcza (H), Lake Szurpiły (S), and Lake Jaczno (J) based on the agglomerative hierarchical cluster (AHC). The numbers behind the symbol of the lake is depth (m) of hydrochemical samples highest density at greater depth. Green algae reached the highest densities in the epilimnion zone (Lake Szurpiły and Hańcza) and in the thermocline zone of Lake Jaczno (Fig. 2). Cyanobacteria were minor component of phytoplankton in the studied lakes.
Vertical distribution of crustacean zooplankton
Maximum concentrations of crustacean zooplankton were found in the thermocline zones of the studied lakes. Vertical distribution of crustacean zooplankton was similar to the phytoplankton distribution (Fig. 1c). Especially in Lake Szurpiły, there were observed two significant increases of crustacean biomass which are similar with the occurrence of phytoplankton (Fig. 1c). The dominant crustacean species was Daphnia cucullata, reaching up to 80% in the total biomass of the crustacean zooplankton in Lake Hańcza and up to 66% in Lake Szurpiły. D. cucullata reaches maximum occurrence within the thermocline zones of the studied lakes (Fig. 3b), which is very similar with the maximum occurrence of phytoplankton (Fig. 3a). Few other crustacean species reached the highest biomass in the thermocline zone, i.e., Thermocyclops oithonoides in Lake Szurpiły and Eudiaptomus spp., Diaphanosoma brachyurum, and T. oithonoides in Lake Jaczno (Fig. 3b). Vertical distribution of the remaining crustacean species showed no significant differences in the lakes' profiles.
Vertical distribution of Rotifera
The biomass of the rotifer species was more or less segregated vertically in Lake Hańcza. In the upper part of the epilimnion, species prevailed in feeding on dinoflagellates (Gastropus stylifer Imhof, 1891), or smaller algae (Polyarthra spp), whereas in deeper part of the zone there was an observed increased of biomass of colonial Conochilus hippocrepis (Schrank, 1803). C. hippocrepis decided also on a strong increase of rotifer biomass in the metalimnion (Fig. 3c).
A different distribution of rotifer species was noted in Lake Szurpiły (Fig. 3c). The highest biomass of rotifers was found below the metalimnion zone. However, it was built mostly by a large predatory species, Asplanchna priodonta Gosse, 1850. The biomass of the remaining smaller rotifers was similar at different depths, with G. stylifer and Polyarthra spp. dominating in the epilimnion, and Keratella cochlearis (Gosse, 1851) dominating in and below the metalimnion.
Domination of K. cochlearis and Polyarthra vulgaris Carlin, 1943 was observed also in the epilimnion of Lake Jaczno. At 4-m depth this community was joined by G. stylifer, which achieved the highest biomass in the middle layer of the metalimnion zone. The biomass of rotifers was similar at all depths down to the sixth meter and doubled at the seventh meter.
Environmental factors affecting the vertical distribution of zooplankton First axes of the CCA ordinations were the most important in the explained distribution of dominant zooplankton species in the vertical profile of the studied lakes (Fig. 4). Correlation values of environmental variables and sites (depth) with the first axes are shown in Table 3. Most of the samples from the thermocline in all lakes were positively correlated with the first axis of the CCA map. Dominant rotifer species were generally less affected by the environmental conditions than the Crustacea species in the studied lakes (Fig. 4). D. cucullata was strongly related to the phytoplankton distribution and water chemistry. Abundance of D. cucullata was associated with cryptophytes and diatoms in Lake Hańcza (Fig. 4a) and Lake Szurpiły (Fig. 4b). In Lake Jaczno, vertical distribution of D. cucullata and Eudiaptomus species was associated with diatoms, green algae, particulate nitrogen, dissolved organic carbon, and dissolved organic nitrogen (Fig. 4c). Abundance of T. oithonoides in Lake Szurpiły could be related to the abundance of diatoms and cryptophytes (Fig. 4b).
Discussion
Our study confirms significant change of hydrochemical parameters in the metalimnion zone of the studied lakes. There was a significant increase of conductivity and concentration of macroelements in the metalimnion zone. Most of the studies describe the thermocline as a nutrient-rich layer (Fee et al. 1996;Wetzel 2001) but there was no significant increase of nutrient concentrations in the metalimnion of Lake Szurpiły and Jaczno. This could be caused by smoother metalimnetic gradients and by the high concentration of algae in this layer. The sharp and deep thermocline in Lake Hańcza was related to the highest concentration of orthophosphates in the metalimnion and high concentration of dissolved inorganic nitrogen in the epilimnion. The large depth of the thermocline makes particles to remain longer in the epilimnetic-mixed layer (Diehl 2002;Berger et al. 2006), and sharp metalimnetic gradients caused some falling particles to be trapped in the metalimnion (Gliwicz 1979;Gliwicz and Kowalczewski 1981). As a result, there was high oxygen concentration in the whole hypolimnion of Lake Hańcza. Deeper vertical mixing of water column in Lake Hańcza can also contribute to greater oxygenation of the hypolimnion (Scully et al. 2000). High similarity of hydrochemical parameters between the metalimnion and hypolimnion in Lake Hańcza and Szurpiły suggests that these layers could be mixed due to small difference in water density.
Smoother thermocline gradient in Lake Jaczno results in faster sinking speed of particles and as a consequence, there was significantly higher nutrient concentration in the hypolimnion zone and strong oxygen depletion just below the thermocline.
The results of our study confirm that the thermocline zone is favorable for phytoplankton. However, the maximum concentrations of phytoplankton were observed in the lower layer of the thermocline and upper part of the hypolimnion. Increased algal densities below the thermocline often were related to nutrient availability (Fee et al. 1996;Fasham et al. 1985), but in our study there was no significant increase of nutrient concentration in the metalimnion. Many studies emphasize the importance of algal sinking velocity for vertical distribution patterns (Jager et al. 2010). Higher density and viscosity of water in the thermocline zone limited sedimentation loss, which is especially important in nonmotile species (Diehl 2002). Due to sedimentation, algae tend to progressively accumulate at a depth where cell density -Eudiaptomus species, Eur.lac -Eurytemora lacustris, The.oit -Thermocyclops oithonoides, Mes.leu -Mesocyclops leuckarti, Asp.pri -Asplanchna priodonta, Col.mut -Collotheca mutabilis, Con.hip -Conochilus hippocrepis, Con.uni -Conochilus unicornis, Gas.sty -Gastropus stylifer, Ker.coc -Keratella cochlearis, Ker.qua -Keratella quadrata, Pol.rem -Polyarthra remata, Pol.vul -Polyarthra vulgaris, Syn.kit -Synchaeta kitina equals water density (neutral buoyancy), or, at least, where water density increases (Wetzel 2001, Camacho 2006. Typically, deep chlorophyll layer is formed by only one or a few algal species, whose population densities are extremely high compared to epilimnetic algal abundance (Gasol et al. 1992;Miracle et al. 1992). Deep chlorophyll layers in our lakes were caused mostly by the diatoms and cryptophytes. Fast sinking algae like diatoms (heavy and nonmotile) could benefit from greater water density (Arvola et al. 1991;Reynolds 1992;Winder et al. 2009). We observed the maximum concentrations of diatoms in the metalimnion of all studies lakes, while maximum density of cryptophytes was observed below the thermocline.
The phytoplankton of Lake Hańcza and Lake Szurpiły was intensively studied (Spodniewska 1978;Hutorowicz and Napiórkowska-Krzebietke 2008;Grabowska et al. 2006;Jekatierynczuk-Rudczyk et al. 2012). The dominant species in 1999-2001 was a cyanophyte Aphanocapsa incerta (Hutorowicz and Napiórkowska-Krzebietke 2008). The diatoms of the genus Cyclotella were the most important group with domination of Cyclotella radiosa (Grunow) Lemmerm, 1900(Jekatierynczuk-Rudczyk et al. 2012. Cryptophytes and chrysophytes are often very motile species and can have mixotrophic feeding strategies (Vincent and Goldman 1980). They can capitalize on higher nutrient levels and bacterial biomass present at the edge of the hypolimnion, because of their low light needs (Ptacnik et al. 2003). Deep chlorophyll layers formed by diatoms and cryptophytes have been reported for many lakes of North America and Europe (e.g., Jackson et al. 1990;Camacho et al. 2001;Stoermer et al. 1996;Camacho 2006). However, some authors reported deep chlorophyll maxima formed by cyanobacteria (e.g., Kasprzak et al. 2000) as well as other algae (e.g., Zvikas 2005).
The results of our studies have shown that different groups of algae reached maximum density at different depths using various niches. Green algae were found in the upper layers of the water, because they are less sensitive to light distribution (Litchman 2000). Diatoms and cryptophytes reached higher densities in lower water layers with maximum densities noted at different depths. Models of vertical phytoplankton distribution for poorly mixed water columns have shown that the phytoplankton maximum occurs at the depth for which they are equally limited by light and nutrients (Klausmeier and Litchman 2001). The models also revealed that the light:nutrient ratio is an important determinant of phytoplankton dynamics (Huisman and Weissing 1995;Diehl 2002).
Relative to the phytoplankton, the vertical distribution of the more motile zooplankton can be linked not only to abiotic forces (temperature, turbulence) but also to the distribution of their prey (phytoplankton) and predators (e.g., Leibold 1990;Pinel-Alloul 1995;Masson et al. 2004). The zooplankton in our study reached the highest density in the thermocline zone. Many of the previous studies showed that zooplankton migrate to the rich food source located in the metalimnion (Jürgens et al. 1994;Gasol et al. 1995;Adrian et al. 2001; (Winder et al. 2003), while these resources are much more abundant in the deep chlorophyll layer (Camacho 2006). Cryptomonas, which are common in deep-water layers, are generally considered as a high-quality food (Barone and Naselli-Flores 2003), and there are many evidences that Cryptomonas are a favorable food for the Daphnia species. We found that vertical distribution of dominant species, D. cucullata was strongly related with phytoplankton distribution. D. cucullata effectively migrated to the deeper zone and could accumulate metalimnetic production via grazing and translocate a large proportion of organic matter from the metalimnion into the water column (Brosseau et al. 2012), whereas smaller species like Bosmina spp. was not affected by the thermocline and vertical distribution of phytoplankton. This could be the evidence that food resources in low trophic lakes are the most important factor affecting vertical distribution of large-bodied daphnids (Dini and Carpenter 1991;Williamson et al. 2011). While the metalimnion offers a rich phytoplankton food source, energetically it may not be profitable to spend time feeding in cold waters where metabolic activity is constrained (Lampert and Grey 2003). Large-bodied species are also susceptible to visual predation (Carpenter and Kitchell 1993) and distribution of these species reflects a trade-off between avoiding predation and maximizing food consumption (Johnsen and Jakobsen 1987). However, some research revealed that Daphnia do not migrate to the metalimnion for feeding on metalimnetic carbon, but it is probably migrating deep enough during the day to avoid visual predation (Dini andCarpenter 1991, Brosseau et al. 2012). Vertical distribution of rotifers is probably under the influence of many different factors, food conditions being among the most important. However, during a day, rotifers may move vertically to avoid both competitors and predators (Karabin and Ejsmont-Karabin 2005). Another factor influencing occurrence of small rotifers is the presence of large Daphnia, which are known to kill and rapidly exclude rotifers (Gilbert 1988a). This interference may affect the species structure of rotifers. However, in Lakes Hańcza and Szurpiły the highest biomass of Rotifera was noted at depths of the highest occurrence of large Daphnia. The explanation is that rotifer biomass was in these tow lakes built mostly by either colonies of C. hippocrepis, or by large predator A. priodonta, which are too large to be suppressed by Daphnia (Gilbert 1988b).
Conclusions
Our study confirms significant change of hydrochemical and biological parameters in the metalimnion zones. The thermocline zone was a favorable place for plankton communities. However, the deep chlorophyll maxima were observed just below the thermocline and were caused by the diatoms and cryptophytes. Such phytoplankton can capitalize on higher nutrient levels and bacteria biomass present at the edge of the hypolimnion because of their tolerance for lower light than in the case for many other phytoplankton. The maximum concentrations of diatoms were found in the thermocline, where heavy and nonmotile algae could benefit from greater water density. While cryptophytes reaches maximum concentration in the upper hypolimnion because they are often very motile and have mixotrophic feeding strategies. Vertical distribution of large crustacean zooplankton was similar to the distribution of phytoplankton. Especially, D. cucullata were strongly related with the phytoplankton distribution and reached maximum densities in deep chlorophyll layers, and, conversely, smaller crustacean species and rotifers were not affected by the vertical distribution of phytoplankton.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. | 2018-04-03T00:00:38.419Z | 2017-07-01T00:00:00.000 | {
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259023808 | pes2o/s2orc | v3-fos-license | Impact of hepatitis virus infection on inpatient outcomes of acute pancreatitis: A population-based study
This study aimed to investigate the associations between hepatitis virus infection and inpatient outcomes of acute pancreatitis (AP). In this population-based, retrospective study, hospitalized patients with AP were identified in the 2005 to 2018 United States Nationwide Inpatient Sample database. Univariate and multivariable regression analyses were conducted to evaluate the associations between hepatitis virus infection, death/discharge against medical advice (DAMA), prolonged length of stay (LOS), and occurrence of life-threatening complications including ischemia/infarction of the intestine, portal vein thrombosis, acute organ failure, systemic inflammatory response syndrome, and hypovolemic shock. A total of 775,416 patients hospitalized for AP comprised the analytic cohort. Amongst, 26,407 subjects (3.4%) had been diagnosed hepatitis virus infection, whereas 749,009 (96.6%) had not. Mean age of the subjects was 51.4 years. After adjusting for relevant confounders, hepatitis virus infection was significantly and independently associated with increased odds of death/DAMA (aOR = 1.33, 95% CI = 1.26–1.40), prolonged LOS (aOR = 1.12, 95%CI = 1.09–1.16), and acute organ failure (aOR = 1.06, 95% CI = 1.01–1.12). In patients with AP, hepatitis virus infection is an independent predictor of worse inpatient outcomes in terms of more death/DAMA, prolonged LOS, and life-threatening complications. The findings may help risk stratification and the development of proper strategies for managing patients suffered from AP.
Introduction
Acute pancreatitis (AP) is inflammation of the pancreas, characterized clinically by acute abdominal pain and elevated levels of pancreatic enzymes in the blood. Incidence is about 36.4 in 100,000 population annually, with women affected more than men (7.9% vs 5.7%, respectively). [1] Incidence of AP is increasing in the US and Europe, making it the leading cause of hospitalization due to gastrointestinal disorders. [1] Gallstones (40-70%) and excess or prolonged alcohol consumption (30%) are the most frequent causes of AP, followed by hypertriglyceridemia, [2] certain drugs (e.g., azathioprine, valproic acid, ACE-inhibitors), [3] infections, trauma and gene mutations and polymorphisms. [2,4] In about 10% of AP cases, miscellaneous causes are responsible, including infection by parasites (e.g., Ascaris lumbricoides), bacteria (e.g., Leptospira and Mycoplasma), and viruses (e.g., mumps-virus, herpes simplex, varicella-zoster, coxsackievirus and hepatitis viruses), [5] with each organism causing AP through different mechanisms. [4] However, to date, studies on the viral causes of AP are limited.
More than half of hepatitis cases in the United States are caused by the presence of a specific hepatitis virus. The most common causes are hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV), any of which may result in acute disease with symptoms such as nausea, abdominal pain, fatigue and jaundice, leading to significant morbidity and mortality worldwide. [6] Acute infection may lead to chronic infection and sequelae, sometimes progressing to cirrhosis and hepatocellular carcinoma. [6,7] In addition, hepatitis D virus and hepatitis E virus (HEV) have been found to cause hepatitis sporadically, primarily in countries where the viruses are Medicine endemic. [7] The surveillance report of the Centers for Disease Control and Prevention reported incidence of HAV (0.4 per 100,000 population), HBV (1.1 per 100,000 population), and HCV (0.8 per 100,000 population) in 2015, but the absence of symptoms in many cases precludes accurate statistics of incidence. [8] While hepatitis viruses infect mainly hepatocytes, tissues of other organs such as the pancreas have also been found to carry antigens for hepatitis viruses A, B, C and non-A/non-B viruses. [9] In particular, AP is associated with fulminant hepatic failure and hepatitis virus antigens have been found in results of histological and serological examination of pancreatic tissue at autopsy. [10] A study of the frequency and characteristics of pancreatitis in acute viral hepatitis found that AP occurs in 5.65% of non-fulminant acute hepatitis-infected patients and that it is usually mild and both diseases can be managed conservatively; among 124 patients, AP was associated with HAV in 2 patients, HBV in 1 patient and HEV in 4 patients. [11] However, no previous population-based study has yet evaluated the impact of hepatitis virus infection on outcomes of AP. Since greater understanding of the role of viral hepatitis in patients with AP may help risk stratification and optimization of treatment, we conducted this study to investigate the prevalence and associations of hepatitis virus infection with outcomes of AP using a nationally representative large cohort.
Data source
Data for this study were extracted from the 2005 to 2018 Nationwide Inpatient Sample (NIS), a database developed by the Healthcare Cost and Utilization Project (HCUP) in the United States that is maintained by the Agency for Healthcare Research and Quality. [12] The NIS database represents a 20% sample of inpatient admissions from 45 states and 1051 hospitals that participated in collecting patient data at discharge. Principal and secondary diagnoses, principal and secondary procedures, admission date and diagnosis, discharge status, patient demographic data, and length of stay are included for each inpatient. Statistical sample weights that allow generalized estimates of national case volumes are also provided in the NIS.
Study design and ethics considerations
This was a population-based, retrospective study analyzing data from the NIS database during 2005 to 2018. The certificate number HCUP-72GWV89J8 was obtained from HCUP-NIS. The study conforms to the data-use agreement of the NIS. [12] Because the NIS received signed informed consent from all included patients before the data collection, and the patients were deidentified in the database, signed informed consent was waived for the present analysis. Accordingly, further approval was also exempt by the institutional review board of our institution.
Study population
Inclusion criteria were: all patients in the 2005-2018 NIS with principal diagnosis of AP, identified through the International Classification of Disease ninth and tenth revision (ICD-9 and ICD-10) (ICD-9: 577.0; ICD-10: K85). Patients with history of pancreatic cancer (ICD9: 157; ICD10: C25) were excluded. We also excluded patients without complete data on age, gender, study outcomes, and sample weights. For further analysis, the study population was further divided into patients with or without hepatitis virus infection (ICD9: 070; ICD10: B15-19).
Study variables
Demographic characteristics including patients' age, gender, race, year of admission, and primary payer (insurance status). Clinical characteristics including etiology of AP (i.e., gallstone/ biliary, alcohol abuse, prior endoscopic retrograde cholangiopancreatography, hypercalcemia, and disorders of lipid metabolism), type of viral hepatitis, and medical comorbidities (obesity, ischemic heart disease, metabolic syndrome, diabetes mellitus [DM], and chronic kidney disease [CKD]) were extracted from the database using relevant ICD diagnostic codes. Data of hospital characteristics including hospital bed size, region, and location/teaching status were also identified as covariates in the analyses.
Statistical analysis
Categorical variables were summarized as frequency and percentage, and also were assessed using the χ 2 test. Numerical variables were presented as mean and standardized error. Logistic regression analysis was performed to estimate the associations between study variables and death/DAMA, prolonged LOS, and occurrence of life-threatening complication. Significant variables having a P value < .05 in the univariate analysis entered the multivariable model, including age, gender, race, year of admission, insurance status, etiology of AP, medical comorbidities, hospital bed size, hospital region, and hospital location/teaching status. All p values were two-sided and a P < .05 was regarded as a statistical significance. Considering the complex sampling design of the HCUP-NIS data, all statistical analyses were performed using PROC SURVEY procedures in the statistical software package SAS software version 9.4 (SAS Institute Inc., Cary, NC). All results reported are weighted unless noted otherwise. Figure 1 shows the flowchart of study selection process. A total of 781,347 patients with the diagnosis of AP were identified in the 2005 to 2018 NIS database. After excluding a diagnosis of pancreatic cancer, missing data for age, gender, study outcomes and data lacking sample weights, a total of 775,416 patients (representing 3,787,043 subjects in the entire US population) hospitalized for AP comprised the analytic cohort ( Fig. 1).
Subjects' demographic and clinical characteristics
Demographic and clinical characteristics of the study cohort are summarized in Table 1. Among the study cohort, 26,407 subjects (3.4%) had been diagnosed hepatitis virus infection, whereas 749,009 (96.6%) had not. Mean age of the subjects was 51.4 years. In the cohort, 52.2% were males and 47.8% females, and most subjects (64.7%) were Whites. In addition, 13.5% patients had obesity, 11.7% had ischemic heart disease, 25.9% had DM and 7.4% had CKD. Among the patients with hepatitis virus infection, 23,857 (90.3%) had hepatitis C infection. Compared to patients who had no viral hepatitis, patients with viral hepatitis were younger (50.9 vs 51.4 years, P < .001) and had higher percentages of male (63.2% vs 51.9%, P < .001), Black race (27.8% vs 16.2%, P < .001), alcohol abuse (52.3% vs 27.8%, P < .001), and CKD (10.4% vs 7.3%, P < .001). In contrast, patients without viral hepatitis had lower percentages of admissions during 2015 to 2018 (28.0% vs 30.5%, P < .001) and insurance covered by private including Health Maintenance Organization (16.7% vs 33.9%, P < .001). With regard to hospital characteristics, higher percentages of patients with viral hepatitis admitted to hospitals of higher bed size (56.7% vs 53.6%, P < .001), urban-teaching hospitals (55.1% vs 46.9%, P < .001), whereas lower percentages of patients with viral hepatitis admitted to hospitals located in the Midwest (17.8% vs 22.6%, P < .001). The frequencies of nearly all outcomes in patients with viral hepatitis were significantly higher as compared with those in patients without viral hepatitis (all P < .001), except for ischemia/ infarction of the intestine, hypovolemic shock and abdominal compartment syndrome (Table 1).
Associations between life-threatening complication and hepatitis virus infection
Associations between life-threatening complication and viral hepatitis in AP patients are summarized in Table 3. Multivariable analyses revelated that hepatitis virus infection was not independently associated with ischemia/ infarction of the intestine, portal vein thrombosis, hypovolemic shock, or abdominal compartment syndrome. In contrast, individuals with hepatitis virus infection were significantly more likely to have acute organ failure (aOR = 1.06, 95% CI = 1.01-1.12, P = .030) ( Table 3). Table 4 shows the results of stratified associations between study outcomes and hepatitis virus infection. Age and status of alcohol abuse did not modify the associations between viral hepatitis, death/DAMA and prolonged LOS. However, after adjustment, viral hepatitis was significantly associated with life-threatening complications among patients < 60 years (aOR = 1.10, 95% CI = 1.04-1.16) but not ≥60 years. Also, after adjustment, viral hepatitis was significantly associated with life-threatening complications (aOR = 1.08, 95% CI = 1.01-1.16) among patients without alcohol abuse (Table 4).
Discussion
Results of the present study show that hepatitis virus infection independently predicts worse inpatient outcomes among patients admitted for AP. In specific, hepatitis virus infection is Table 2 Associations between viral hepatitis, death/AMA and prolonged LOS in hospitalized patients with AP. independently associated with greater risk for death/DAMA, prolonged LOS, and acute organ failure. These findings may help risk stratification and development of proper strategies in the management of patients with AP. Previous studies have also reported increased deaths and organ failure in AP patients with viral hepatitis. Viruses are, in fact, the largest group of infectious organisms associated with AP, and HBV is most often the causative hepatitis virus, especially in those who have undergone liver transplantation. [4,5] In a meta-analysis of AP patients, risk of death was found to double when severity was marked by the presence of infected pancreatic necrosis and organ failure; when either is present, the diagnosis is critical AP and when both are present the mortality rate is as high as 43%. [13] In AP that develops in conjunction with fulminant viral hepatitis, mortality is contingent upon severity of the hepatitis infection; while AP with HAV responds to conservative measures, HCV and HEV are associated with necrotizing pancreatitis, multiple organ failure and death. [14] DAMA, in particular, has been linked with higher rates of 90-day mortality and 30-day readmission in general population who admitted to the hospitals. [15] Also, it is believed that DAMA patients often readmitted in critical condition and were more likely to have organ failure leading to death. [15,16] Post-discharge interventions is needed for 1% to 2% of DAMA patients to help reduce the high risk of adverse outcomes. [16] Although readmission may be important when evaluating the short-term outcome of AP, such data are not available from the NIS dataset. In the present study, nevertheless, we combined deaths and DAMA as a composite outcome, and found hepatitis virus infection independently predicted higher risk for deaths/DAMA.
Outcomes
In our analysis, after adjustment, hepatitis viral infection did not significantly raise the risk for ischemia/infarction of the intestine or portal vein thrombosis. Specifically, colonic necrosis and splenic vein thrombosis are noted complications of AP, [17,18] and colonic involvement especially is associated with increased mortality due to erosion or inflammation of the large bowel and progression to life threatening necrosis, hemorrhage or perforation. [17] The predictive factors identified for splenic vein thrombosis in severe AP are alcoholism, smoking and male gender; however, in that study, viral causes were listed with other possible causes of AP but were not associated with severe AP. [18] Other authors have also reported colonic stenosis following severe AP. [18,19] Aswani et al [19] reviewed venous complications of AP, noting that splenic vein thrombosis may lead to splenic complications such as rupture or subscapular hematoma, and that in portal vein thrombosis, cavernous transformation of the portal vein occurring within three weeks after obstruction is expected to maintain hepatopetal flow in about 70% to 100% of patients. In the present study, however, no significant associations were found between colonic necrosis or splenic vein thrombosis in AP patients with hepatitis virus infection compared to those without viral hepatitis.
Among the subtypes hepatitis virus in the present study, HCV was predominant in AP patients, followed by HBV and HAV. In the United States, 2.4 million people are estimated to be living with hepatitis C. [20] On the other hand, inactive carriers are the largest group (300 million) among patients infected with HBV, and the diagnostic workup can mask positive HBV status because it is based on negative HBeAg and positive anti-HBeAg, with normal alanine aminotransferase levels and only slight liver fibrosis. [21] In a study of the frequency and characteristics of pancreatitis in acute viral hepatitis, HAV and HBV were present among 5% of patients with AP, and a small percentage had HEV. [11] While none of the hepatitis virus-infected AP patients in the NIS database had HEV, at least 14 case studies of HEV infection associated with AP have been reported in the literature, mainly describing young adults in areas endemic for the virus. [22] A review study identified 55 cases of HEV-associated AP, among which about one-fifth were severe and overall mortality was 3.2%. [23] Kamar et al [24] reported that HEV, despite its association with AP, was also associated with extrahepatic disorders, including hematologic disorders and autoimmune myocarditis and thyroiditis.
Our analysis revealed that hepatitis virus infection is independently associated with greater risk for acute organ failure. A previous systematic review summarized the data on pancreatitis associated with viral hepatitis. Pancreatitis was reported as severe in 32.88% of cases. Moreover, the review documented that respiratory system was affected in 2.74% of patients, and 6.85% experienced renal failure, while 5.48% experienced a multiorgan dysfunction syndrome. [25] Most cases of acute pancreatitis due to hepatitis viruses had been reported in association with acute liver failure. [11] In addition, about 60% of all acute liver failure patients fulfill the criteria for SIRS irrespective of presence or absence of infection and often contributes towards multi organ failure. [26] This partly addressed the potential mechanisms on the greater risk of acute organ failure posed by hepatitis virus infection among patients with AP.
Strengths and limitations
An important part of our strategy for this study was using the comprehensive, nationally representative patient data in the 2005 to 2018 NIS, the largest inpatient sample in the United States containing data from more than 1000 participating hospitals in the United States. Nevertheless, this study had several limitations. First, the study variables were identified based on medical records of inpatient admissions using ICD-9 diagnostic codes from discharge data. Therefore, the time sequence of individual exposure to hepatitis virus infection and outcomes is not totally clear, that is, it is not known in every case whether the hepatitis virus was active during hospitalization. Second, the diagnosis relied on the accuracy of hospital coders who assign the codes based on discharge data and pathology reports. Nevertheless, errors in ICD coding are reported to be limited. Unmeasured confounders such as lifestyle, behavior, environmental exposure, family medical history and clinical lab data were not included in the NIS database and could not be accounted for. Because the NIS database includes discharge data only, the present study also could not evaluate long-term health status of the patient population. Long-term prospective study is required to confirm results of the present study and to further explore associations between AP outcomes in the presence of acute and chronic viral hepatitis. Lab data and severity of viral hepatitis were not available in the NIS, thus could not be controlled for.
Conclusions
This nationally representative large study found hepatitis virus infection independently predicts worse inpatient outcomes in patients admitted for AP. The findings may help risk stratification and development of proper strategies in the management of patients suffered from AP. | 2023-06-03T06:17:50.064Z | 2023-06-02T00:00:00.000 | {
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226960806 | pes2o/s2orc | v3-fos-license | Primary colon adenocarcinoma with choriocarcinoma differentiation: a case report and review of the literature
Background Choriocarcinoma is an aggressive malignancy of trophoblastic tissue, typically of gestational etiology. Sporadic, nongestational cases are rarely found outside of the gonads. There are only 31 cases of primary choriocarcinoma of the colon reported in the literature. As a consequence of their rarity and aggressive nature, timely diagnosis and effective treatment have proved challenging, and prognosis is very poor. For that reason, we present a rare case with prolonged survival in the youngest reported patient . Case presentation A 26-year-old Caucasian woman presented with abdominal cramping and rectal and vaginal bleeding. Elevated serum human chorionic gonadotropin and an 8-cm right-sided mass seen on ultrasound suggested ectopic pregnancy. The patient was treated with methotrexate; however, her symptoms persisted, and her human chorionic gonadotropin levels continued to rise. Further workup showed a large mass of the sigmoid colon with multiple hepatic lesions suggestive of metastases. Preliminary pathology showed adenocarcinoma. Despite surgical resection and initiation of FOLFOX chemotherapy (folinic acid, fluorouracil, oxaliplatin), the patient had significant clinical deterioration, and her human chorionic gonadotropin increased exponentially. Further pathological review showed two distinct phenotypes: adenocarcinoma merging with choriocarcinoma. The result of evaluation of the metastatic lesions was also positive for choriocarcinoma. Treatment was promptly changed to a choriocarcinoma-targeting chemotherapy regimen of EMA/CO (etoposide, methotrexate, actinomycin D, cyclophosphamide, vincristine), resulting in rapid and dramatic response. The patient had mild progression after 1 year and was switched back to FOLFOX with bevacizumab. After five cycles, scans showed further progression, and the patient was started on third-line therapy with FOLFIRI (folinic acid, fluorouracil, irinotecan) and bevacizumab. Eighteen months after her diagnosis, the patient was alive and maintaining an overall response. Conclusions Our patient achieved a marked response and prolonged survival. Although a comprehensive review of the literature showed that survival with these tumors has improved over the past 10 years, prognosis remains poor. Currently, there is no established algorithm for the management of these rare tumors, but both the literature and our patient’s case indicate that a choriocarcinoma-targeted regimen is critical for survival. Further evaluation of these rare tumors is warranted in order to identify pathological patterns that may help in the diagnosis, management, and survival of these malignancies.
(Continued from previous page)
Conclusions: Our patient achieved a marked response and prolonged survival. Although a comprehensive review of the literature showed that survival with these tumors has improved over the past 10 years, prognosis remains poor. Currently, there is no established algorithm for the management of these rare tumors, but both the literature and our patient's case indicate that a choriocarcinoma-targeted regimen is critical for survival. Further evaluation of these rare tumors is warranted in order to identify pathological patterns that may help in the diagnosis, management, and survival of these malignancies.
Keywords: Choriocarcinoma, Colon, Adenocarcinoma, Choriocarcinoma metaplasia, Extragonadal choriocarcinoma, Tumor dedifferentiation, Nongestational choriocarcinoma Background Nongestational choriocarcinoma is a rare malignancy of trophoblastic tissue. These tumors most often arise in the uterus or gonads, but they have also been observed to arise from extragenital sites, typically in midline structures such as the retroperitoneum, mediastinum, and pineal gland. Even rarer is their occurrence in parenchymal organs such as the liver, lungs, or gastrointestinal tract. Within the gastrointestinal tract, these malignancies are most commonly found in the stomach. Extragonadal, nongestational choriocarcinomas of the bowel are extremely rare, with only 31 cases reported in literature . Their diagnostic elusiveness, aggressive nature, and lack of established treatment management make prognosis for these malignancies extremely poor, with an average survival of only 8 months. We present a case of colon choriocarcinoma in the youngest documented patient whose treatment regimen resulted in marked response and longer-than-average survival.
Case presentation
A 26-year-old gravida 2, para 2 Caucasian woman presented with a 2-day history of bright red rectal and vaginal bleeding with abdominal cramping. Her reported last menstrual period was approximately 8 weeks prior. The patient's history was notable for intermittent rectal bleeding for the past 2 years that had been attributed to postpartum hemorrhoids. Her serum human chorionic gonadotropin (hCG) was elevated at 1138 mIU/ml, and an in-office ultrasound revealed an 8-cm right-sided mass, strongly suggestive of an ectopic pregnancy. Diagnostic laparoscopy did not reveal an ectopic pregnancy; however, follow-up laboratory tests 1 week later showed a rise in β-hCG to 5511 mIU/ml. Repeat ultrasound showed an 8.9-mm mass but did not identify any intrauterine or extrauterine gestation. The patient was given a dose of methotrexate; however, she continued to experience intermittent lower abdominal cramping and vaginal bleeding. Follow-up laboratory tests 1 week later showed that her β-hCG had tripled to 16,326 mIU/ml. Repeat ultrasound was consistent with previous examinations showing a nonspecific heterogeneous right-sided mass but no gestational sac. The patient was given a second dose of methotrexate. She failed to follow up with repeat laboratory work as scheduled, and she continued to experience right lower quadrant pressure and intermittent vaginal bleeding. When she presented 15 days later, her repeat β-hCG had risen to 101,290 mIU/ml, at which time she was emergently admitted to the hospital. Computed tomography (CT) showed numerous hepatic masses measuring up to 2.3 cm scattered throughout the liver, strongly suggestive of widespread metastatic disease (Fig. 1a). An exploratory laparotomy revealed a sigmoid mass that almost completely obstructed the colon. A segmental resection of the sigmoid colon was performed with colostomy, as well as excisional biopsy of one of the hepatic nodules. Preliminary pathology showed high-grade adenocarcinoma of the colon.
The patient tolerated surgery well and was discharged to home to recover while arrangements for treatment were made, pending final pathological results. Contrary to expectations, the patient's β-hCG continued to rise postoperatively. Two weeks after surgery, the patient returned to the hospital with new right upper quadrant and right chest wall pain with shortness of breath. Her β-hCG had markedly increased to 792,770 mIU/ml, and repeat CT showed new scattered pulmonary nodules and an interval increase in the size and number of hepatic lesions. Additional laboratory tests showed anemia, hyperbilirubinemia, and elevated lactate dehydrogenase. She was febrile and hypotensive and experienced episodes of tachycardia and dysrhythmia. The following day, updated pathological analysis noted invasive, high-grade colonic adenocarcinoma merging with a very poorly differentiated high-grade malignancy showing positivity for hCG, concerning for choriocarcinoma. Multiple medical colleagues, specialists, and pathologists were consulted, and specimens were sent to the Mayo Clinic for further analysis.
The patient continued to deteriorate clinically, with continued fevers, leukocytosis, and anemia, which were managed with antibiotics and transfusions. The results of blood cultures and a chest x-ray were negative, and the patient's symptoms were ruled to be secondary to her malignancy. Given her rapidly worsening clinical picture and her tumor burden burgeoning in only 2 weeks, the decision was made to initiate FOLFOX therapy (folinic acid, fluorouracil, oxaliplatin). The patient received one dose and was discharged to home 1 week later. The following week, final pathology from the Mayo Clinic confirmed colon adenocarcinoma mixed with choriocarcinoma. Repeat laboratory tests showed continued increase in β-hCG to 916,335 mIU/ml. In light of the confirmed pathology and continued rise in β-hCG, the decision was made to modify therapy to a choriocarcinoma-targeted EMA/CO regimen (etoposide, methotrexate, actinomycin D, cyclophosphamide, vincristine) [32,33].
Two weeks after initiation of EMA/CO, the patient's β-hCG levels had dramatically dropped to 14,981 mIU/ ml. After only five cycles, her β-hCG was 17 mIU/ml, and imaging showed a significant decrease in the size and number of her innumerable hepatic lesions (Fig. 1b), with almost complete resolution of her pulmonary disease. She continued to have marked radiographic response to EMA/CO therapy ( Fig. 1b-d) and a continued decline in β-hCG, with the last quantitative measurement showing 2.7 mIU/ml after nine cycles.
The patient had completed a total of 14 cycles of EMA/CO when CT showed mild interval progression of some hepatic lesions (Fig. 2a). A core needle biopsy of one of the lesions showed metastatic adenocarcinoma consistent with colon cancer. The patient also began to experience abdominal pain, diarrhea, and fevers and was found to have gram-negative bacteremia. Following treatment with antibiotics, the patient was initiated on a FOLFOX + bevacizumab regimen. After five cycles of FOLFOX + bevacizumab, scans showed further progression of her lung lesions and mixed response of the liver lesions (Fig. 2b). Subsequently, her treatment regimen was changed to FOLFIRI (folinic acid, fluorouracil, irinotecan) + bevacizumab. The patient received one cycle of FOLFIRI + bevacizumab and was alive 18 months after her initial diagnosis.
Pathology
This case was noted to be very difficult, requiring additional input from Mayo Medical Laboratories, where it was shared with multiple pathologists. Within the 18.5-cm segment of resected sigmoid colon submitted for analysis, a lesion measuring 8.5 × 3.8 × 3.0 cm was identified as invasive, high-grade colonic adenocarcinoma merging with a very poorly differentiated high-grade malignancy showing positivity for hCG ( Fig. 3a-c). The lesion was found to arise within a preexisting tubulovillous adenoma with high-grade dysplasia and extended completely through the muscularis propria and serosal surfaces. The nodular lesion consisted of enlarged, cytologically malignant glands consisting of tall columnar cells exhibiting dysplastic nuclear features such as prominent enlargement, extensive stratification, nuclear hyaline, and total loss of polarity with large nucleoli and focal mitoses. These features became more prominent in the sheets of highly dysplastic cells seen in the intraserosal component of the tumor as it extended outward. Rare giant nuclei and occasional multinucleate cells were also identified with a somewhat complex cord growth pattern focally. There were also focal areas of necrosis, as well as slight cytoplasmic vacuolization observed within some cells. The serosal fat and overlying serosa both exhibited extensive hemorrhage. Additionally, three of five serosal lymph nodes exhibited metastatic high-grade carcinoma.
Analysis of the submitted liver specimen showed poorly differentiated carcinoma that was morphologically similar to the poorly differentiated component in the colon. Fragments showing extensive necrosis with recent hemorrhage were observed. Within the necrotic areas were irregular foci consisting of sheets of cohesive cells exhibiting highly pleomorphic nuclei with large nucleoli, irregular membranes, and loss of polarity. Some areas were noted to have larger cells with giant nuclei and occasional multinucleated cells.
Immunohistochemical stains were done on both the glandular and undifferentiated components of the colon malignancy, as well as the liver lesion ( Table 1). The results echoed histopathological findings in that the malignancy progressively exhibited fewer features of adenocarcinoma and increasing features of choriocarcinoma as the tumor spread and metastasized.
Genomic testing was significant for TP53-pR175H with a 72% estimated variant allele frequency. Microsatellite instability testing showed intact nuclear enzymes for MLH1, MLH2, MSH6, and PMS2 in all specimens. Tumor mutational burden was low (two mutations/megabase). Several targeted genomic alterations were tested, including BRAF, KRAS, MYC, NRAS, ROS, and RET. No mutations were identified.
Of note, there were multiple tubular and tubulovillous adenomas found to be present within uninvolved areas of the patient's colonic mucosa. Certain architectural features were focally suggestive of malignancy and noted to be arguably classified as intramucosal carcinoma (versus high-grade dysplasia). For example, multiple polypoid fragments of tubulovillous colonic mucosa were seen to exhibit foci of dysplasia consisting of increased cellularity associated with prominent nuclear stratification, enlargement, loss of polarity, large centers, and nucleoli. Some of these foci were found to be cytologically malignant. Some of the glands exhibited a very complex cribriform and microglandular pattern and had frequent mitoses.
Discussion
Choriocarcinoma is an aggressive, hCG-secreting tumor that rarely arises in extragenital tissues. Upon review of the literature (Table 2) , most colon choriocarcinomas arise in the sigmoid (n = 11) or ascending (n = 9) colon. They are highly invasive with early hematogenous metastatic spread. Of the reported cases, all but two had confirmed metastases, most commonly to the liver (n = 22) and/or lungs (n = 15). Consequently, patients with these tumors frequently present with signs of significant bleeding and often die of complications of liver or respiratory failure. Per the literature, the average age of presentation is 50.5 years, and the female-to-male ratio is 1.2 to 1.
Of the cases reported, primary colon lesions were predominantly biphasic tumors, comprised of adenocarcinoma and choriocarcinoma (n = 23); yet, 76% of metastases with reported histology were solely choriocarcinoma. In our patient's case, there were features of choriocarcinoma and adenocarcinoma in both the primary and metastatic lesions. However, it was noted by pathology that the cellular architecture became poorer and less differentiated as the tumor extended and spread, with the metastatic liver lesion more closely resembling choriocarcinoma than the adenocarcinoma of its primary source. This histological pattern is consistent with other reported cases and congruent with Pick's [34] widely accepted theory that these malignancies are the result of a primary adenocarcinoma undergoing retrodifferentiation, or trophoblastic metaplasia [35], resulting in transformation into progressively more primitive cellular forms until they are indistinguishable from choriocarcinoma. The process of retrodifferentiation, or dedifferentiation, can be clearly demonstrated in cases where the choriocarcinoma merges with the adenocarcinoma. As observed in our patient's case, there is often a transitional zone [36] between the two tumor types that exhibits certain histologic features of both malignancies. Examination of these transitional forms allows the direct visualization of the dedifferentiation process, also referred to as "opisthoplasia," occurring sequentially in situ. In cases where there is solely choriocarcinoma without any adenomatous component identified in the primary lesion, it is believed that trophoblastic metaplasia has occurred to the extent that the more aggressive choriocarcinoma has essentially overtaken the adenomatous tumor, resulting in complete tumor dedifferentiation [37]. Genetic instability is believed to be responsible, at least in part, for the dedifferentiation of adenocarcinoma and its divergence into multiple phenotypes [5]. Interestingly, it has been demonstrated that up to 43-52% of morphologically typical adenocarcinomas have positive immunohistochemistry for hCG [38,39], suggesting some shared functional commonality. Upon reviewing the literature, only a few cases were determined to be pure primary choriocarcinoma of the colon without any adenomatous component. Of those, at least 30% were initially diagnosed as adenocarcinoma on the basis of histology but were ultimately ruled to be choriocarcinoma on the basis of immunohistochemical profiles showing strong positivity for hCG, with mixed responses for cytokeratin 7 (CK7), caudal type homeobox-2 (CDX-2), and villin, among other stains [21,30,31]. Taking into account the frequency with which otherwise normal colorectal adenocarcinomas display hCG positivity, it is reasonable to wonder whether these cases were actually pure choriocarcinomas or rather examples of genetic instability in action resulting in the divergence of adenocarcinoma cells into different subpopulations, including a transitional subgroup showing mixed histological and immunohistochemical features. Although they may have been pure choriocarcinomas as initially diagnosed, it is possible they are simply additional variations of the transitional zones described previously in the literature.
Despite a common functional component to choriocarcinoma, the dedifferentiation of colorectal adenocarcinoma to choriocarcinoma is extremely rare. Additionally, these particular carcinomas tend to occur in a younger population than other colon malignancies or even choriocarcinomas elsewhere in the gastrointestinal tract, raising the question if there could potentially be an underlying genetic component or hereditary cancer syndrome involved. To that point, the biopsy in this case noted multiple adenomas with malignant features in uninvolved tissue. However, genomic testing showed no microsatellite instability or gene alterations aside from p53. The patient had no personal or family history of gastrointestinal disease or malignancy. Furthermore, of all the reported cases of primary colon choriocarcinoma, there were only four cases with any prior history of gastrointestinal pathology. These included a single rectal polyp removed 10 years prior to diagnosis [9], a patient with diverticular disease who was 31 months postsurgical resection of an annular carcinoma of the rectum [12], a patient with Lynch syndrome who had gastric cancer treated 25 years ago [26], and a perineal abscess in a patient with Crohn disease [31]. There were two additional patients with a remote history of cancer: one was 2 years postlobectomy for squamous cell lung carcinoma [2], and the other was 3 years postmastectomy for breast cancer [20]. Ultimately, the lack of a strong oncological or gastrointestinal history or identifiable genomic mutational pattern in these patients attenuates the theory of a hereditary component in the tumorigenesis of this particular malignancy.
Identifying potential genetic drivers of these tumors, whether inherited or sporadic, should continue to be pursued because it could provide valuable insight regarding the detection and management of an aggressive malignancy with a very poor prognosis. The average survival from diagnosis is only~8 months; however, it should be noted that this is longer than previously reported in the literature, possibly reflecting more effective treatment over the last 10 years. Although there is currently no consensus or standard treatment regimen for this rare malignancy, standard chemotherapy for gestational trophoblastic neoplasias such as EMA/CO, VIP (etoposide, ifosfamide, cisplatin), or BEP (bleomycin, etoposide, platinum) have most commonly been used. The literature suggests that the presence of choriocarcinoma has the greatest impact on prognosis, even more than delay in diagnosis, metastatic disease burden, or the coexistence of adenocarcinoma [14]. Review of cases with longer-than-average survival, including our patient's case, shows that prompt initiation of chemotherapy directed at choriocarcinoma rather than adenocarcinoma is essential to survival.
However, it is important to recognize the heterogeneity of these tumors. Although the presence of choriocarcinoma is observed to be the predominant factor influencing prognosis, these tumors are noted to be more aggressive than traditional gestational trophoblastic neoplasias [33]. In our patient's case, even with effective treatment with EMA/CO, the adenocarcinoma component of the tumor eventually progressed. It is suggested that the differences in cellular origin could be associated with differences in chemosensitivity in these tumors [22]. Consequently, the hybrid population of these tumors necessitates that management include multiagent therapy, ideally targeting both choriocarcinoma and adenocarcinoma [20].
Conclusion
Our patient was a woman with primary choriocarcinoma of the sigmoid colon. She is the youngest reported patient with this malignancy and achieved longer-thanaverage survival secondary to radical surgical resection, a single dose of FOLFOX, prolonged EMA/CO therapy, and multiple cycles of FOLFOX with bevacizumab, followed by FOLFIRI with bevacizumab. The patient is maintaining her overall response and remained alive 18 months after her initial diagnosis.
Colorectal choriocarcinoma is an extremely rare malignancy with a very poor prognosis. It disproportionately affects younger patients and has no established standard treatment. Given that timely diagnosis has proved challenging and its aggressive course, one should maintain a high index of suspicion in younger patients presenting with symptoms of colon malignancy or in patients with elevated β-hCG for whom gestation cannot be confirmed. Further investigation of molecular, genetic, and immunohistochemical characteristics of these tumors is encouraged because identification may lead to more timely and accurate diagnoses, development of a standard treatment regimen, and prolonged survival in this unique group of patients. | 2020-11-16T14:39:50.318Z | 2020-11-16T00:00:00.000 | {
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236677684 | pes2o/s2orc | v3-fos-license | HARMONIZATION OF EUROPEAN LEGISLATION IN THE FIELD OF SAFETY AND HEALTH AT WORK IN THE LEGAL SYSTEM OF THE REPUBLIC OF NORTH MACEDONIA
Occupational safety and health is one of the most important international labor standards of ILO. This means that ILO member states should ensure encouraging and maintenance at the highest level of safe working conditions in order to avoid accidents and occupational diseases. Achieving this goal means that employers must make a continuous risk assessment at the workplace, and at the same time decide whether appropriate measures and activities are necessary in order to create the highest level of safety and health at work, i.e. to avoid side effects. In order to achieve this goal, a systematic approach is necessary in preventive action and connection of all entities that are bearers of certain obligations and activities at the national level, but also beyond the international institutions in this area. By application for membership of the Republic of Macedonia in the European Union on 22 of March 2004, the process of harmonization of the domestic with the European labor legislation begins, and thus inevitably harmonization of the domestic legislation for safety and health at work with the European. Thereby, this process takes place pursuant to article 32 of the Constitution of the Republic of Macedonia, according to which the protection of workers is of primary importance, i.e. health and safety at work is a constitutional-legal category and every individual has the right to work, free choice of employment and work protection. In the harmonization process the basic concept is the Framework Directive 389/391/EES for safety and health at work, according to which the national legislation on safety and health at work should be harmonized exactly according to the principles of this directive.
Introduction
Approaching the national legislation to the law of the European Union in the process of European integration is an inevitable step for each country that wants full membership. One of the Copenhagen criteria 1 that the Republic of Macedonia needs to meet is precisely harmonization of its national legislation with the acquis commmunautarieie 2 with the law of the European Union.
The term harmonization, i.e. approximation can be defined as adoption of the European law and mutual harmonization of the legislation of the EU member states, i.e. harmonization of the law of the candidate or aspirant countries for membership with the EU law and the legislations of the member states and their incorporation into the national law with a special procedure and methods of harmonization 3 .
The methodological significance of the harmonization refers to the EU legal regulations with which the domestic legislation should be harmonized, i.e. the harmonization establishes a goal towards achieving internal unity and avoiding internal conflicts between the different parts of the legal systems that aim at eliminating internal contradictions and differences between national legislations based on commonly defined standards 4 .
For transition countries, aspirants or candidates such as the Republic of Macedonia, the harmonization of legal systems is a process that should establish a reform of legal systems followed by radical political, economic and social changes, which mean abandoning the former basic principles and setting a new legal framework for the transition to democracy, the rule of law, human rights and freedoms and market democracy.
By ratification of the Republic of Macedonia of the Stabilization and Association Convention in the EU 5 in 2001, our country committed to harmonization of the legal system with the one in the EU, where among other things it was emphasized that one of the top priorities will be meeting the Copenhagen criteria defined from the European Council since 1993. The harmonization process is defined in Chapter VI, article 68 paragraph 1 which states: "The Parties recognize the importance of adapting the existing and future laws of the Republic of Macedonia to those of the Community. The Republic of Macedonia shall strive to gradually ensure compatibility of its laws with those of the Community. By receiving the candidate status on December 17, 2005 the 4 Ibidem page 13 5 Decree on promulgation of the law on ratification of the Stabilization and Association Settlement of the Republic of Macedonia and the European Communities and its member states - 12.04.2001 Republic of Macedonia, the obligation for harmonization of the legal system is raised to the level of a specific obligation for harmonization of domestic law in 33 areas, determined by the so-called "White Paper" according to which the harmonization process of legal acts can be divided into three stages: 1. Transposition of the legal measures of the Community into national legislation; 2. Implementation of the measures into state institutions; 3. Implementation of harmonized national acts. 6 For that purpose, the Republic of Macedonia prepares a National Program for Adoption of the Approximation of EU Legislation (NPAA) 7 , where are listed all legal and bylaw acts that need to be adjusted, i.e. harmonized with EU legal acts.
International Occupational Safety and Health Regulations
Several international organizations and institutions such as ILO, the UN, the Council of Europe and the EU pay respectable attention to occupational safety and health as evidenced by the adoption of several Charters, Conventions, Recommendations, Declarations, etc. in this area. These international legal instruments represent wider legal framework and oblige member states to implement into the national legislations. 6
Occupational safety and health in UN acts
The Constitution of ILO (International Labor Organization), adopted in 1919, establishes the basic goals of ILO, which are expressed in article 427 of the Treaty of Versailles of 1919, which are implemented in the ILO Constitution. According the Preamble of the ILO Constitution, ILO objectives can be divided into three main groups. One of them refers to the working conditions where it is said that "poor working conditions bring injustice, misery and poverty to a large number of people, which results in great dissatisfaction that threatens world peace and harmony." 8 These goals are also conveyed in the Philadelphia Declaration of June 10, 1944, where in Chapter III, as one of the main goals of ILO is stated "achieving adequate protection of life and health of employees in all occupations." 9 The Universal Declaration of Human Rights, adopted by Resolution 217A (xxx) of 10 December 1948 by the UN General Assembly, also explicitly emphasizes the right of each employee to "fair and satisfactory working conditions and protection against unemployment". 10 The UN International Covenant on economic, social and cultural rights obliges the contracting states to take maximum steps in order respect the economic, social and cultural rights of each citizen, where the right of everyone to enjoy fair and favorable working conditions, which provide "safety and hygienic working conditions" is very important. 11 . We have previously mentioned that all international organizations and institutions have implemented this goal by 8 Dr. B Bukuljash and m-r Adrijana Bilikj, Меѓународно радно право со посебним освртом на МОР, Сплит, 2006 page29. 9 Philadelphian declaration, Chapter 3, st. g. 10 See article 23 paragraph 1 of the UN Universal declaration of human rights 11 See article 7 of the UN International pact for economic, social and cultural rights. adopting several Charters, Conventions, Recommendations, Directives and others.
Occupational safety and health in ILO acts
Although ILO is an integral part of the UN, i.e. it represents specialized UN agency, we still believe that the acts (Conventions and Recommendations) adopted by this organization should be separated in a special segment due to their tangibility, specificity and specialty. As more important ILO Conventions related to safety and health at work we would mention the following: Convention No. 16 According to Article 4 of the Convention, each member, based on national conditions and practices, and in consultation with the major part of the representatives of (representative) organizations of employers and employees, should formulate, implement and periodically review a coherent national safety and health policy. 12 This means that every ILO member state that has ratified this convention should adopt a National Safety and Health Policy (Program). In this context, the main goal of such a policy should be protection against accidents and injuries of health whose occurrence is related to the workplace, by reducing the risks associated with the work environment within reasonable limits. 13
Regulation on safety and health at work in the acts of the Council of Europe and the EU
Occupational safety and health, as one of the international labor standards, in addition to having an important place in the international legal instruments of the UN, it has also provided its place in the acts of all major regional organizations, especially the European Union. The most important EU acts relevant to occupational safety and health are the Single European Act of 1986, the European Social Charter of the Council of Europe of 1961. The European Social Charter was adopted on 18 October 1961 by the Council of Europe and entered in 1965. The Charter was revised twice in 1988 and 1996, in which the contracting parties as goal to their policy aim to "implement the principle that all employees have the right to safe and sound working conditions (Part 1, item 3). Part 2 of the Charter in article 3 states that in order to ensure the effective exercise of the right to safe and sound working conditions, the contractual 12 Article 4 paragraph 1 of the Convention no.155 of ILO that refers to the safety and health of employees 1313 Ibid. article 4 paragraph 2 parties obliged, in consultation with the organizations of employers and employees, to: formulate, implement and periodically monitor the single national policy on occupational safety and health and the work environment. The primary purpose of this policy is to improve occupational safety and health and to prevent injuries that may occur at work and related to work, especially by reducing the causes and risks that inevitably occur in working environment.
1. to adopt safety and health regulations; 2. to ensure application of such regulations by appropriate supervision measures and 3. to promote the development of the occupational health service for all employees who would have primarily preventive and advisory functions.
The Contract for foundation of the European Union, in Chapter XI, Part 1, that refers to the provisions on social issues, namely articles 136 and 137, states that the Community and the country members, having regard the social rights provided in the European social charter and the Charter of the Union for fundamental rights of employees, it is stated that their goal among other is improving living and working conditions,.... 14 that is, improving, in particular, the working environment for health and safety protection". 15 Article 118A of the Single European Act of 1986 states that "Member states oblige to promote improvement, especially in the field of working environment, protection and safety and health of employees, and achieving harmonization and progress with the existing conditions in this are." 1416 See article 136 and 137 of the Contract on foundation of the European Community 1517 Ibid www.japmnt.com In addition, article 11 of the ESP obliges contractual parties to take appropriate measures to eliminate, to a large extent, the causes of bad health, provide advisory and educational benefits for promotion of health and encouraging individual responsibility for health matters; as well as to prevent as much as possible epidemiological, endemic and other diseases, and also accidents.
The Union Charter of fundamental social rights of the employees, adopted at the meeting of the Heads of states or governments of the EEZ Member States held in Strasbourg on 9 December 1989, also gives an important place to protection of health and safety at work. Article 19 of the Charter stipulates that "every employee must benefit in his own work environment from the satisfactory conditions of protection of his health and safety", i.e. to take "adequate protection measures, especially for involvement of employees from information and consulting on current risks and measures taken to overcome or reduce them.
Directive no. 83/391 of 16 June 1989, which is based on article 118A, regulates the general principles that produce a number of other recommendations in this area. This Framework Directive aims to improve the safety and health of employees at work. It establishes the general principles that are systematized in two types: action against risks (evaluation, elimination, prevention, adaptation to technical evolution), as well as general obligations of employers and employees in relation to health and safety at work.
Of the above legal instruments of the EU we can conclude that normatively this area is relatively well regulated which is a good basis for legal regulation of this problematic and at the national level of EU member states and candidate countries for EU membership.
National regulation for safety and health at work in the Republic of Macedonia
Very important element in the system of safety and health at work is the regulation. Elaborating on the importance of the abovementioned international legal instruments of the UN, ILO, EU in regulating the right of employees to safety and health at work, we can establish that our country RM, as a member of the UN, ILO and EU candidate country has ratified and accepted the most important international legal instruments (conventions, recommendations, charters, declarations) for safety and health at work. This means that RM has implemented the same in its own national labor legislative, which is basic precondition for out further integration in the international community, especially in the EU.
"Employees in the Republic of Macedonia exercise their right to a safe and healthy working environment with the employer in accordance with the Constitution of the Republic of Macedonia, the Law on Labor (Official Gazette of RM no. 62/05), the Law on Safety and Health at Work (Official Gazette of RM no. 92/07), and other laws from labor area, bylaw acts-rulebooks, as well as collective agreements." 16 We mentioned above that the protection and safety and health of employees is a constitutional category. The Constitution of the Republic of Macedonia explicitly mentions the right to a healthy and safe working environment in article 32, where is stipulated that "Everyone has the right to work, free choice of employment, protection at work ....." 17 , and this right implies incorporation in its content not only rights but also obligations on the part of both the employee and the employer. The employee is obliged to comply with the prescribed conditions and measures for work protection (article 32 of the Labor Law). In that context, the employee is obliged to perform the work in the workplace with care, in order to ensure not only his life and health, but also the life and health of other employees, as well as the citizens in general. If the employee does not comply with the prescribed measures for safety and health, or does not use the means and equipment for work protection according to the established regulations, by the Labor Law this is considered as violation of work discipline or non-fulfillment of obligations stipulated by law, collective agreement and an employment contract, due to which the employee is terminated the employment by dismissal from the employer without notice. This provision of the Law is undertaken in almost all special (branch) collective agreements, where certain specifics of the activity are preserved." 18 The Settlement on foundation of the Economic social council of the RM stipulates that "The Government of the
Republic of Macedonia, the Federation of Trade Unions and the Economic Chamber of Macedonia, in order achieving the fundamental values of the Constitution of the Republic of Macedonia for democratic and social state and providing social justice for more harmonious development of social processes, for the promotion of social dialogue, three-partism and the application of international conventions and recommendations, form the Economic and Social
Council." 19 "The Council has an advisory-consultative function, through which the social dialogue takes place and a significant part of the negotiating function of the social partners for economic and social issues that are of interest to the RM." 20 "In accordance to these functions, the Council reviews issues and gives opinions, proposals and recommendations on certain issues in the field of working conditions and occupational safety." 21 4. Law on Safety and health at work "Given the great importance of the Law on Safety and health at work (Official Gazette of RM no. 92/07) we believe that it is necessary to elaborate this law more widely. In fact, this law determines: 1) measures for safety and health at work; 2) general and other obligations of the employer; 3) rights and obligations of the employees in the field of safety and health at work; 4) preventive measures against occupational risks, elimination of risk factors for accidents; 5) informing and consulting the social partners about safety and health of the employees; 6) establishment and functioning of the Council for safety and health at work; 7) authorization; 8) supervision and 9) misdemeanor provisions.
The representative of the employees for safety and health also has an important place in the system of safety and health at work. The rights and obligations of the representative of the employees for safety and health at work are regulated by articles 28, 29 and 30. According to them, the representative of the employees for safety and health at work is elected by the employees from their rank, at a union meeting, or meeting of employees, taking into consideration the representation of all parts of the operational process and mandatory for those where the working conditions require (increased danger to the safety and health of workers, work in separate places, etc.). The employee representative enjoys special protection from employment, as does the representative of the trade union organization in the specific company, in accordance to the law and the collective agreement. The number of representatives depends on the number of employees, with: over 10 employees one representative is being elected; two representatives of 101-500 employees and three representatives of employers who have over 501 employees.
According the Law, the representative of the workers for safety and health at work has the right: to tour the workplaces in order to perceive the situation regarding occupational safety and health; to communicate to the employer about the needs and problems of safety and health at work; to inform the labor inspection and request its mediation or supervision; to inform the labor inspection about its possession, to attend, to give its observations and to have an insight in the minutes compiled by the inspectorate during mediation or supervision; request information from the employer and have access to occupational safety and health assessment statements; special training for prevention and safety and health at work, specific to the activity of the employer, which is determined by a collective agreement." 22
Methodology for harmonization of national legislation with EU legislation
Due to the success of the process of harmonization of the legislation of the Republic of Macedonia with the European law, for consistency, coordination and clear image and monitoring of the process of approximation of the legislation in 2000, the Methodology and structure for harmonization of the national legislation with the EU legislation was prepared.
"According to the Methodology, the harmonization process is divided into four stages: 23 3. Transposition phase -operational elaboration of new legislation in accordance with a previously defined plan. At this stage, the real approximation of the Macedonian legislation with that of the EU takes place. Namely, this is the stage where national experts and EU experts must prepare new draft laws or proposed amendments to existing laws or bylaws, in order to achieve compatibility between the legal order in the Republic of Macedonia and EU legislation and finally 4. Implementation phase -not only the adoption of new laws in the Assembly or amendments to existing ones, but also their proper implementation in practice and management of the effect they have on the existing institutional infrastructure." The legal basis for adoption and harmonization of the legislation in the Republic of Macedonia is established based on the monistic approach, i.e. the supremacy of international law which is regulated in article 118 of the Constitution of the Republic of Macedonia (Official Gazette of RM no. 52/91, 1/92, 31/98, 91/2001) which states: "International agreements that are ratified in accordance to the constitution are part of the internal legal order and cannot be changed by law", i.e. Article 8, paragraph 1, line 10: "The fundamental value of the constitutional order of the Republic of Macedonia is ... (10) the observance of the generally accepted norms of international law".
The harmonization of the Macedonian legal system with the EU law is a complex process, which can be efficient and take place with the necessary speed if it respects certain methodological postulates such as: 1. Determination of the right goals, choosing the appropriate methods and priorities; 2. Determination of the appropriate phases and rigorous planning and conducting the harmonization process, as opposed to the dynamics of the development of the legal system; 3. Establishing effective application of harmonized regulations, not just formal compliance.
The harmonization process refers to the entire acquiscommunitaire which includes much more than legal regulations. The harmonization refers to 35 chapters 24
Incorporation of European legislation in the Law on Safety and Health at Work of the Republic of N. Macedonia
By application for membership of the Republic of North Macedonia in the European Union on March 22, 2004, begins the process of harmonization of domestic with European legislation, and thus inevitably harmonization of domestic legislation on safety and health at work with European. As a framework directive on occupational safety and health 89/391/EES (Council Directive of 12 June 1989), it inevitably followed that national legislation on occupational safety and health should be harmonized exactly according to principles of this directive. 24 By beginning of the process of harmonization of the existing Macedonian Law on Occupational Safety (Official Gazette no. 13/98), which arose from the Constitution of the Republic of Macedonia, following article 32, with the Framework Directive on Occupational Safety and Health (89/391/EES) set a precedent for the entire area known as "Occupational Safety" to be renamed "Occupational Safety and Health". Namely, the experts who were involved in the process of harmonization of the legislation in the Republic of Macedonia with the one of the European Union, came to conclusion that the existing regulation does not correspond to the one required by the framework directive and decided to fully translate the directive and its adaptation into national law, which would not treated the field of occupational safety, but the field of occupational safety and health.
In order to improve the protection of safety and health at work, labor experts have undoubtedly asked for establishment of a new Law that will protect these principles. Thus, on 1 August 2007, the Law on Occupational Safety and Health 25 was adopted, which was fully harmonized with Framework Directive 89/391/EES. The law was designed in order introducing measures and activities aimed at improving the safety and health of workers at work. "At the same time, the Legislator by this law defines the measures for safety and health at work, the obligations of the employer and the rights and obligations of the employees in the field of safety and health at work, as well as the preventive measures against occupational risks, elimination of risk factors for accident, information, consulting, training of 25 Basic principle according the Law is "the principle of prevention of injuries at work, occupational diseases and workrelated diseases". The Law on Safety and Health at Work introduces for the first time the institute "risk assessment", which introduces a new approach in preventing activity for raising preventive approach in creating safe and healthy working conditions, all in order to protect the health of employees.
The provisions of this law are applied in all activities of the public and private sector, for all persons insured against injury at work or occupational diseases according the regulations for pension, disability and health insurance and for all other persons involved in the work processes. 27 The provisions of this law do not apply to the activities if this matter is regulated by a special regulation (armed forces, police, in some special activities of the protection and rescue forces) as well as to home assistance.
In order to implement the requirements related to occupational safety and health, arising from the Law on Occupational Safety and Health, it is necessary to conduct a comprehensive and constant education of both employers and employees, also to all other participants that have an impact on the decision making process in this area and spreading culture of work. www 10.2011) and a group of bylaws that overall organize, regulate and ensure safety and health at work 26 .
CONCLUSION
As a summary of the above, we can conclude that although legislation in the field of occupational safety and health follows all European and international regulations and constantly complies with them, still the biggest problem is the lack of implementation.
The analysis of the European Commission Progress Reports 27 of the Republic of Macedonia, primarily Chapter 4.19, also indicates that our country is committed to the process of harmonization of national labor legislation related to safety and health. However, the reports note the need to strengthen the capacity of the labor inspectorate and collective contracting and re-point out the situation of inadequate enforcement of protection regulations and the large number of workrelated injuries and deaths. For further harmonization with EU standards, it is necessary to have in consideration the directives of the European Agency for Safety and Health at Work (EU-OSHA). 28 The EK Reports also emphasize that our legal system has successfully and almost completely transponder the law of the European legislation in the field of safety and health at work, but lacks proper, correct and precise implementation of the legal regulations, i.e. the state through the institutions insufficiently promotes the application of the practice of good legal solutions.
As a major remark in the reports is stated that the administrative capacity, still not sufficiently staffed and does not have adequate capacity for normal operation, is not adequate to ensure proper implementation and enforcement of legal provisions, as well as poor interinstitutional coordination and cooperation.
Nevertheless, according to EK Reports, as well as the Reports conducted by state and non-governmental organizations, there is no reduction in accidents at work, but they are constant, which indicates that not only harmonization of European legislation but also greater diligence is not enough and action by all concerned institutions, as well as employers in order to improve the protection of safety and health at work of workers. The EK has repeatedly noted in the Reports that better consolidation of accident data collection is needed.
According to above, we can conclude that the Republic of Northern Macedonia is a country that is moderately prepared for compliance with EU legislation in this area, i.e. if it should be assessed with a score from 1 to 5, in the field of occupational safety and health, the same according to EU is scored with 3. | 2021-08-03T00:06:34.280Z | 2021-01-01T00:00:00.000 | {
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233794297 | pes2o/s2orc | v3-fos-license | Analysis of physics concept of newton’s laws on the dadhak merak dance in the reogponorogo cultural arts
Indonesia is a multicultural country with a variety of cultures, one of which is Reog Ponorogo. Reog Ponorogo is a traditional dance passed down from generation to generation as a culture and identity of the Ponorogo society. Unfortunately, many people considered that Reog Ponorogo only as entertainment that does not contain scientific concepts. This research investigates physics concepts, especially Newton’s Law in Dadhak Merak dance of Reog Ponorogo. This research used the content analysis method involving unitizing, sampling, recording, reducing, inferring, and narrating. The result indicated that Newton’s First Law came up in the inertial frame of the Dadhak Merak. Newton’s Second Law is shown in the acceleration changes influenced by the mass and force of Pembarong. Newton’s Third Law is related to the dancer’s reaction force and the ground that raises a lateral force to the Dadhak Merak dance. This research is expected to contribute as a source of information on the development of contextual teaching and learning physics material.
Introduction
Culture is a way of life that develops in a group of people or community and is passed down from generation to generation as a way of living. Culture is formed based on several elements such as religion, politics, lifestyle, architecture, works, fashions, and languages. Reog Ponorogo is classified as traditional art, and it is prevalent in East Java. Reog art can be found in around 17 districts in East Java involving Mojokerto, Kediri, Surabaya, Jombang, Pacitan, Ngawi, Nganjuk, Trenggalek, Tulungagung, Sidoarjo, and Ponorogo itself, where the Reog Ponorogo art developed [1]. The popularity of the Reog Ponorogo art in the East Java Region makes this traditional art still subsist today.
Indigenous knowledge needs to introduce to the school. Students need to be aware of Indonesian culture and preserve the cultural diversity that has become the nation's identity [2]. The introduction of culture can be conducted by integrating the indigenous culture's scientific process into school science by providing scientific rationalization on the existing indigenous knowledge. The integration of indigenous knowledge into school science is known as ethnoscience [3]. Students' everything can be used as a scientific knowledge source to build and construct their fundamental knowledge [4]. One of the subjects included in the realm of ethics is physics concepts [5]. Physics is part of natural science to explain and investigate various natural phenomena and their interactions. In daily activities, Physics studies matter more related to motion and behavior within space and time and the related actualities of an object's energy and force. Based on the description above, it is necessary to investigate the physics concept in the Reog Ponorogo to make physics learning more contextual and based on local wisdom to preserve indigenous culture. This study aims to identify the concept of Newton's Laws in the art of Reog Ponorogo, especially on the components of Dadhak Merak.
Method
The method used in this study was qualitative content analysis. This research was a semiotic content analysis that is usually used in cultural research by understanding the meaning. The objects of semiotic research are mass media, films, comics, cartoon literature, and music [6]. In this study, the researcher used an acceptable method, such as interviews, documents, and observation, to gather various comprehensive information. Interviews were conducted with experts and the Reog player or Pembarong to support researchers' opinions regarding the Reog Ponorogo. The document analysis sheet is based on the theoretical basis of Newton's laws. At the same time, observation is conducted carefully by analizing the Reog Ponorogo videos and art images. The main instrument used in this study was the human instrument, namely the researcher himself with, the accuracy, criticality, and knowledge of the researcher researching and searching for data [7]. The validity of the data in this study is based on the validity of semiotics. The semantic validity is implemented to determine the suitability of the analysis results' in the chosen context [8]. Experts carried out this validation by checking the suitability of the data interpreted with the actual concept. Following this research design.
Figure 1. Research Design
The data analysis technique used in this study, According to Krippendorff, 2004 follows six stages as follows: (1) unitizing in this stage the researcher collects data relating to research that is calm Reog Ponorogo art, (2) sampling this stage was done to facilitate research, the data entered only data relating to Dhadak peacock Reog Ponorogo. (3) The recording was done with the findings on the Reog Ponorogo video or image. (4) 'Reducing was done to eliminate things that are not relevant to the study. (5) Inferring (conclusion drawing) was done by analyzing the data further by searching for existing units' meaning. (6) Narrating this stage was decision making based on research results-some text.
Result
This study analyzed the physics concepts that exist in Reog Ponorogo of Dadhak Merak dance. The result of the data analyzed in the interviews is presented in the following table. If there are people who rise above the Dhadak Merak it will be heavier because the initial weight (Dhadak Merak) 50 Kg can be doubled, so the movement is not long because it is easier to be tired. Menawi muter e ngiwo kakinipun dijorokake nengen nggeh?
When rotating to the left, does the foot press to the right?
informants Iyo, arah e sikil walikane Yes, the direction is the opposite Informants said that performing Dhadak Merak requires energy or a trainer to give a force to move Dadhak Merak. According to the informants, a style was given shortly after performing the Kebat movement, which makes Dhadak Merak move beautifully after Pembarong stood up. This is showing that Newton's First Law came up in the inertial frame of the Dadhak Merak. The faster the movements made by the Pembarong, the easier the Pembarong will feel tired. If the mass of Dhadak Merak increases due to the presence of someone riding on top of Dadhak Merak, then what Pembarong feels will be heavier, because the burden carried is getting heavier, so doing this movement is usually not too long. This proves the Newton's Second Law is demonstrated in the changes of acceleration in dance influenced by the mass and force of Pembarong. When the Pembarong makes a circular motion to the left, then his feet will press the ground to the right. Then his feet will press the ground to the right. This shows the existence of Newton's third Laws
Discussion
The Dadhak Merak dance starts with the performer wearing a mask that has the shape of a lion's head completed with a crown made of peacock feathers. This mask has a mass more than 50 kg. This heavy mask will be carried by dancers using teeth [9]. The movement on the Reog dance using the Dhadhak Merak is not something that is easily needed by its thoughts to regenerate because the scene cannot be IOP Publishing doi:10.1088/1742-6596/1832/1/012038 4 played by just anyone [10]. Dadhak Merak dance is inseparable from the concept of physics, one of which is Newton's law. Newton's Law provides explanations related to the position's, speed, acceleration, and time [11].
Newton's First Law
Newton's first law states that the resultant force acting on the Dadhak Merak is zero. In this movement shows the application of Newton's 1st Law: if the object is stationary, then the object will remain stationary [12]. Based on the recognition from the guest speaker named Supriyono as follows "In using the Dadhak Merak mask, it requires stronger energy." (07/25/2020). Dadhak Merak moves due to the force performed by the dancer. As shown in Figure 2 below beautifully and supply. This shows the strength, flexibility, and balance of the Pembarong body. This movement shows the application of Newton's first Law in which the Dhadhak Merak, which was originally standing still when given a backward force quickly it will be seen at the top of the Dhadhak Merak maintains its state. The situation is proof of the nature of inertia. Newton's 1st Law is often called the law of inertia [13].
Newton's Second Law
In the Reog Ponorogo dance, the dancer moves with certain speeds according to the tempo of the musical accompaniment. So that the dancer/performer moves with an uncertain speed. This change in speed results in an acceleration/deceleration of movement. When a dancer or performer wants to make a quick move, a great style is needed. This is in accordance with Newton's 2nd Law, namely, "The acceleration of an object is directly proportional to the total force acting on it and inversely proportional to its mass. The direction of acceleration is the same as the direction of the total force acting on it" [14]. Mathematically written
= ∑
Where the force that works is directly proportional to the magnitude of speed. So when the Pembarong wants to make movements quickly Pembarong must provide a great force so that the Dhadak Merak moves in accordance with the wishes of the Pembarong. Additionally, the magnitude the force given by the Pembarong to make movements is influenced by the mass of the Dhadak Merak. In the Reog Ponorogo dance performance, there are not infrequently scenes wherein someone is riding a Dadhak Merak. Figure 2 the mass received by the Pembarong certainly increased not only the Dhadhak peacock mass, but also the mass of the person. Accordance with Newton's Second Law, where mass and force are directly proportional when the mass plus the force required will be even greater. Addition, there are several forces in the opposite direction to the forces carried out by the Pembarong namely the force of gravity and air friction. Therefore, from that in one show usually provided 2-3 Barongan players who will play the dance alternately so as not to fatigue.
Newton's Third Law
In the Reog dance, there is also a movement to shake the Dhadak Merak by moving forward and simultaneously rotating the body from right to left. Every movement, such as to wag Barongan there are rules about the position of the feet, neck and hand movements according to Supriyono "The position of the foot is one step forward, with the neck shortened. While both hands are used to shake the peacock's Dadhak ..." (04/18/2020) "When making a circular motion must balance the legs and pressed" (07/25/2020) According to Newton's Third Law "When an object exerts a force on both objects, the second object exerts an equal force, but in opposite directions to the first object" [15]. When the Pembarong makes a circular motion to the right, then his feet will press the ground to the left. The ground will react and press the spacer to move right so the trajectory moves to the left. Vice versa if you want to make a circular motion to the left, then his feet will press the ground to the right. This shows the existence of Newton's 3 Laws that "for every action, there is an equal but opposite direction" reaction. Mathematically written as follows: The action force is the compressive force performed by the dancer / Pembarong against the ground. So that the ground will give a reaction force so that the dancer / Pembarong moves in the opposite direction.
Conclusion
The dance in the Reog Ponorogo art can give an actual picture related to the application of Newton's Law to explain the acceleration relationship of the Dadhak Merak with the style that works on the Dadhak Merak. Newton's first law explains the inert nature of Dadhak's Merak in the Kebat movement. Newton's Second Law explains changes in acceleration or deceleration in dance as well as the influence of the mass of the style issued by the Pembarong. While Newton's third Law relates to the reaction force of the dancer and the ground to give a sideways force on the Dadhak Merak. Reog Ponorogo still has many other movements that need to be studied scientifically. So, further research is needed to examine other concepts in Reog Ponorogo art as a source of information in the development of contextual physics learning | 2021-05-07T00:04:30.115Z | 2021-03-01T00:00:00.000 | {
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234338448 | pes2o/s2orc | v3-fos-license | Android based information system for eco-site destination
With the rapid development of information and communication technology, the internet has become an essential medium for obtaining or exchanging information without the obstruction of distance, time, and place. The purpose of developing this application is to design the nearest Tourism Location Search Application in South Aceh district based on Android. This application was created using Android Studio. This application provides information about existing tourist objects from location information, facilities, and guidance to reach the location using the Maps method. Users of the application can choose attractions based on categories and the system will provide a guide to the tourist sites that can be visited.
Introduction
With the rapid development of information and communication technology, the internet has become an essential medium for obtaining or exchanging information without the obstruction of distance, time, and place. The internet network is widely available and the growth of its distribution is fast so it is very easy to use it in any location quickly and accurately. This is apparent from the increasing number of Android designs that have arisen because of the rapid development of an Android-based virtual world that can provide various conveniences in everyday life.
Likewise in the tourism sector which is one of the potential sectors in Indonesia. One of them is South Aceh Regency which is located in Aceh Province. South Aceh Regency has very varied tourist locations, such as beach tourism, nature tourism, and historical tourism. However, the spread of information about the charm of Tapaktuan tourism is still done manually through the distribution of brochures, posters, newspapers, and others. This is quite peculiar in increasingly rapid technological developments, and therefore it is time for this manual information organization system to be replaced with an information administration system using technology.
This needs to get attention from the local government because the development of the tourism sector will have an impact on increasing the number of domestic and foreign tourists coming to the South Aceh Regency. So that it can improve the productivity of the local community and provide substantial viability for the region and country. Therefore, this research will design an android-based information system that can introduce the potential of beach tourism for tourists visiting Tapaktuan. This Android is designed to be used by the public in search of information about South Aceh Regency tourism.
Materials and method 2.1. System
According to [1], the system is something that consists of objects or elements, components related to and related to each other in such a way that the elements constitute a particular processing unit. In a structured approach, a system is defined as a network of interrelated procedures, gathered together to carry out an activity or complete a specific goal.
Android
According to [2], Android is an operating system application for cellular phones based on Linux. Android provides open programs for developers to create their applications for use by a variety of mobile devices.
Google maps
According to [3], Google Maps is a popular free service provided by Google. Google Maps is a map of the world that we can use to observe an area. In other words, Google Maps is a map that can be viewed using a browser. Google provides various APIs (Application Programming Interface) that are very useful for web developers. Desktop applications to take advantage of various features provided by Google such as AdSense, Search Engine, Translation, or display Google Maps can also be selected, based on original photos or route map images only.
Java
According to [4], Java is a programming language that can be run on various computers including mobile phones. This language was created by James Gosling while still joining Sun Microsystems when it is part of Oracle and was released in 1995. This language adopted many of the syntax found in C and C ++ but with a simpler object model syntax and minimal down-level routine support [5].
Google maps API
According to [6], Google Maps is a free service provided by Google. Google Maps is a map of the world that we can use to observe an area. In other words, Google Maps is a map that can be viewed using a browser. We can add Google Maps features on the web that we have created or on our blogs that are paid or free even with the Google Maps API. Google Maps API is a library in the form of JavaScript [7].
Information system
According to [8], an information system is a system in an organization that meets the needs of daily transaction processing that supports the organization's operational functions that are regulated with the strategic activities of the organization to be able to provide certain external parties with the reports they need. So, an information system is a collection of data that has been organized along with its use procedures that cover much more than just presentation. This term implies an intention to be achieved by selecting and organizing data and arranging its use procedures [9].
Data collecting techniques
The author conducted a survey directly to the tourist sites, and interviewed representatives of the South Aceh tourism office, and recorded the coordinates of tourism spots. Then take a photolocation to complete the data collected.
Research method
The research site was conducted in tourist spots and supported by information obtained from the Tapaktuan tourism service for 1 (one) month. This research uses research and development (R&D) methods because this research is a development and making a new prototype. The data taken is a 3 source or raw material that is very valuable for the process of producing information. Therefore in taking data it is necessary to handle it carefully so that the data obtained has quality and benefits.
While the method of data collection in this study consisted of Field Studies (observation and interviews) and taking references from books that fit the theme of this study. Engineering In this stage, the author collects the needs of all system elements such as the data used. Where these elements can be extended in the development of tourist information systems in Tapaktuan.
Analysis
Then the stage of analysing system requirements is done by gathering requirements with a focus on software, including information, each function. Then provide tools and techniques that can help the writer to determine the needs through a system that has been running on a tourist information system in processing data. The device in question is the use of Data Flow Diagrams (DAD) to compile data input, process, and output vocational functions. Design At this stage, data structure design, software architecture, procedural details, and interface characteristics are carried out. And designed display screens such as input forms and output forms from the system to be designed. Testing Before an Android-based tourist information system (Tour Guide) can be used properly, testing must be done first. This series of testers is run together with the actual data from an existing system.
System Design
Flowchart is a chart that shows flow in the program or system procedure logically [10].
Figure 1. Flowchart admin
From the flowchart above, the main menu of the program will appear until there is a response from the program when the user chooses the options on the main menu. By using symbols and information, the flowchart above describes the steps that can be done by the user as follows: a) Before entering the system, the admin input username, and password. b) Admin presses the login button to enter the system. c) After the main menu page appears, several menu options can be selected by the admin d) Admin enters the data menu to access tourist sites.
Figure 2. Flowchart user
From the flowchart image above, the main menu of the program will appear until there is a response from the program when the user chooses the options on the main menu. By using symbols and information, the flowchart above describes the steps that can be done by the user as follows: a) After opening the main menu page, there are several menu options for users b) Users can choose the desired menu, according to the existing program.
Use Case is a functional description of a system that will be built so that it can be learned by the following users [11]. Use Case diagram of Android-based Tourist Information System in Tapaktuan.
Figure 3. Use case diagram
From the use case diagram above it can be observed that in this system the admin has access rights in the form of login, see all tour data, whereas the user only has access rights to choose the desired tourist location.
Map display
When the map item is clicked on by the user, the map will be displayed, the map contains the number of tourist attractions. On this map page, there are coordinate points of attractions, this makes it convenient for tourists to see the location of attractions, on this page the map also displays the name of the tourist location. This display shows the type of tourist destination. In this view, there are two tourism options namely, nature tourism and historical tourism. Choose one of them. In this category This menu displays various natural attractions that can be visited by tourists. The information provided is the tourist information display, tourists can choose the type of tour, namely natural attractions, and historical tourism. There is a description of the distance in kilometers from the location of Tapaktuan to tourist sites. This displays the author's bio-data containing info about the application maker, if there are the latest tourist attractions can contact the application maker. In this view, there is a history of the application owner, by displaying the full name, place of birth date, address, gender, marriage status, religion, phone Number, email, university, and study program.
Admin interface
The login menu is used as system security from abuse of access rights, so data security can be guaranteed. Here the user is asked to enter a username and password. In this admin login, the admin enters "username" and "password" that have been registered to enter. This main menu is the initial admin display which contains the home menu, category, location, user, and logout. Admin can choose the menu icons. On the main web page display, there is a slide menu including, home, category, location, user, and logout, each menu has different data. The menu displays data in the form of tourism categories in the form of nature tourism and historical tourism, in this menu the admin can add, edit, and delete data in the category. In this category view, admin can add tourism categories in it, we can see as the picture above there is no category, image category, and action, this action intends to edit category and delete options.
Figure 9. Category menu display
The admin can add data on the tourism category if there is a new tourism destination. In the display add a new category, as contained in the previous image, to add this new category that is filled in is the category name and image. In the categorical name, it can be adjusted to the desired name, as well as images, first select the image in the choose file then save if it is correct, and can be edited again. The menu displays tourism location data that already exists in the system, in this menu the admin can add, edit, and delete data at the location. In this location view, display location no, place name, latitude address, longitude, category, image, edit and delete location data.
Figure 11. Location menu display
In the menu, the admin can add, edit, and delete users. In this view, User Data or Username and Password can be edited and deleted.
Conclusions
The conclusions that can be drawn from developing information systems in Android-based Tapaktuan are as follows: a) This application able to display routes. b) This application successful to display eco-site information. c) This application can display distance and travel time. d) This application also faster and accurate. | 2021-05-11T00:06:12.763Z | 2021-01-01T00:00:00.000 | {
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226384699 | pes2o/s2orc | v3-fos-license | Technology Implementation in SMEs
Purpose - This study aims to examine conceptually the implementation of the use of technology in SMEs, what is the driving force, and inhibiting factors based on theoretical and empirical studies. Design / Methodology / Approach - this research method is a literature study or literature study that contains theories that are relevant to research problems Findings-technology has a significant role in the development of SMEs, arguing that there are six main areas to develop entrepreneurial competencies, namely opportunity, relationship, conceptual, organizing strategic, and commitment skill. Research Limitations / Implications - This research only discusses research and studies related to aspects of SME information technology, learning organizations, drivers of implementation as well as E-Wom factors in SMEs within specific period time. Practical Implications-It could serve as a basis for future research on technology in SMEs Originality-This research has originality by comparing several aspects of research so that it produces some interesting aspects to be studied in technology implementation research in SMEs. Latest-Value Information and can understand in the framework of the implementation of SME technology concerning business performance .
Introduction
Indonesia is a very potential country for SMEs, with a large population and high consumer interest in SME products. A large number of internet users in Indonesia provides an opportunity for SMEs to promote their products through electronic media (N. A. Hamdani & Maulani, 2018) and (Permana, 2019). The use of information systems in SMEs is also growing with the changes and technological developments (A. Hamdani & Fatah, 2018). Technology also plays a role as a reliable business strategy and can be used by all lines of the business organization(N. A. Hamdani, Herlianti, & Maulani, 2019;. The number of SMEs in 2015 is estimated to reach 3,615,650 business units. In other words, there was an increase of 2.6% from 2014; that is, 3,522,851 business units. Regarding the development of SMEs in Indonesia, there has been an increase every year in the period 2010-2015 although it is always below 5% (BPS, 2016). This study aims to examine conceptually the implementation of the use of technology in SMEs, what is the driving force and inhibiting factors based on theoretical and empirical studies. In this study, we will try to compare previous studies regarding the implementation of technology in SMEs.
Literature Review
Some studies say that Business is very dynamic in its changes, to meet the needs of consumers and meet them with environmental changes very quickly (Kalibatiene, Vasilecas, Savickas, Vysockis, & Bobrovs, 2016). Furthermore, mentioning that the key to success in analyzing a business situation is how the company's ability to capture market capability and be able to identify internal capabilities (Teece, Pisano, & Shuen, 1997). While (Putten, Schief, Berlin, & Darmstadt, 2012), requires companies to be able to identify factors that can affect the sustainability of a business. While (Foster, 2013) in his thesis explains that the ability to apply Information Systems and Decision Support Systems is the key to success in analyzing the competition. The strategy is the steps that must be achieved by the company to achieve the targets or objectives set based on the analysis of the company's resources and capabilities (Hamdani, N.A dan Rhamdani, 2019).
Several studies have shown that the use of information technology will benefit SMEs. SMEs will have good competitiveness if they have adopted IT in carrying out their business activities (Apulu & Latham, 2010). While (Zoroja, 2016), also has a similar opinion that SMEs who have IT will be more innovative to be able to compete with other MUs. Information technology will have a positive impact on the company (N. A. . The company will have the ease of entering the market, Volume 1 Issue 1 (April) 2020 © 2020 Study Program of Entrepreneurship, Universitas Garut, Indonesia All rights reserved.
diversifying the types of products produced, and the efficiency of production costs. The positive impact of IT on the company will be able to improve company performance in the end (Maulani & Hamdani, 2019b). So it can be said that the use of IT in companies is a strategic step that will bring the company to an increasingly increasing profitability, is that is one of the financial performance indicatorsn (Ling, 2017), (A. Hamdani & Fatah, 2018) dan (Lingesiya, 2012).
The ability of information technology as the company's ability to mobilize and disseminate resources based on information technology in combination or combination with other resources and capabilities (Ling, 2017). Technology capability refers to the ability to develop and design new products and process and enhance knowledge of the physical world in a unique way, thereby transforming this knowledge into designs and instructions for creating expected outcomes . More concretely, technological capability is a collection of pieces of knowledge that includes practice and theoretical know-how, methods, procedures, experience, and physical devices and equipment. The ability of IT as the ability of a computer system, a collection of computers and related technology in an organization to store, process, and convey information (Al-Rajhi, Majed and Liu, 2010).
Therefore, to measure information technology capability, dynamic information technology capability, intergrating information technology capability, utility information technology capability indicators are used (Ling, 2017).
Methodology/Materials
The research method is a literature study or literature study that contains theories that are relevant to research problems. This section reviews the concepts and theories used based on the available literature, especially from articles published in various scientific journals.
A Literature study serves to build concepts or methods that form the basis of study in research. Literature study or literature study is an activity that is required in research, especially academic research, whose main purpose is to develop theoretical aspects and aspects of practical benefits.
Result and Findings
The use of information technology systems at this time is needed by SMEs, at some SMEs, the level of information system needs and the ability of learning organizations is needed so that SMEs can survive (N. A. Hamdani & Susilawati, 2018) and Further research explains that SMEs need technology to develop and have a positive impact on SMEs (Albesher, 2012;N. A. Hamdani, Maulani, Tetep, & Supriyadi, 2020). On the other hand, the application of technology provides benefits. Still, most SMEs have constraints.
Some of the results of this study indicate information technology and co-creation have a significant influence on business performance. Information technology is used by SMEs to promote their products. In addition, SMEs also use information technology for business management even though information technology is not implemented optimally because of financial limitations (N. A. Hamdani, Solihat, & Maulani, 2019) and (Fadil, 2015;N. A. Hamdani, Maulani, & Solihat, 2020).
Companies that have no competitiveness will be abandoned by the market. Because not having competitiveness means not having excellence, and not superior means there is no reason for a company to survive in the competitive market for the long term (Kabue & Kilika, 2016). Competitiveness relates to how the effectiveness of an organization in the competitive market, compared to other organizations that offer the same or similar products or services. Companies that are able to produce good quality products or services are effective companies in the sense that they will be able to compete. Several studies have measured the competitiveness of SMEs by using indicators containing factor conditions, demand conditions, related and supporting industries and business strategies (Anton, Muzakan, & Muhammad, 2015). The measure of competitiveness in SMEs in this study uses organizational capability, competency (Nota & Santander, 2012). Some studies in the last 10 years include several studies on SMEs as follows: Volume 1 Issue 1 (April) 2020 © 2020 Study Program of Entrepreneurship, Universitas Garut, Indonesia All rights reserved. Table I.
Conclusion and Implication
The Adoption of technology is needed to support the business performance and competitiveness of SMEs. The use of information technology has a huge role, supported by government policies that support the technology sector. Based on this, it was also stated that technology has a major role in the development of SMEs, said that there are six main areas to develop entrepreneurial competencies, namely opportunity, relationship, conceptual, organizing strategic and community skills. In this study, there are four topics in the SME technology study, namely E-Wom, information technology, ICT driving organizations, and external factors driving IT implementation. | 2020-10-28T19:20:32.196Z | 2020-04-21T00:00:00.000 | {
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218760399 | pes2o/s2orc | v3-fos-license | Using Machine Learning to Predict Bacteremia in Febrile Children Presented to the Emergency Department
Blood culture is frequently used to detect bacteremia in febrile children. However, a high rate of negative or false-positive blood culture results is common at the pediatric emergency department (PED). The aim of this study was to use machine learning to build a model that could predict bacteremia in febrile children. We conducted a retrospective case-control study of febrile children who presented to the PED from 2008 to 2015. We adopted machine learning methods and cost-sensitive learning to establish a predictive model of bacteremia. We enrolled 16,967 febrile children with blood culture tests during the eight-year study period. Only 146 febrile children had true bacteremia, and more than 99% of febrile children had a contaminant or negative blood culture result. The maximum area under the curve of logistic regression and support vector machines to predict bacteremia were 0.768 and 0.832, respectively. Using the predictive model, we can categorize febrile children by risk value into five classes. Class 5 had the highest probability of having bacteremia, while class 1 had no risk. Obtaining blood cultures in febrile children at the PED rarely identifies a causative pathogen. Prediction models can help physicians determine whether patients have bacteremia and may reduce unnecessary expenses.
Introduction
Fever is one of the most frequent reasons for visits to the Pediatric Emergency Department (PED) [1,2], and estimates say that up to 10% to 25% of cases with febrile illness had bacterial infection [3,4]. Determining the appropriate method for evaluating febrile children remains a challenge, especially due to fears regarding occult bacteremia in febrile children who appear well without an obvious infection focus [5]. Bacteremia is a severe bacterial infection that can be detected by a blood culture, one of the most frequently ordered microbiological tests in the PED [6]. Blood cultures remain the gold standard test for detecting patients with bacteremia. Isolating the organism from the blood can confirm a diagnosis, which helps physicians identify the cause of the infection and then administer the appropriate antimicrobial agents. Upon receiving a blood culture result, physicians must decide whether the organism represents a clinically significant infection [6][7][8]. However, seeing a high rate of negative, false-positive, or contaminated blood cultures is quite common in children visiting the PED [9]. All of the above conditions result in the unnecessary use of healthcare resources and costs, including additional invasive testing, the inappropriate use of antibiotics, prolonged hospital admission, and parental anxiety [10][11][12][13].
Over the past few decades, several algorithms have been developed to identify children at a higher risk of severe bacterial illness [3,4,14]. In addition to clinical findings, some studies have suggested that laboratory findings, such as white blood cell count (WBC), absolute neutrophil count (ANC), C-reactive protein (CRP), and procalcitonin (PCT), may be useful in helping physicians recognize children with severe bacterial infection [4,[14][15][16][17][18]. Although these parameters can help pediatric clinicians identify febrile children at high risk of severe bacterial infection, are they capable of predicting bacteremia in febrile children? Many studies also debate the role of appropriate blood cultures in the PED. Obtaining blood cultures is only recommended for children with extensive infections or immune-compromised patients or for those with moderately or severely ill children, according to published guidelines and studies [19,20].
In this study, we tried to build machine learning models to predict bacteremia in children with fever who visit the PED. Our findings can be clinically important as they may help physicians in the PED either order the appropriate blood culture or manage treatment depending on whether bacteremia is predicted.
Study Population
Patients younger than 18 years of age who presented to the PED of the Kaohsiung Chang Gung Memorial Hospital in Taiwan with fever during the period of January 2008 through December 2015 were evaluated. Febrile children from whom a blood test and blood culture were obtained formed our retrospective cohort. In this cohort, each febrile child with true bacteremia was randomly matched with 10 febrile children without bacteremia according to gender and age in order to form a case control study. All the blood cultures were collected by nurses in accordance with our institution's standard procedures. Obtaining two sets of blood cultures was quite difficult in pediatric patients, so one set of blood culture for children with fever was generally practiced in our PED. This study was approved by the Institutional Review Board of Chang Gung Medical Foundation.
Blood Culture Criteria
The following organisms that were isolated from the blood sample represent a true pathogen: Staphylococcus aureus, Streptococcus pneumoniae, Salmonella enterica, group A streptococci, Pseudomonas aeruginosa, Haemophilus influenzae, Escherichia coli (E. coli), and Candida species, among others [21][22][23][24]. Certain organisms isolated from blood samples have been found to represent contamination. These pathogens include coagulase-negative staphylococci, Staphylococcus epidermidis, Corynebacterium spp., Gram-positive Bacillus, Micrococcus, etc. [21][22][23]. In the case of any doubt related to the potential pathogenicity of one of the isolated species, the research coordinator reviewed the case to determine whether the corresponding blood culture was an actual infection.
Statistical Analysis
Continuous variables were expressed as mean ± standard deviation, and categorical variables were reported as percentages. To compare clinical characteristics between children with and without bacteremia, Student t tests and Fisher's exact test or x 2 test were used for continuous variables and categorical variables, respectively. Univariate and multivariate binary logistic regression analyses were used to identify the significant risk factors. A P-value less than 0.05 was considered statistically significant. IBM SPSS statistical software for Windows, version 22.0 (Chicago, IL, USA) was used for statistical analyses.
Experimental Methodology
Age, gender, and laboratory values obtained from medical records were evaluated as predictive variables by the models. For every laboratory value variable, only values obtained simultaneously with the blood culture sample were used. In addition to gender and age, 17 laboratory values were included in our study population. The normal reference of these values may differ in different ages or genders, so case control study was used to eliminate these confounding factors. Due to the hugely different case numbers in the bacteremia group and non-bacteremia group, each case with bacteremia was randomly matched by age and gender with ten cases without bacteremia. To avoid sample selection bias, the matching procedure was repeated 100 times for the characteristic variable analysis. The features selected for machine learning were determined according to the 100 times of characteristic variable analysis. The selected features were further used to establish the predictive model.
Python language, R language, and machine learning methods-logistic regression (LR) and support vector machines (SVM) were used to establish a predictive model for bacteremia. Cost-sensitive learning was applied to make a trade-off between false negative predictions and cost reduction to increase the usability of the predictive model. In the dataset, 97.5% of the cases were used for training, and 2.5% were used as testing data. A risk value of each case was calculated using the SVM or LR predictive model. Since the dataset is generally imbalanced, cost-sensitive learning is used for imbalanced classification. The costs of prediction errors (and potentially other costs) are considered when training a machine learning model. We set bacteremia as positive and non-bacteremia as negative. The amounts of positive and negative cases were quite different. Considering the confusion matrix of inference, the costs of true positive and true negative were set to zero. That is, those cases for which we can correctly predict the result had no cost. In this study, the false negative was more important, which means it will cost more when we predict bacteremia as non-bacteremia. Therefore, we must pay more attention to false negatives. If we assign the cost of false positive as 1, then we must carry out experiments to dynamically adjust the cost of a false negative (an important parameter that can significantly affect the performance of prediction) and find the optimal one. The optimal value is in the range of 7~13. The risk is defined as the cost of negative (the cost of predicting a non-bacteremia result) minus the cost of a positive (the cost of predicting a bacteremia result) (i.e., risk = negative cost -positive cost). If cost > 0, predict bacteremia, otherwise, predict non-bacteremia. However, this risk-based binary prediction (i.e., cost > 0 and cost < 0) does not have good performance. Instead of predicting by plus or minus of risk value, we partition the range of risk value into a couple of segments, such as the quartile, and we make a prediction for each segment; therefore, the performance can be obviously improved.
Patient Characteristics
Among a total of 266,679 children that visited the PED during the 8-year period, 16,967 febrile children with blood culture tests were enrolled for further analysis. The mean age of the total study population was 5.16 ± 3.97 years old. Of these 16,967 children, 55.3% (n = 9388) were male. We observed three kinds of blood culture results: bacteremia indicating true infection, contamination indicating a false-positive result, and negative culture (no growth of wither aerobic or anaerobic pathogens). Patients were categorized into two groups according to the blood culture results: the bacteremia group and the non-bacteremia group (including contamination and negative results). In this study cohort, 146 (0.86%) febrile children had true bacteremia, 405 (2.39%) children had a contaminant result, and 16,416 (96.75%) children had a negative result. The gender, age, and laboratory tests were obtained for further analysis and compared according to the blood culture results, as shown in Table 1. Age, percentage of neutrophil and band, ANC, Hb, platelet, and CRP were statistically different between bacteremia and non-bacteremia encounters. The three most common pathogens in the bacteremia group were Salmonella entericae (28/146, 19.18%), Escherichia coli (22/146, 15.07%), and Streptococcus pneumoniae (14/146, 9.59%). The three most common pathogens isolated from blood culture and considered contamination were Staphylococcus epidermidis (137/405, 33.83%), coagulase-negative staphylococcus (129/405, 31.85%), and Micrococcus (45/405, 11.11%). The age distribution of the bacteremia group is shown in Figure 1. More than half (55.7%) of the febrile children with bacteremia were under the age of 3 years old. 33.83%), coagulase-negative staphylococcus (129/405, 31.85%), and Micrococcus (45/405, 11.11%). The age distribution of the bacteremia group is shown in Figure 1. More than half (55.7%) of the febrile children with bacteremia were under the age of 3 years old.
Feature Selection and Risk Classification
After repeating the characteristic variable analysis 100 times, WBC, MCV, MCH, MCHC, Monocyte, Eosinophil, CRP, Band percentage, Segment + Band percentage, and ANC were all positively or all negatively correlated with bacteremia (Table 2) and their odds ratio are shown in Table 3. These 10 features were selected to establish the predictive model. Among these 10 features, significant risk factors were also picked up for multivariate binary logistic regression analysis and their coefficients are shown in Table 4. We used the cost-sensitive approach in machine learning to tackle the imbalance dataset which we have collected in this study. The curves of recall (sensitivity), true-negative rate (specificity) and AUC (area under the ROC curve) for different cost we applied are shown in Figure 2 (each point represents the average result of 100 times analyses). These three performance indexes were considered in our study. Thus, we can choose the best cost value to balance these performance indexes.
The maximum values of different performance indexes by LR are shown in Table 5. With cost-sensitive learning, the maximum areas under the curve (AUC) of LR and SVM to predict bacteremia were 0.768 and 0.832, respectively. Table 2. The maximum value, minimum value, mean value, and standard deviation of logistic regression coefficients of characteristic variables.
Diagnostics 2020, 10, x FOR PEER REVIEW 5 of 13 After repeating the characteristic variable analysis 100 times, WBC, MCV, MCH, MCHC, Monocyte, Eosinophil, CRP, Band percentage, Segment + Band percentage, and ANC were all positively or all negatively correlated with bacteremia (Table 2) and their odds ratio are shown in Table 3. These 10 features were selected to establish the predictive model. Among these 10 features, significant risk factors were also picked up for multivariate binary logistic regression analysis and their coefficients are shown in Table 4. We used the cost-sensitive approach in machine learning to tackle the imbalance dataset which we have collected in this study. The curves of recall (sensitivity), true-negative rate (specificity) and AUC (area under the ROC curve) for different cost we applied are shown in Figure 2 (each point represents the average result of 100 times analyses). These three performance indexes were considered in our study. Thus, we can choose the best cost value to balance these performance indexes. The maximum values of different performance indexes by LR are shown in Table 5. With cost-sensitive learning, the maximum areas under the curve (AUC) of LR and SVM to predict bacteremia were 0.768 and 0.832, respectively.
Variable
Minimum Considering the bacteremia samples in the dataset, we calculated a risk value of each sample and their quartile ranges are shown in Figure 3. We did the same for the risk value of non-bacteremia samples and showed its quartile range. According to the range of the minimum risk value of bacteremia −0.640) and maximum risk value of non-bacteremia (0.644), it is divided into three categories. Further analyzing the quartile range, bacteremia data are located between [−0.014, 0.15], while the non-bacteremia data are located between [−0.061, 0.132]. Using the 1st quartile of bacteremia and 3rd quartile of non-bacteremia, we can divide the range of risk value to five blocks (as shown in Figure 4). With these five classes, we can predict class 1 as being non-bacteremia, class 2 as low risk of bacteremia, class 3 as medium risk, class 4 as high risk, and class 5 as being bacteremia.
Diagnostics 2020, 10, x FOR PEER REVIEW 7 of 13 categories. Further analyzing the quartile range, bacteremia data are located between [−0.014, 0.15], while the non-bacteremia data are located between [−0.061, 0.132]. Using the 1st quartile of bacteremia and 3rd quartile of non-bacteremia, we can divide the range of risk value to five blocks (as shown in Figure 4). With these five classes, we can predict class 1 as being non-bacteremia, class 2 as low risk of bacteremia, class 3 as medium risk, class 4 as high risk, and class 5 as being bacteremia.
Subgroup Study
Most febrile children with bacteremia were those under the age of 3 years old. Therefore, we conducted a subgroup study that included only these younger children. Among this subgroup, 1.58% (n = 81) febrile children had true bacteremia, 4.7% (n = 238) children had a contaminant result, and 93.8% (n = 4795) children had a negative result. Gender, age, and laboratory tests were obtained for further analysis and compared according to the blood culture results, as shown in Table 6. Age, percentage of band and eosinophil, hemoglobin and CRP differed statistically between bacteremia and non-bacteremia encounters in the subgroup study. Of these young children, the most common isolated pathogens were Salmonella entericae ( The AUC ranged between 0.616 to 0.750 for predicting bacteremia in children under the age of 3 years old in the model we developed.
Variable
Bacteremia Non-bacteremia p-value Figure 4). With these five classes, we can predict class 1 as being non-bacteremia, class 2 as low risk of bacteremia, class 3 as medium risk, class 4 as high risk, and class 5 as being bacteremia.
Subgroup Study
Most febrile children with bacteremia were those under the age of 3 years old. Therefore, we conducted a subgroup study that included only these younger children. Among this subgroup, 1.58% (n = 81) febrile children had true bacteremia, 4.7% (n = 238) children had a contaminant result, and 93.8% (n = 4795) children had a negative result. Gender, age, and laboratory tests were obtained for further analysis and compared according to the blood culture results, as shown in Table 6. Age, percentage of band and eosinophil, hemoglobin and CRP differed statistically between bacteremia and non-bacteremia encounters in the subgroup study. Of these young children, the most common isolated pathogens were Salmonella entericae ( The AUC ranged between 0.616 to 0.750 for predicting bacteremia in children under the age of 3 years old in the model we developed.
Subgroup Study
Most febrile children with bacteremia were those under the age of 3 years old. Therefore, we conducted a subgroup study that included only these younger children. Among this subgroup, 1.58% (n = 81) febrile children had true bacteremia, 4.7% (n = 238) children had a contaminant result, and 93.8% (n = 4795) children had a negative result. Gender, age, and laboratory tests were obtained for further analysis and compared according to the blood culture results, as shown in Table 6. Age, percentage of band and eosinophil, hemoglobin and CRP differed statistically between bacteremia and non-bacteremia encounters in the subgroup study. Of these young children, the most common isolated pathogens were Salmonella entericae (26/81, 32.10%), Escherichia coli (19/81, 23.46%), and Group B Streptococcus (6/81, 7.41%). After repeating the characteristic variable analysis 100 times, the significant factors and their ORs are shown in Supplemental Table S1. The multivariate binary logistic regression coefficients of significant risk factors are shown in Supplemental Table S2.
The AUC ranged between 0.616 to 0.750 for predicting bacteremia in children under the age of 3 years old in the model we developed.
Discussion
The main findings of this study were as follows: (1) the bacteremia rate in febrile children that presented to the PED was low, (2) CRP was significantly higher and hemoglobin was significantly lower in children with bacteremia, (3) younger children (<3 years of age) with fever are more likely to have bacteremia than older children, and (4) machine learning can help us classify the risk of bacteremia in febrile children.
As many as 3-10% of well-appearing children under the age of 3 years old with fever without a source were found to have an occult bacteremia in the prevaccine era. Due to the concern of bacteremia becoming an invasive illness, many practitioners recommended routine blood tests, including blood culture, and then antibiotic therapy based on WBC results, as part of the management strategy for these children [3,14]. However, since the introduction of the Haemophilus influenzae type b (Hib) vaccine in the late 1980s and the pneumococcal conjugate vaccine (PCV) in the 2000s, a dramatic decline in bacteremia was observed as low as 0.25% to 1.43% in children [25][26][27][28]. Irwin et al. also reported an annual reduction of 10.6% in vaccine-preventable bacteremia and found that PCV was associated with a 49% reduction in pneumococcal bacteremia between 2001 and 2011 [29]. The Hib vaccine and PCV were first introduced to Taiwan in 1996 and 2005 respectively. In our current study, the overall bacteremia rate was about 0.86% in all febrile children and about 1.58% in younger febrile children (less than 3 years) that presented to the PED. Furthermore, only <0.1% (n = 14) febrile children were identified to have bacteremia with Streptococcus pneumoniae, and none of the blood culture results yielded Hib. This result is in agreement with the low bacteremia rate in the post-Hib vaccine and post-pneumococcal vaccine eras. Although adequate aseptic techniques can substantially reduce the risk of contaminating blood culture specimens, contamination rates of 2% to 3% are considered acceptable [30]. The overall contamination rate was 2.39% in our study, and the isolation of contaminant organisms from a blood culture has a significant negative impact on patient management, including misdiagnosis, unnecessary antibiotics, performance of additional and unnecessary diagnostic tests, additional costs, and prolonged hospital stays [11][12][13]. A low positive blood culture result with a high rate of contaminant results has made physicians doubt the usefulness of blood cultures in children with fever that visit the PED. How to reduce unnecessary blood cultures will become an important issue for healthcare systems in the postvaccine era.
CRP is an acute-phase reactant protein synthesized by the liver in response to elevated cytokine levels and has been studied as a sensitive marker of bacterial infection [31,32]. Many studies have proposed that high CRP concentration may be associated with severe bacterial infection in febrile infants and children [15,18,[33][34][35]. In both the complete study population and the younger age subgroup (<3 years) in our study, elevated CRP concentration was significantly higher in patients with bacteremia. Our results support the finding of high CRP levels in children with bacterial infection. Using CRP to properly manage children with fever may help identify true bacteremia and reduce unnecessary antibiotic therapy. Some studies have used CRP together with other parameters to predict children with severe bacterial infection. In two recent studies, CRP with extreme leukocytosis was proposed to be useful in predicting severe bacterial infection in children [33,34]. Buendia et al. showed that Rochester criteria plus CRP testing was the most cost-effective strategy for detecting serious bacterial infections in children one to three months old with fever without a source [36]. However, the Rochester criteria is especially applied to young infants, not older infants and children. To the best of our knowledge, no study used more than two clinical parameters together with CRP to predict bacteremia in febrile children. We proposed a useful model for predicting bacteremia in febrile children, not only those with CRP but also other common laboratory parameters in the PED setting.
Anemia due to disease is often seen in various inflammatory states, including acute or chronic infections, autoimmune problems, chronic kidney disease and inflammation, and certain cancers [37]. Anemia has commonly been associated with infections that are typically seen in a pediatric primary care setting [38]. In 2009, Ballin et al. also reported that bacteremia and pyelonephritis are accompanied by a significant drop in hemoglobin levels without evidence of hemolytic anemia [39]. When infection occurred, the inflammatory cytokine could induce hepcidin production in the liver, increase macrophage activation and red blood cell (RBC) destruction, and suppress erythropoiesis. Therefore, inflammation-related anemia may result from hepcidin-induced hypoferremia combined with the cytokine-mediated suppression of erythropoiesis and decreased lifespan of erythrocytes [40]. This phenomenon can explain the findings of lower Hb in children with bacteremia in our cohort.
Significant differences in the percentage of neutrophil, ANC, platelet and eosinophils were also observed in our study. However, most reference intervals of pediatric hematology analytes are age-dependent, especially WBC and its differential count [41,42]. The changes in either absolute count or percentage of neutrophils are dynamic, particularly in the young infants and during the first years of life. The mean percentage of neutrophils may as low as 31-33% and the mean count of neutrophils also achieves its nadir at an age of 6 months to 2 years old [42]. Both lower percentage of neutrophils and ANC in children with bacteremia may due to the younger age (81 of 146 cases are younger than 3 years of age) in our study population. Reactive thrombocytosis has diverse etiologies, including inflammatory, neoplastic and infectious diseases [43]. In most patient series, acute infections represent the most common cause of reactive thrombocytosis [44,45]. In addition to CRP induction, interleukin-6 also plays a pivotal role in thrombocytosis of inflammation [46]. In children with bacteremia, the inflammation-associated cytokines produced primarily by WBC at inflammatory sites may further cause the elevation of CRP level and induce reactive thrombocytosis. This can explain the finding of higher platelet count in our bacteremia group. The pathophysiology of eosinopenia is related to the migration of eosinophils to the inflammatory site, presumably as a result of chemotactic substances secreted during the acute phase of inflammation [47]. A decreased number of circulating eosinophils is regarded as a consequence of acute bacterial infection and several studies have used eosinophil count as an indicator of bacteremia [48][49][50][51]. Our finding supports the view of low eosinophil count in patients with bacteremia.
Zeretzke et al. reported that the children most at risk for occult bacteremia are those younger than 36 months of age with a fever of 39 • C or higher [52] due to the high probability of developing serious bacterial infections, such as meningitis, sepsis, pneumonia, septic arthritis, osteomyelitis, and pyelonephritis [5,14,53]. Therefore, obtaining blood cultures for febrile children with a young age is reasonable. However, febrile children with bacteremia are mostly seen in children under the age of 3 years old with a bacteremia rate of 1.58% versus 0.55% in children more than 3 years old in our study. In other words, these older febrile children with lower probability of bacteremia may have more unnecessary blood cultures, which may waste medical resources. How to reduce the frequency of blood culture in febrile children without misdiagnosis of bacteremia becomes an important issue.
The quality and cost of the healthcare being provided has become an increasing issue worldwide. This concern has led to a focus on how we can achieve equal or better quality outcomes with fewer health resources or less money. Segal et al. described contaminant blood cultures in 85 children that added more than $78,000 in unnecessary charges [13]. A recent study also demonstrated a yearly savings of ∼$250,000 in hospital charges when the blood culture contamination rate was reduced from 3.9% to 1.6% [11]. Some guidelines for inpatient community-acquired pneumonia (CAP) management recommend considering blood culture testing for inpatients with moderate to severe bacterial pneumonia [19,20]. However, obtaining blood cultures in children hospitalized with CAP rarely identifies a causative pathogen, which makes blood cultures less useful [54,55]. The high rates of negative culture results can also represent overuse. In our current study, we have also found a high negative culture rate and a low bacteremia rate, which indicates an overuse of blood culture and a waste of healthcare resource in febrile children. Therefore, how to reduce the over-use of blood culture without missing patients with dangerous bacteremia is important. With the "cost-sensitive learning" model that we proposed with machine learning, we can identify those febrile children with no risk of bacteremia (class 1 risk value) and avoid unnecessary blood cultures to save healthcare resources.
Blood culture remains the gold standard to diagnose bacteremia, but it is a time-consuming diagnostic tool. After the blood sample being collected, it may take couple days to have the initially result of Gram stain, such as Gram-positive cocci, Gram-negative bacilli, etc. Physicians may be informed the final result of blood culture further few days later. Those features used in our prediction model are laboratory data which can be available within one hour after blood sample being collected. In clinical practice, we can implement a decision-making application program running over personal computer as a decision support tool. Those variables of laboratory findings are input data, and this tool can give a report to illustrate a risk probability of bacteremia for clinical reference. In addition, the report will also come out with the distribution of each variable of patients in the database in a way of data visualization for comparison. Therefore, our prediction model can be a part of clinical decision support system to help physicians determine whether patients have risk of bacteremia and thereafter arrange adequate medical treatment.
The results of this study should be interpreted with respect to certain limitations. First, procalcitonin (PCT), a useful biomarker proposed for predicting bacterial infection [56], was not commonly used in our hospital during the study periods. Therefore, PCT use was noted as a variable in our models. Second, those febrile patients who visited out-patient departments and were hospitalized were not included. These patients may also have bacteremia. Third, the models that we used mostly relied on laboratory tests, and the information contained within medical notes were not used. Fourth, some information recorded in medical notes, such as clinical symptoms, location of infection, vital sign (heart rate, respiratory rate, blood pressure, and oxygen saturation), respiratory pattern, and general appearance of the patient, etc. are important to help physician to determine the severity of a febrile patient. However, these data were not available in our database to improve our prediction model. These might have limited our model's performance. Natural language processing techniques to get bacteremia-relevant information from unstructured medical notes are expected to improve the predictive models.
Conclusions
Obtaining blood cultures in febrile children at the PED are definite diagnosis of bacteremia but they rarely identify a causative pathogen. Moreover, overuse or waiting the result of blood culture has been described in relation to a financial burden to healthcare system. Our machine learning prediction model can be a part of clinical decision support system and help physicians determine whether patients have risk of bacteremia and may reduce unnecessary expenses.
Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4418/10/5/307/s1, Table S1: The odds ratio of each variable and its 95%CI after repeating 100 times univariate logistic regression in subgroup study, Table S2: The maximum value, minimum value, mean value, and standard deviation of multivariate binary logistic regression coefficients of significant risk factors in subgroup study. | 2020-05-21T09:07:47.378Z | 2020-05-01T00:00:00.000 | {
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219164563 | pes2o/s2orc | v3-fos-license | THE LEADERSHIP STYLE TOWARD STUDENT’S PERFORMANCE AS EVENT ORGANIZER OF ECONOMIC CARE DAY
: The aims of the study was to see the leadership style toward students’ performance as event organizer of 'Economic Care Day' held by students. Quantitative becomes a method in this study. Meanwhile, data was analyzed with SPSS 19 tools using a simple linear regression test. Research Data was collected through observations, questionnaires and documentation. The research sample was students of Semester IV (four) class B in the economic education study program of STKIP YPM that contracted leadership courses. Based on data, it was known that the research results show that: there was a significant influence between the leadership style toward the student's performance in the events in the Economic’s Care Day. It can be seen from the ability of team leaders in establishing cooperation and coordination relationship with various parties, especially among the organizer team, in this matter was based on the ability of effective communication and team leader behavior. The regression coefficient was obtained at 0.77, meaning that any change in team leadership appears to clearly affect student performance as the event organizer by 77.0%.
INTRODUCTION
The realization of the theory in a field project done in the fourth semester of Class B, will make them understand directly the theory they have had. The combination of theoretical and practical learning models was applied for better mastery of student skills. In mastery of such skills not only on the theoretical level but get the direct experience of the field will enrich the knowledge and experience with their teamwork to be trained in finding solutions to the problem they encounter in the real world. So in the working world they are not awkward dealing with the community. This concept of learning, then known as active learning, which is currently directed by the Ministry of Technology Research and Higher Education to face the era of Industrial Revolution 4.0. The Era of Industrial Revolution is characterized by using high technology in almost all aspects of life. Nowadays, various fields of work have successfully replaced by high-tech systems, such as; ticket sales, online shopping and other daily necessities.
Leadership that has emerged from a variety of literature shows an almost uniform definition, which is the ability to influence other people or groups that aim to do something in achieving a particular goal. In this case, often the leadership is linked to the manager, but there are many fundamental differences in the concept of leadership with the manager, as expressed Luthan in the Organizational Behavior that was hired from Managing the dream: Leadership in the 21st Century (Warren G Bennis, in Journal of OCD).
Students' activities in human beings who have the ability to communicate are the responsibility of education, especially in preparing the maintenance of the event's care day students into subjects that have communication, both inter-team have a strong, creative, independent, professional in their respective fields.
In the success of an organization strongly influenced by the performance of the team, performance is a working achievement, which is a comparison of the work that is real with the standards of the work set. Each organization will strive to always improve student communication in order to achieve a defined goal. Various ways can be taken by organizations to improve student LEADERSIP skills.
Then according to Blancard and Hersey (in Reni Yesi, S 2017:29) expressed leadership is the process of influencing the activities of individuals and groups in order to achieve the objectives in certain situations.
According to Fiedler (in Reni Yesi S,29), no one can be a successful leader by simply applying a variety of leadership styles to all situations. Therefore, successful leaders must be able to apply different leadership styles according to different situations.
According to Lord & Maher (in Susilo Toto Raharjo, 2007:72), a person is a leader because of the perceived other party as a leader. The leader is an object of perception, whether to be a credible person, it also depends on the perpetrator (perceiver) in selecting, organizing, and interpreting the information it receives.
According to Wukir (2013:29) leadership may be defined as individuals who work to influence changes in the actions or behaviors of follower followers. The approach to leadership that comes from before that focuses only on the individual as a leader is a concept that includes leadership and follower relationships as separate units.
According to Suwanto (2013:140) leadership is the ability to influence the group towards goal-reaching. Kouzes and Posner (2004:3) say leadership is the creation of a way for people to contribute to the realization of something extraordinary.
Early observations were done by researchers related to the performance of students in the find some fact that in the implementation of the students' duties have different traits and behaviors. Some of them were passionate and full of responsibilities, there is also a laboratories that in doing the job party a sense of responsibility, but also there are students who are often irresponsible for the courses that are in.
According to Susilo Toto Raharjo (2006:70) leadership is one of the issues in management that is still interesting enough to be talked about until today. Mass Media, both electronic and print, often display opinions and talks discussing leadership. A very strategic and important leadership role for the achievement of the Mission, vision and purpose of an organization, is one of the motives that encourages people to always investigate the subtleties associated with leadership.
Then according to Muhaimin (2009:29) The leadership is the existence of a process in the leadership to give the social influence of other people, so that the other person runs a process as the leader was exiled.
Power, time the team's performance is necessary. The team leader is still in the game with students. With the performance of the team, this activity can run well with the presence of students who have low team performance, students' ability is very difficult to achieve if the lecturer does not know the development of the era. Thus the ability is not as expected.
The problem in this research is to contribute to the influence of the leadership of the team leader in the performance of students in event's Economic Care Day activities at SDN 114 Merangin? ". In this research aims to know how the team's leadership influence on student performance.
RESEARCH METHODS
The research uses quantitative approach with a descriptive method, with the intent to look for influences between the variable independent (X) with the dependent variable (Y) using the statistical formula. In this study, the respondent was a semester IV student who took 28-person leadership courses. The data collection techniques were done through observations, questionnaires, documentation and interviews.
FINDINGS AND DISCUSSION
From the results the authors want to discuss the influence of leadership styles led to student performance in the event of the activity economic activities of Ecomonic's Care Day, can be explained in School principal regression coefficient of 0.77. It means that each one of the values of a team leadership style will gradually increase the student's performance by 77.0%, thus the higher the leadership of the team will be relatively Improve student performance.
The ability to be held by lecturers in managing human resources based on competencies defined in order to achieve the objectives that have been determined. The future of students is very important for students in to bring about a good future.
Based on the results of a hypothesis test it appears that Ho was rejected, meaning a simple regression coefficient is a positive and significant influence over the team leadership style towards student performance.
In this study it was revealed that leadership leaders demonstrate a significant influence on student performance with a correlation coefficient of 0620 and a t count value of 6,993 at a significant level of α = 5% then obtained a value of 1,701.
So that the influence of both variables is expressed significantly. These coefficients include very strong. In other word, the team leadership style demonstrates a strong influence on student performance. This can be seen from the contribution given by the team leadership style to the student performance of 34.3% while the remaining 15.5% is determined by another variable.
Simanjuntak (2005) performance is the level of achievement of employees working. Personally, employees know their strengths and weaknesses so that they can spur their progress in careers.
Performance characteristics students characteristics of people who have high performance are the following (Mangkunegara, 2002:68): Have a high personal responsibility. Dare to take and bear the risks faced. Have a realistic goal. It has a thorough work plan and struggles to realizes its objectives. Feedback that is concrete in all work activities it does. Looking for opportunities to realize a plan that has been programmed b y lecturers and students.
In an effort to meet expectations, leaders use the ability to and intelligence by having the environment and potential in the organization. In other words, leaders strive to involve members of their organization to achieve their goals. The ability to move, direct and influence organizational members in an effort to achieve organizational objectives as a form of leadership.
Leaders can determine the attitude to social relationships where leaders are sometimes superiors, companions, coaches and advices. This can be done by the leadership face-to-face, through work procedures and other conditions. If this social connection can run well will cause a working spirit that can ultimately increase the productivity of the work.
Then to strengthen this study according to Fiedler (in Reni Yesi S,29), no one can be a successful leader by simply applying a variety of leadership styles to all situations. Therefore, successful leaders must be able to apply different leadership styles according to different situations. Based on findings in this research it is clear that the team leadership style is very influential on student performance. It is secause the ability of students to improve their performance due to the ability of team leadership in establishing cooperation relationship between teams is indispensable to the existence of the ability of the efficacy, the negation will run well What to expect.
CONCLUSION
From the research results To inform that the student leadership style has a positive connection to the student's performance. Thus the ability of students in teamwork will run according to what is expected especially the team leadership is very influential in student performance. Students ' ability to improve teamwork in establishing cooperation and coordination relationship with various parties based on conducive team leadership skills and behaviour.
The magnitude coefficient of the school principal's leadership variable is 0620, the contribution given by the team leadership style variables to the student performance of 34.3%, While the remaining 15.5% is determined by other variables not used in this study. This information gives a description that the team leadership style variables have a strong influence on student performance in the event of the activities of the event's care day.
The head of the school's leadership regression coefficient (X2) amounted to 0.77. means that each addition of one score or value is leadership will provide a score increase of 0.77 or 77.0% of the team's leadership style values, thus the higher the value of team leadership forces will be more likely to improve student performance. | 2019-12-10T22:01:25.422Z | 2019-10-25T00:00:00.000 | {
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211035239 | pes2o/s2orc | v3-fos-license | The role of platelet function analyzer-200 in predicting perioperative bleeding risk
Background/Aims Various preoperative screening tests, such as platelet count, prothrombin time, activated partial thromboplastin time, and bleeding time, have been widely used to evaluate the risk of bleeding during surgery. Use of platelet function analyzer (PFA)-100/200 for assessing platelet function instead of bleeding time is increasing. However, its role in predicting the perioperative risk of bleeding remains controversial. Methods Data of 703 patients who underwent surgery under general anesthesia were retrospectively analyzed. Preoperative platelet function was measured using PFA-200 system and the association with intraoperative bleeding was assessed. Additionally, other variables that could affect PFA-200 results were assessed by logistic regression analysis. Results Collagen/epinephrine (COL/EPI) test was prolonged in 199/703 (28.3%) patients (EPI group), while 99/212 (46.7%) patients showed COL/adenosine diphosphate test abnormalities. Bleeding over 300 mL during surgery occurred in 14.3% and 20.1% of patients in the normal and EPI groups, respectively (p = 0.058). In addition, red blood cell transfusion within 72 hours after surgery rate was significantly higher in the EPI group than in the normal group (31.7% vs. 23.4%, p = 0.024). In multivariate logistic analysis, prolongation closure time with COL/EPI (p = 0.068) was marginally associated with risk of bleeding during surgery. Furthermore, PFA-200 results were influenced by various factors, such as nonsteroidal anti-inflammatory drug use, blood group, hematocrit, and time of blood collection. Conclusions Preoperative PFA-200 test may be helpful in predicting the risk of perioperative bleeding. However, its results should be carefully interpreted because they are affected by several factors.
INTRODUCTION
Excessive bleeding is one of the most important complications of surgery. In case of neurosurgery or cardiac surgery, bleeding complications can lead to devastating consequences. Thus, it is very important to evaluate patient's baseline bleeding tendency and to detect impaired hemostasis prior to invasive procedures such as surgery. In fact, it is common to have hemorrhagic complications that lead to a fatal outcome after surgery without proper evaluation of the hemostasis abnormalities. In order to identify patients with impaired hemostasis, The Korean Journal of Internal Medicine. Vol. 35, No. 5, September 2020 it is necessary to assess bleeding history and family history and perform physical examination. In addition, various preoperative laboratory test parameters, including platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT), and bleeding time (BT), can be used to evaluate the risk of bleeding during surgery [1]. However, their role in assessing the risk of bleeding is still controversial and routine preoperative testing is not recommended in patients without a bleeding history [1,2].
Various coagulation screening tests, such as PT and aPTT, are used to identify defect of coagulation pathway. To evaluate the adequacy of platelet function, BT and platelet aggregation tests has been used for many years. However, the use of BT test is gradually decreasing due to several limitations: low sensitivity, high operator dependency, clinical usefulness, and invasiveness [3]. Platelet aggregation assessed by light transmission aggregometry or whole blood aggregometry is much more sensitive methods for detecting platelet dysfunction compared to BT test. However, these tests are relatively complicated, expensive, and time-consuming [4]. Recently, platelet function analyzer (PFA)-100 has been widely used as an alternative to overcome the limitations of prior tests [5][6][7][8]. The PFA-100 is a test device capable of measuring platelet adhesion and aggregation function under high shear-stress blood flow conditions comparable to those that occur in vivo [5]. Platelet adhesion and aggregation occur following exposure to platelet agonists, such as epinephrine (EPI)-or adenosine diphosphate (ADP)-coated membranes. As a result, the time until the membrane aperture occlusion (closure time) is measured by PFA-100. The recently introduced PFA-200 is an upgraded version of the PFA-100.
The most potent role of PFA-100/200 is that of a screening tool for detection of primary hemostasis abnormalities, such as von Willebrand disease (vWD) and various platelet disorders [9,10]. In addition, recent studies have shown its utility in monitoring antiplatelet therapy (including aspirin) and desmopressin therapy in both vWD and platelet disorders [5,11,12]. The use of PFA-100/200 test is increasing because it is easy to use and has a high sensitivity to detect hemostasis abnormalities [8]. However, its role as a preoperative screening tool to predict the risk of bleeding during invasive procedure, such as surgery, remains controversial. Some studies have reported that PFA-100/200 results were associated with perioperative bleeding risk, while other studies found no correlation between the calculated blood loss and either preoperative or postoperative PFA-100 values [13][14][15][16]. Another problem of PFA test is that the closure time (CT) of PFA-100 is influenced by several other factors not actually related to bleeding. To date, blood group, time of blood collection, plasma vWF level, citrate concentration, low platelet count, and hematocrit have been reported to affect CT [8,17].
In this study, we aimed to identify the variables that affect the results of PFA-200 test. Furthermore, we estimated the usefulness of PFA-200 test as a preoperative test to predict the risk of bleeding in patients undergoing various types of surgery.
Patients
Data of 703 patients older than 18 years who underwent surgery under general anesthesia at Korea University Guro Hospital between October 2015 and April 2016 were retrospectively analyzed. Various real-world types of surgery, ranging from simple surgery, such as appendectomy, to cancer surgery or open-heart surgery, were analyzed in this study. Patients with a history of bleeding disorder or bleeding symptoms at the time of preoperative risk evaluation were excluded. Comprehensive chart reviews of patients' demographic information and previous history, including major comorbidities and the use of anticoagulant and non-steroidal anti-inflammatory drugs (NSAID), were assessed. The Korea University Guro Hospital Institutional Review Board approved this study (2019GR0113). Informed consent was waived by the board.
Measurement of PFA and other variables
The whole blood sample for platelet function analysis was collected in a 3.2% sodium citrate anticoagulant bottle. After the collection, samples were kept at room temperature (15°C to 25°C) and transferred to the laboratory. Innovance PFA-200 system (Siemens Healthcare Diagnostics Products, Munich, Germany) was used to assess platelet function. The test was performed at least 15 minutes after the collection of samples for blood www.kjim.org https://doi.org/10.3904/kjim.2019.112 equilibration and up to 5 hours considering the sample stability according to the manufacturer's recommendation [8]. In our institution, platelet count, PT, aPTT, and PFA are tested in all patients scheduled for surgery to determine the risk of bleeding according to the anesthesiologist's policy. In case of the PFA test, COL/EPI test was performed first and the COL/ADP test was additionally confirmed in patients with an abnormal initial EPI test. However, both COL/EPI and COL/ADP tests were performed simultaneously in some patients. The laboratory results of preoperative evaluation, including complete blood count, PT, aPTT, liver function test, renal function test, electrolytes, and blood group, were collected.
Perioperative bleeding risk evaluation
Included patients were categorized according to the type of surgery. The severities of surgery were classified to major surgery and minor surgery according to the following criteria. Major surgery was defined as an operation in which a body cavity was entered, a mesenchymal barrier was crossed, a facial plane was opened, an organ was removed, or normal anatomy was altered. Minor surgery or procedure was defined as an any operative procedure in which only the skin, mucous membranes, or superficial connective tissue was manipulated, such as an endoscopic procedure, biopsy, incision & drainage, or similar procedure [18]. In addition, to assess perioperative bleeding complications, we investigated each patient's blood loss volume during surgery recorded in the operation note, hemoglobin (Hb) concentration before and after the surgery, and the amount of red blood cell (RBC) transfusion performed during and 72 hours after surgery.
Statistical analysis
All the data were statistically analyzed using IBM SPSS version 20 (IBM Co., Armonk, NY, USA). Patient demographics, preoperative laboratory findings, and bleeding outcome of surgery were compared between two groups according to the results of PFA closure time. Independent t test and Mann-Whitney test were used to detect statistical differences between the two groups for continuous variables. Chi-square test or Kruskal-Wallis test was applied for comparisons of categorical variables. To identify predictive factors for bleeding events during surgery, univariate and multivariate logistic regression analyses were performed. Cox proportional hazards regression analysis was conducted using statistically significant variables in univariate analysis (p < 0.1). A p values < 0.05 was considered statistically significant.
Patients characteristics
A total of 703 patients who underwent surgery were evaluated in this study. Of them, 223 (31.7%) patients showed prolongation of PFA CT just before surgery. Baseline characteristics of patients, abnormal results of PFA test (PFA group), and normal results (normal group) are detailed in Table 1. The proportions of younger and male patients were significantly higher in normal group than in PFA group. The proportion of patients with hypertension or cardiovascular disease was significantly higher in the PFA group than in the normal group. In addition, the use of antiplatelet drugs or NSAID were also higher in the PFA group. Table 2 demonstrates preoperative laboratory results of patients. The proportion of patients with Hb level < 120 g/L was higher in the PFA group than in the normal group (41.3% vs. 29.4%, p = 0.002). Other factors were not significantly different between the two groups. Table 3 demonstrates the types of surgery assessed in this study. No significant difference was found in the proportion of major surgery between the two groups (63.5% in normal group vs. 62.3% in prolongation group, p = 0.757). Abdominal surgery (32.3%) was the most common type of surgery, followed by orthopedic surgery (31.4%), and neurosurgery (9.7%). The categories of surgery applied in the two groups of patients were not significantly different (p = 0.939).
PFA closure times
CT prolongation as revealed by COL/EPI test was detected in 199/703 (28.3%) patients (EPI group), while 99/212 (46.7%) patients had abnormalities in the COL/ADP test (ADP group). Seventy-five patients (35.4%) showed CT prolongation by both tests. Median CTs with COL/ EPI test were 135 seconds (range, 35 to 249) in the normal group and more than 300 seconds (range, 250 to more than 300) in the EPI group. The COL/ADP test was performed in 212 patients, with median CTs of 93 The Korean Journal of Internal Medicine. Vol. 35, No. 5, September 2020 seconds (range, 43 to 110) in the normal group and 138 seconds (range, 110 to 300) in the ADP group. Of the factors affecting the results of PFA test, medication history, such as antiplatelet or NSAID, was the most significant. The CT results with COL/EPI of patients taking aspirin (250 seconds) were found to be significantly longer than those patients who did not take anti-platelet drugs (154 seconds) or other anticoagulants (177 seconds) (p < 0.001) ( Table 4). In case of NSAID, median CT results with COL/EPI were 215 sec for patients who received NSAID, and 147 sec for patients who had no medication (p < 0.001). However, the CT results of the COL/ADP test were not affected by antiplatelet (p = 0.391) or NSAID (p = 0.249) drug use. Patients with low Hb level (< 120 g/L) in preoperative test showed prolonged CT results in both COL/EPI (p = 0.027) and COL/ADP test (p < 0.001). Similar results were obtained in patients with low hematocrit (< 35%). PT and aPTT test were excluded from the anal-ysis because the number of patients who had abnormalities in the preoperative examination was too small. CT results with COL/EPI test were found to be influenced by blood sampling time, with the lowest (148 seconds) in the morning (6:00 to 12:00) and the most prolonged (186 seconds) at night (18:00 to 24:00). CT prolongation in patients with type O blood group was revealed only by the COL/ADP test (p = 0.022) and not by the COL/EPI test (p = 0.916).
Bleeding outcomes with surgery
To confirm the effect of the abnormal PFA results on the operation, several parameters, such as the volume of blood loss during operation, amount of RBC transfusion, and amount of Hb decrease, were assessed. Table 5 demonstrates postoperative bleeding outcomes between two groups. The rate of patients with bleeding over 300 mL during surgery was 14.3% in the normal group and (Table 6). CT prolongation in COL/EPI test (HR, 1.55; 95% CI, 0.97 to 2.47; p = 0.068) was marginally associated with increased risk of bleeding during surgery. To exclude the confounding effects of anti-platelet drugs and NSAID, subgroup analysis was performed only on patients who did not receive anti-platelets and NSAID. In this subgroup, multivariate logistic analysis revealed that sever-ity of surgery (HR, 3.82; 95% CI, 1.74 to 8.37; p = 0.001), preoperative aPTT (HR, 3.29; 95% CI, 1.05 to 10.32; p = 0.041), and serum albumin level (HR, 8.03; 95% CI, 3.70 to 17.45; p < 0.001) were predictive factors for bleeding. In addition, COL/EPI test prolongation was found to be a significant predictive factor for intraoperative bleeding (HR, 1.16; 95% CI, 2.62 to 5.93; p = 0.021).
DISCUSSION
Unselected preoperative screening tests, such as PT, aPTT, and PFA-100/200, are generally not recommended for all patients without bleeding history because they may delay surgery inappropriately and cause unnecessary increases in medical costs [2,8]. However, unselect- Preoperative screening tests to identify patients with primary or secondary hemostatic disorder is very important because intraoperative bleeding in these patients can cause fatal outcomes. However, until now, it is impossible to predict these diseases only by application of specific tests, so the bleeding history, family Values are presented as median (range). Prothrombin time and activated partial thromboplastin time were excluded because the number of abnormal results was too small. PFA, platelet function analyzer; COL/EPI, collagen/epinephrine; COL/ADP, collagen/adenosine diphosphate; NSAID, nonsteroidal anti-inflammatory drug. a Dawn, 0:00-6:00; Morning, 6:00-12:00; Afternoon, 12:00-18:00; Night, 18:00-24:00.
history, and physical examination should be performed. PFA-100 was first introduced in 1995 as a screening test for the diagnosis of vWD and platelet disorders [19]. In addition, the use of PFA-100 instead of BT to assess platelet function has also raised expectations for the role of PFA-100 as a preoperative screening test for primary hemostatic disorders. Koscielny et al. [13] reported in a large cohort study with preoperative questionnaire that the vast majority of patients with impaired hemostasis could be identified by PFA-100. The sensitivity of the PFA-100 COL/EPI CT was the highest (90.8%) in comparison with the other screening tests (BT, aPTT, PT, vWF:Ag). Fattorutto et al. [14] observed a weak correlation between preoperative COL/EPI CT and second-hour mediastinal blood loss in 70 patients with cardio-pulmonary bypass (r = 0.34, p = 0.01). However, Lasne et al. [15] and Cammerer et al. [16] reported no correlation between calculated blood loss and either preoperative or postoperative PFA-100 values. The worldwide prevalence of vWD is estimated to be 1%, but in Korea, the number of patients registered in the Korean Hemophilia Foundation by 2017 is only 130, which is much lower than prevalence of this disease in other countries. In case of platelet disorders, such as Bernard-Soulier syndrome and Glanzmann thrombasthenia, the prevalence is difficult to estimate, and only a few cases have been reported. Therefore, it is even more controversial to use PFA-100/200 as a preoperative screening tool for all unselected surgical patients to identify platelet disorders in Korea, where the prevalence of platelet disease is very low. This is consistent with the results of studies reported in Korea that preoperative PFA-100 screening test is less relevant to actual bleeding outcomes [20][21][22]. However, these studies had limitations to evaluate the role of PFA as a preoperative screening tool in unselected patients because the number of included patients was small and only specific operations were included. The advantage of our study was that we included a relatively large number of patients and analyzed various types of surgery performed during the study period. Different surgeries may act as confounding factors for the amount of bleeding. However, the effect of variety of surgeries on the amount of blood loss was very small, because the proportion of patients according to the category of surgeries was not different between the two groups even though the category of operation was separately assessed. Another advantage of the present study is that various factors were identified to accurately assess perioperative bleeding. Because of the limitations of retrospective studies, it is difficult to accurately assess the amount of intraoperative bleeding and the amount of delayed blood loss after surgery. We reviewed both the intraoperative anesthesia notes recorded by the anesthesiologist and operation notes written by the surgeon, because surgeons tend to record less blood loss. In addition, the amount of blood transfusion during operation, the amount of transfusion within 72 hours after surgery, and the amount of Hb decrease within 72 hours after surgery were compared. In our study, PFA-200 prolongation was associated with the rate of intraoperative bleeding and RBC transfusion within 72 hours postoperatively. Although multivariate logistic analysis showed borderline significance, prolonged CT as revealed by COL/EPI was also associated with intraoperative blood loss (HR, 1.55; 95% CI, 0.97 to 2.47; p = 0.068). In addition, these results were more prominent in the subgroup analysis of patients without medication history in whom the bias related to antiplatelet drugs and NSAID use was excluded (HR, 1.16; 95% CI, 2.62 to 5.93; p = 0.021). These results suggest that preoperative PFA abnormality may increase the risk of perioperative bleeding regardless of the use of antiplatelet agents or NSAID. Therefore, in patients with these abnormalities, accurate history and physical examination and laboratory test findings should be checked to exclude primary hemostatic disorders.
It is widely known that the use of aspirin and NSAID can affect the results of PFA-100. In addition, it has been reported that the blood group, hematocrit, platelet count, and time of blood collection can affect the results of PFA-100 [8,[23][24][25]. In our study, the use of aspirin and NSAID only affected the results of COL/EPI and did not affect the results of COL/ADP. Low Hb and hematocrit levels also affected the results of both COL/EPI and COL/ADP tests, which is consistent with the results of previous studies. Platelets count only affected the result of COL/ADP, which is difficult to interpret precisely because the number of patients with low platelet counts included in our study was too small. In particular, since our study was performed on patients who underwent surgery, most patients had platelets counts > 100 × 10 9 /L and very few patients had platelet counts < 100 × 10 9 /L. It has been reported that time of blood collection also affects the results of PFA-100 [17,24]. In accordance with the results of previous results, our study showed that the fast CT of COL/EPI occurred in the morning and the CT results became prolonged during the day. The results of COL/ADP showed a similar tendency, but without statistical significance, which is thought to be because of a diurnal variation in platelet function, which is more dependent on epinephrine than on ADP [26]. In addition, the increased platelet aggregation is estimated to be because of the higher in vivo epinephrine level in the morning, which induces platelet activation [27]. It was The Korean Journal of Internal Medicine. Vol. 35, No. 5, September 2020 reported that the CT of PFA-100 was prolonged in the blood group O compared to other blood groups. Our study also showed that the PFA-200 results of group O were the most prolonged, but the statistical significance was not confirmed. Previous studies have shown that CT of PFA-100 is prolonged in the order of group AB, group B, group A, and group O, and our results are also exactly the same [25,28,29]. However, the association between blood group and PFA results did not have statistical significance, as the difference between the results of group A and group O was small. The prolongation of PFA in blood group O can be explained by the fact that the CT of PFA-100/200 is influenced by the plasma level of vWF and the vWF concentration, which is lower in blood group O.
In conclusion, preoperative PFA-200 test may be helpful in predicting the risk of bleeding in patients with surgery. However, the results of PFA-200 is influenced by various factors, such as use of aspirin and NSAID, blood group, hematocrit, and time of blood collection, the results of PFA-100/200 should be carefully interpreted after precluding the effects of these confounding factors. | 2020-02-06T09:09:54.916Z | 2020-02-06T00:00:00.000 | {
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14865845 | pes2o/s2orc | v3-fos-license | Symmetric cube L-functions for GL_2 are entire
The purpose of this paper is to prove the long awaited holomorphy of the third symmetric power L-functions attached to nonmonomial cusp forms of GL_2 over an arbitrary number field on the whole complex plane.
Introduction
The purpose of this paper is to prove the long awaited holomorphy of the third symmetric power L-functions attached to nonmonomial cusp forms of GL 2 over an arbitrary number field on the whole complex plane.
To be more precise, let F be a number field whose ring of adeles is A = A F . Let π = ⊗ v π v be a cuspidal (unitary) representation of GL 2 (A). Let S be a finite set of places of F such that for v ∈ S, π v is unramified. For each v ∈ S, let denote the semisimple conjugacy class of GL 2 (C) defining π v (cf. [Bo], [La4]). We recall that if π is attached to a classical modular form of weight k on the upper half-plane for which the Fourier coefficient at p is a p , then Fix a positive integer m. Denote by r m = sym m (ρ 2 ), the m th symmetric power representation of the standard representation ρ 2 of GL 2 (C) = L GL 0 2 , an irreducible representation of dimension m + 1 (cf. [De], [La4], [Se], [Sh5,7], [T]). Set *The first author was partially supported by NSF grant DMS9610387. The second author was partially supported by NSF grant DMS9622585.
is the Langlands L-function attached to π v and r m . Recall that if O v and P v are the ring of integers of F v and its unique maximal ideal, then q v = card(O v /P v ).
A cuspidal representation π of GL 2 (A) is called monomial if π ∼ = π⊗ η for some nontrivial grossencharacter η, necessarily quadratic. Let K/F be the quadratic extension of F attached to η by class field theory. Then there exists a quasicharacter χ = ⊗ w χ w of K * \A * K which does not factor through the norm so that if σ v = Ind(W Fv , W Kw , χ w ), then π v = π(σ v ), the representation attached to σ v by Langlands correspondence or the Weil representation attached to χ w . Here W * denotes the corresponding Weil group, w|v, and π = ⊗ v π v (cf. [L-La], [S-Tan]).
Throughout this paper we shall assume m = 3. By Langlands [La1], L S (s, π, r 3 ) is a meromorphic function of s on C. If π ∼ = π⊗ η, η = 1, then the completed L-function L(s, π, r 3 ) = L(s, χ 3 )L(s, χ 2 χ ′ ), where the L-functions on the right are those of Hecke attached to quasicharacters of K * \A * K (cf. Proposition 2.3). The quasicharacter χ ′ is the conjugate of χ by the nontrivial element of the Galois group. Without loss of generality we may assume the central character ω π = η · χ|A * is trivial on R * + , where A * = I = I 1 · R * + with I 1 ideles of norm 1 and R * + the multiplicative group of positive real numbers. Observe that this is equivalent to χ|R * + ≡ 1. Thus L(s, π, r 3 ) for such π has poles only at s = 0 and 1 if and only if χ 3 = 1. Moreover, the poles are simple. Observe that since π is cuspidal χ = 1. In this paper we prove (Theorem 4.9): Theorem 1. Suppose π is not monomial. Then the partial symmetric cube L-function L S (s, π, r 3 ) is entire.
In fact, what we prove is a much stronger result: the holomorphy of the full (completed) L-function on all of C, stated in this introduction as Theorem 3, from which Theorem 1 follows.
The interest in this L-function and such a result has a long history and has attracted the attention of number theorists (cf. [D-I], [De], [H-Ra], [Sh5,7], for example). Ever since Shimura [Shi] and Gelbart-Jacquet [Ge-J] established similar results for L S (s, π, r 2 ) which led to the so-called Gelbart-Jacquet or adjoint square lift of π to GL 3 (A) with numerous important applications (cf. [B-D-H-I], [H-Ra], , , [La3], [Ra], [Sh4,7], to name a few), similar questions were posed for L S (s, π, r 3 ) with the hope that, beside its arithmetic consequences, it could lead to a lift of π to GL 4 (A), a result of great importance (cf. [Sh7] and in particular the estimate 3/22 for Fourier coefficients of Maass forms).
It was Langlands who first established the meromorphy of L S (s, π, r 3 ) on C by expressing it as the constant term of an Eisenstein series on an exceptional group of type G 2 as one of many examples in his Euler Product monograph [La1], [Go]. His ideas about their functional equations which were explained in a letter to Godement, were taken up by Shahidi [Sh2,3,1], [C-S] who established their functional equations in general. This led to a new approach to the study of automorphic L-functions which is now referred to as the , [M-W2], [Sh1,3,4,5], [K3], [F-Gol]. In the case of L S (s, π, r 3 ), similar results were observed by Deligne who used the Gelbart-Jacquet lift Π of π together with the properties of L(s, π×Π) (cf. [J-S], [M-W2], [Sh5], and Proposition 2.2).
The first instances where the holomorphy of L S (s, π, r 3 ) on all of C were established were proved in [Mo-Sh] and [Sh5], and it was in [Sh5] that the local factors ε(s, Π v , r 3 , ψ v ) and L(s, π v , r 3 ) were defined for all v and the full functional equation was stated. Moreover the adjoint cube representation r 0 3 = r 3 ⊗ (Λ 2 r 1 ) −1 was defined and the machinery for applying the converse theorem using L(s, π, r 0 3 , ρ), ρ a grossencharacter, was set up (Theorem 4.1 of [Sh5]). But the results were conditional (cf. Proposition 2.2.2 here and Theorem 4.1 of [Sh5]) and one purpose of the present paper is to establish the result with no restriction (Theorem 2 below).
Although several efforts were made, leading to new and interesting Lfunctions, it was not until 1994 (date of the preprint) that the first integral representation for L S (s, π, r 3 ) was found by Bump, Ginzburg, and Hoffstein [B-G-H]. Their integral generalized that of Shimura [Shi] and Gelbart-Jacquet [Ge-J] and therefore required a 3-sheet cover of GL 2 , necessitating that F contain 3rd roots of 1. It was there that holomorphy of L S (s, π, r 3 ) up to Re(s) > 3/4 was established, extending the bound Re(s) > 1 of absolute convergence in [Sh5], a consequence of a similar statement for L(s, π × Π) due to Jacquet and Shalika [J-S]. Uncompromising complications at archimedean places have stopped the extension beyond 3/4.
Our method is that of Langlands-Shahidi and is therefore quite representation theoretic. It starts with an observation of Kim (Observation 4.1), used first in [K3] to prove that the exterior square L-functions for GL n , n odd, are entire, and a consequence of Langlands' theory of Eisenstein series [La2], [M-W1], that if L(s, π, r 0 3 ) has a pole for 1/2 < s < 1, then the corresponding residual representation must be unitary [K2]. We recall that arguments of this type, but in the opposite direction, were used by Speh [Sp] and later by Tadic [Ta] to determine unitary duals of local groups. Since the unitary dual of G 2 over a p-adic field (for real groups see [V]) is now completely determined (see a recent article of Muić [Mu]), this gives a contradiction, but not until we bring an indispensable global element into our proof. (See the discussion before Theorem 4.3.) This is provided by Ramakrishnan's recent result [Ra] which states that many of the π v , v ∈ S, are tempered (true for all π v by Deligne, if π is holomorphic). We remark that the possibility of poles on Re(s) = 1/2 needs to be eliminated by other, all global, means (cf. [Sh5], [La2], and Proposition 2.2 and Lemma 4.7 here).
We give several proofs for Theorem 1 (Theorem 4.9), the first two relying in varying degrees on [Ra]. The first relies more on global information [Ra], while the second, which uses the hermitian property of each J(s, π v ) if s is a pole (Observation 4.4), relies more on the classification of Muić [Mu]. As expected, neither proof is completely local and in fact the first proof uses the existence of tempered components at a set of (lower) density > 1/2. The possibility of a pole at s = 1/2 in the first proof is eliminated by using a quadratic base change [La3], the holomorphy at s = 1/2 if the central character is trivial [Sh5], a result also used to eliminate the pole at s = 1/2 in the second proof, and the functional equation [Sh2,5]. The theorem now follows from the functional equation. (See Section 2.) We should remark that having even one unramified tempered component π v for an arbitrary cuspidal representation π of GL 2 (A) which was used in the second proof, is highly nontrivial. In fact, if n ≥ 3, although expected, nothing as such is known, even though it appears that the methods of [Ra] will give the existence of tempered components for self contragredient forms on GL (3).
Third, we sketch a proof in which our global ingredient is much weaker than the two others, namely: Given a cusp form π and any ε > 0, there exists a local component [K3]). But, we then need to make much deeper use of the unitary dual of G 2 coming from the conjugacy class of Borel subgroups ( [Mu,Th. 5.2]). This is the approach pursued in [K3] as one does not expect strong results such as those in [Ra] to be available when more general cases are treated.
b) The standard functional equation One still needs to twist with arbitrary cusp forms on GL 2 (A) (cf. Theorem 8.2 of [Sh7]), but with this theorem we have hopefully taken a major step in establishing the symmetric cube lift from GL 2 (A) to GL 4 (A), i.e. the existence of an automorphic form on GL 4 (A) whose standard L-function is equal to L(s, π, r 3 ).
More precisely, if Π is the adjoint cube lift of π, which as explained one expects to establish using a converse theorem, then the symmetric cube lift of π is simply Π⊗ ω π , where ω π is the central character of π (cf. [Sh5]).
b) The standard functional equation is satisfied.
As a consequence of Theorem 3, there is another proof of the following result. We refer to [Ga], [Ik], [PS-Ral] for original proofs (cf. Remarks 1.1 and 4.12).
We should point out that Theorem 3 can also be used to supplement the necessary holomorphy condition needed in the main Theorem C of [H-Ra] which was provided by [B-G-H] there. One can then dispose of Lemma 5.4 of [H-Ra].
The authors hope to take up related arithmetic questions in future papers. We would like to thank Dinakar Ramakrishnan for his careful reading of the manuscript and many useful comments.
Preliminaries
Let F be a number field and let G be a split group of type G 2 defined over We follow App. 2]. In what follows the roots are those of T in U . Let β 1 be the long simple root and β 6 the short one. Let denote the other positive roots.
Let P the maximal parabolic subgroup generated by β 1 (the long root). Then we have a Levi decomposition ( [Sh5] Here X(M ) is the group of F -rational characters of M . If ρ P is half the sum of roots generating N , then ρ P = 5 2 β 4 . Letα = β 4 and identify s ∈ C with sα ∈ a * C . Then sα corresponds to the character | det(m)| s . Let π = ⊗ π v be a cusp form on M (A) = GL 2 (A). We may and will assume that the central character ω π of π is trivial on R * + , where A * = I = I 1 · R * + with I 1 ideles of norm 1. Given a K-finite function ϕ in the space of π, we extend ϕ to a functionφ on G(A) as in [Sh3] and set Here H P is the usual Harish-Chandra homomorphism of M into Hom(X(M ), R). We define an Eisenstein series by It is known that E(s,φ, g) converges for Re(s) >> 0 and extends to a meromorphic function of s on C, with a finite number of poles in the plane Re(s) > 0, all simple and on the real axis.
It is also known that L 2 dis (G(F )\G(A)) (M,π) is spanned by the residues of Eisenstein series for Re(s) > 0. We note that for each s, the representation of G(A) on the space of Φ s is equivalent to I(s, π) = Ind G P π⊗ exp( s, H P ( ) ), where H P is the Harish-Chandra homomorphism. We know that the poles of Eisenstein series coincide with those of its constant terms. So it is enough to consider the constant term along P , which is given for each f ∈ I(s, π) by Weyl group representatives are all chosen to lie in K ∩ G(F ). Here where U α is the one parameter unipotent subgroup. Then Then r = r 0 3 ⊕ ∧ 2 r 1 , where r 1 is the standard representation of GL 2 (C), r 3 is the symmetric cube representation of GL 2 (C), and r 0 3 = r 3 ⊗ (∧ 2 r 1 ) −1 is the adjoint cube representation of GL 2 (C) (see [Sh5]).
Then it is well-known ( [La1], [Sh4], equation (2.7)) that for w = ρ 6 ρ 1 ρ 6 ρ 1 ρ 6 where S is a finite set of places of F , including all the archimedean places such that for every v / ∈ S, π v is a class one representation and if f = ⊗ v f v , then for v / ∈ S, f v is the unique K v -fixed function normalized by f v (e v ) = 1. The functionf v is the K v -fixed function in the space of I(−s, w(π v )).
A review of earlier global results
We will continue with our assumption that ω π |R * + ≡ 1. As mentioned before we will lose no generality and therefore we resume this assumption throughout our proof. We know the following facts about the symmetric cube L-functions. Let S be a finite set of places of F such that every π v , v / ∈ S, is of class one, i.e., it has a vector fixed by GL 2 (O v ). Then the class of each π v , v / ∈ S, is uniquely determined by the conjugacy class of a diagonal matrix Then given v / ∈ S, the local Langlands' L-functions are [Sh2,3,5]). (1) Fix a nontrivial additive character ψ = ⊗ v ψ v of F \A, unramified for v / ∈ S. Then at every place v ∈ S, a local L-function L(s, π v , r 3 ) and a local root number ǫ(s, π v , r 3 , ψ v ) (which equals 1 if π v and ψ v are unramified) can be defined so that if L(s, π, r 3 ) = v L(s, π v , r 3 ) and ǫ(s, π, r 3 ) = v ǫ(s, π v , r 3 , ψ v ), then L(s, π, r 3 ) = ǫ(s, π, r 3 )L(1 − s,π, r 3 ), whereπ is the contragradient of π. Similarly for L(s, π, r 0 3 ).
(3) They can be extended to meromorphic functions of s on C.
Proposition 2.3. Suppose π is a monomial cuspidal representation of GL 2 (A), i.e., π ⊗ η ≃ π for some nontrivial character η of F * \A * ; then L(s, π, r 0 3 ) has poles possibly only at s = 0, 1 and nowhere else. It has a pole at s = 1 and 0 if and only if π corresponds to the two-dimensional Galois representations of the Weil group of F with the image isomorphic to the symmetric group S 3 . The poles are simple.
This has a pole for Re s > 0 if and only if π(χχ ′ −1 ) is cuspidal and π(χχ ′ −1 ) ≃ π(χ). This is equivalent to χ 3 = 1, χ = 1 and χ| A × = 1. Representations in this class correspond to the two-dimensional Galois representations of the Weil group of F whose images are isomorphic to the symmetric group S 3 .
Remark 2.1. Note that the central character of π(χ) is η · χ| A × . Therefore in the above case, the central character of π = π(χ) is ω π = η and it is a nontrivial quadratic character.
So from this section on, we may assume that π is a nonmonomial cuspidal representation of GL 2 (A). Our goal is to prove that L(s, π, r 3 ) and L(s, π, r 0 3 ) are both entire.
Therefore it is enough to show that L(s, π, r 0 3 ) is holomorphic for the region 1 2 ≤ s < 1. Our proof will not apply to s = 1/2 and we therefore treat that separately.
Analysis of local intertwining operators
In this section we prove two useful propositions. We normalize the local intertwining operators by means of root numbers and L-functions as in [Sh1] (cf. Section 2 here). Notation will remain as in [Sh1]. In both propositions π v is a local component of π, although the results and the proofs are valid for any local unitary representation.
Proposition 3.1. The normalized intertwining operator N (s, π v , w) is holomorphic for Re s > 0.
Proof. For π v tempered and Re(s) > 0, the local factors L(s, π v , r i ) and M (s, π v , w) are holomorphic. Moreover L(s, π v , r i ) is never zero. Next assume π v is a complementary series; π v = π(µ| | r , µ| | −r ), 0 < r < 1 2 . Here we have suppressed the dependence of µ and r on v. We follow [K2] and [Za]. Under the identification M ≃ GL 2 , We can identify the operator M (s, π v , w) with M (Λ, χ, w).
The following proposition is due to [Za]. In [K2], only the case Re(s − 3r) > 0 was treated.
Proposition 3.2. Suppose 1/2 ≤ Re(s) < 1. Then for each v, the image of N (s, π v , w) is irreducible. We denote this image by J(s, π v ). If π v is tempered, it is the ordinary Langlands' quotient of I(s, π v ).
Suppose Re(s − 3r) = 0. Then by inducing in stages, we have: (1, µ) and P ′ is the other maximal parabolic subgroup of G. We note from [K2] that P ′ = M ′ N ′ , M ′ ≃ GL 2 andα ′ = β 3 and χ(1, µ) is the character of T (F ) with respect to this identification. Here B 0 = M ′ ∩ B. Again by Langlands' classification, the image of N (s, π v , w) is irreducible.
The fact that J(s, π v ) is the Langlands quotient when π v is tempered follows from the fact that M (s, π v , w) and N (s, π v , w) are proportional if π v is tempered (cf. proof of Proposition 3.1).
Proof. By [La2], [M-W1] the global intertwining operator M (s, π, w) gives the poles of the Eisenstein series. In view of the results in Section 2 and Proposition 3.1, they are precisely those of L(s, π, r o 3 ) if 1/2 ≤ s < 1. Moreover the residue at a given (simple) pole s on this interval is J(s,π) = ⊗ v J(s,π v ), an irreducible representation (Proposition 3.2) which by [La2], [M-W1] appears in L 2 and is therefore unitary. More precisely by [La1], [M-W1], the kernel of the residue of the Eisenstein series, a map from I(s,π) into the L 2 -space, is exactly that of the normalized operator N (s,π, w) = ⊗ v N (s,π v , w) whose image is the irreducible representation J(s,π).
The new local result which we need to rely on is in a recent article of Muić [Mu] in which he has determined the unitary dual of G 2 over a non-archimedean field completely. Here, we recall a part of the classification needed.
In view of this observation it is clear that to eliminate the poles between 1/2 and 1 (Theorem 4.9), beside those of Section 2 and Observation 4.1, one still needs another global ingredient since a priori local components π v could all be in the complementary series, i.e. nontempered. For this we use a recent result of Ramakrishnan [Ra] that allows an arbitrary cuspidal representation to have tempered components at a subset of finite places of F with a fairly large lower Dirichlet density, a very deep result. In both proofs that we give for our main result (Theorem 4.9), we need his result, but to differing degrees. One requires his density, but not fully, while the other uses the fact that the cusp form has at least one nondiscrete (non-supercuspidal is enough) tempered component, still a fairly deep global result. Consequently the second proof relies more on local results.
Next, let J(ν, σ) be the Langlands quotient for an irreducible unitary representation of G. Let P 1 = M 1 N 1 be the corresponding standard parabolic subgroup, N 1 ⊂ U , so that σ is a tempered representation of M 1 (F v ) and ν ∈ a * M 1 , where a M 1 is the real Lie algebra of the split component A 1 of M 1 . Then since J(ν, σ) is unitary, it is hermitian [K3]. This means that there exists an element w 1 in the Weyl group of A 1 in G satisfying w 1 (σ) ∼ = σ and w 1 (ν) = −ν, where ν is real. We record this observation from [K3] as: Observation 4.4. Any unitary representation is hermitian.
Proof. Since π v is tempered and J(s, π v ) is unitary, it must be hermitian by Observation 4.4. Consequently w(π v ) ∼ = π v . By the discussion in the last part of the proof of Proposition 6.1 of [Sh5] Lemma 4.6. Suppose L(s, π, r 0 3 ) has a pole for 1/2 ≤ s < 1. Then ω 2 π = 1.
Proof. By Ramakrishnan's Theorem 4.3 and Lemma 4.5, ω 2 πv = 1 for a set of primes whose lower Dirichlet density is at least 9/10. One can now apply either the equidistribution of values of ω π , or as pointed out to us by Ramakrishnan, use the simpler result of Hecke that two idele class characters agreeing at all the places in a set of Dirichlet density larger than 1/2 are equal, to conclude that ω 2 π = 1.
The following lemma relies more on local results of Muić [Mu], and Propositions 3.1 and 3.2 here. It reduces our use of [Ra] to π having a single nondiscrete tempered component, still a fairly deep global result. We will use this lemma to give a second proof of our main result.
We are now ready to prove the main result of our paper: Theorem 4.9. Suppose π is not monomial. Then the symmetric cube L-function L(s, π, r 3 ) and the adjoint cube L-function L(s, π, r 0 3 ) are both entire.
Proof. We shall give three proofs. The first one relies more on global results [Ra] than local ones [Mu]. Besides the results in Section 2, it requires Lemmas 4.5, 4.6, and 4.7. The second one is more local and uses more of classification of the unitary dual of G 2 over a non-archimedean field [Mu]. But it still needs Ramakrishnan's global result [Ra], although not as strongly. Besides the results in Sections 2 and 3, it requires Lemma 4.8.
First proof. In view of the results in Section 2 and Lemma 4.7 we only need to show that the L-functions are holomorphic for 1/2 < s < 1. Suppose L(s, π, r 0 3 ) has a pole for 1/2 < s < 1. Then by Lemma 4.6, ω 2 π = 1. By equidistribution of values of ω π , ω πv = 1 for a set of (finite) primes v with a Dirichlet density at least 1/2. On the other hand by Theorem 4.3, the lower Dirichlet density for primes with π v tempered is at least 9/10. By Observation 4.1 we therefore have a finite place v and a nondiscrete tempered component π v with a trivial central character such that J(s, π v ) is unitary. By parts (4) and (5) of Theorem 4.2 this is a contradiction, which completes the theorem.
Second proof. Assume that s (with 1/2 < s < 1) is a pole of L(s, π, r 0 3 ). Then by Lemma 4.8, π ∼ =π = π⊗ ω −1 π . Unless ω π = 1, π is monomial. We therefore need to assume ω π = 1. By Theorem 4.3 there is at least one unramified tempered component π v . Since ω πv = 1, Parts 4 and 5 of Theorem 4.2 show that J(s, π v ) is not unitary, contradicting the pole at s. The pole at s = 1/2 is eliminated by Proposition 2.2.1 and the theorem is now a consequence of the results of Section 2, particularly the functional equation. | 2014-10-01T00:00:00.000Z | 1999-09-01T00:00:00.000 | {
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252505969 | pes2o/s2orc | v3-fos-license | Formula development of sizing for basalt fiber. Part I: Role of film former
Abstract Sizing is a multi-element liquid system consisting of film former, coupling agent and other functional additives. In this article, Taguchi method with orthogonal array design was used to get an optimal formula of sizing for basalt fiber, and the role of film former in sizing was studied. The results showed film former promoted the sizing to spread on fiber surface, which increased the interaction area between the fiber and sizing. With the assistance of film former in sizing, a uniform layer was formed on fiber surface, which repaired the surface defect on basalt fiber with reduced stress concentration. The sizing with an appropriate concentration of film former had the capability to increase the tensile strength of a single filament with the best effect of forming a film on fiber surface.
Introduction
Basalt fiber (BF), an environment-friendly high-performance filament made from natural basalt rock, has attracted much attention in both academia and industry in recent years (Yan et al., 2021). Besides the high tensile strength and elastic modulus, BF possesses high resistance to alkalis, acids and environmental attacks (Overkamp et al., 2018;Yan et al., 2021). One of the main applications of BF is for fiberreinforced polymers (FRPs). FRPs have been widely used in many fields, such as aerospace, automotive, marine, recreational equipment and construction, due to the advantages of high stiffness to weight ratio and ease of processing (Kocaman et al., 2017;Wan et al., 2019;Zhou et al., 2020), and so on.
Sizing is a kind of surface coating that is applied to nearly all types of artificial fibers during manufacture. Applying a thin layer of sizing on fiber surface can protect the substrate from damage and friction during the production and processing and promote the fiber-matrix adhesion in composites through the coupling effect. Sizing can also regulate the distribution of surface defects, such as scratches and voids, which are commonly found in brittle fibers (Moosburger-Will et al., 2018). Generally, sizing consists of film former, coupling agent, antistatic material, lubricant, and each component has its specific function (Thomason, 2020;Thomason, 2019).
By retrieving the literatures in this field, it was found that massive researches put effort into revealing the function of sizing for BF. Our recent work (Xing et al., 2019) investigated the effects of sizing on the tensile strength of commercial BF. The results showed that sizing, functioning as 'clothing' for fiber, could effectively enhance the strength of BF by reducing the stress concentration in crack tips. It illustrated the importance of such post-treatment for developing high-performance fiber. Ralph et al. (2019) investigated the role of different fiber sizing, including sizing formulated by epoxy and polypropylene, on the properties of BF-reinforced polypropylene composites. They found that sizing changed the topography and chemistry of fiber surface significantly, responsible for the increased mechanical properties and chemical bonding between the fiber and polymer matrix. Wang et al. (2020) prepared starch-based sizing to improve the mechanical properties of BF. The results demonstrated that a uniform and compact coating layer successfully covered the fiber surface. The breaking force of BF was more than 45% higher than that of nonsized fiber. While much progress has been achieved in this field, the function of sizing was not clear in the reported papers, mainly because of the differences in the constituents of reported sizing for BF. In our recent paper (Xing et al., 2021), the effect of type and concentration of constituents in sizing on the mechanical properties of BF was investigated. The results revealed that the sizing brought a positive effect on enhancing the tensile strength of both single and bundle BFs, and the synergy arising from the main film former (Epoxy) and auxiliary one (Polyurethane) in sizing contributed much for such enhancement. Film former, making up the bulk of sizing, is one of the essential components in sizing. It can form a thin layer of film on fiber surfaces to protect the substrate from damage and promote the bonding of single filaments. Unfortunately, there is barely any clear vision of the role of this component on the performance of sizing and corresponding filament. Definitely, this field needs more participation from the composite communities to achieve the high impact it deserves.
In this study, sizing with different components and concentrations was formulated by Taguchi method with orthogonal array design, aiming at optimizing the formulation of sizing for BF. Based on the optimized result, we specifically studied the effect of film former and its content on the mechanical properties of BF, and the role of film former in governing the overall performance of sizing was illustrated, which was the innovation of this article compared with the previous research (Xing et al., 2021
Preparation of sizing and application on fiber surface
E-51 epoxy resin (200.0 g), span-80 (12.0 g) and SDS (6.0 g) were put into a beaker, and the mixture was stirred and dispersed at 2000 rpm using the high speed mixer (HSM-100L, Ross, China) in a 55 C water bath. During this process, water (282.0 g) was added dropwise to the system to obtain epoxy emulsion with a concentration of 40% (Liu et al., 2012). The coupling agent (KH-550) was added into the water with a pH value of 3.5 adjusted by glacial acetic acid and stirred at room temperature for 2 h until the solution was transparent. The lubricant (PEG-MS) and antistatic agent (CTAB) were dissolved with deionized water at 80 C. After the above solutions were prepared, an appropriate amount of deionized water was added to the film former emulsion. Then the antistatic agent, lubricant and coupling agent were added in order and stirred for 1 h. Finally, the solution was thoroughly mixed and the required sizing was obtained.
BF was coated with sizing using a continuous coating system, which consisted of an unwinding device, a set of rollers, hot air dryers, consolidating and enwinding devices.
The temperature in the dryers was set at 120 C, as reported in our previous paper (Xing et al., 2021). The final sample with different sizing was dried at 120 C for 24 h to remove the moisture.
Characterization
The tensile strength of a single filament was measured on a fiber tensile tester with a load cell of 1 N (XQ-1A, Shanghai Xinxian, China) according to the ASTM C1557-14 standard. The gauge length of the sample and loading speed were fixed at 25 mm and 2 mm/min, respectively. At least 40 specimens of each fiber sample were tested and the mean value with standard deviation was reported. The tensile strength of the fiber bundle was determined on a universal testing machine with a load cell of 10 kN (C43-104, MTS, US) according to the ISO 3341-2000 standard. The gauge length of the sample and the loading speed were fixed at 500 mm and 200 mm/min, respectively. At least 10 specimens of each fiber sample were tested. The morphology of BF with different sizing was characterized by scanning electron microscopy (SEM, Phenom XL, Thermo Scientific, US). The micro-mechanical properties of sizing after drying on glass substrate were tested by a nanoindenter (NanoTest Vantage, Micro Materials, UK; Zhu et al., 2015). The surface tension of sizing was obtained by a micro-electromechanical balance system (DCAT 25, Data-Physics, Germany) at room temperature. The viscosity of sizing was measured by a rotational viscometer (LVDV-1, Shanghai Fangrui Instrument Co., Ltd, China). The content of sizing on fiber surface was evaluated by measuring the weight change of sample before and after thermal treatment at 500 C for 30 min using an electric furnace (Yingkou Jianke, China) .
Design of BF sizing
The sizing formula was optimized by Taguchi method with orthogonal array design. Most commercial sizing is an aqueous system with 0.05-10% solid and generally consists of several multi-purpose components (Thomason, 2020). Epoxy resin has excellent physical, mechanical and electrical insulation properties, and the material shows excellent compatibility and reactivity with various substrates. KH-550 is a typical silane with amino-terminated groups, which is widely used as a coupling agent for sizing (Thomason, 2019). PEG-MS has prominent properties for emulsification, dispersity, lubricity and thickening effect. CTAB has excellent permeability and antistatic performance. Based on these advantages, in the current study, Epoxy was used as a film former, KH-550 was employed as a coupling agent, PEG-MS was adopted as a lubricant agent, and CTAB was used as an antistatic agent. Table 1 summarizes the factors and levels used in the experiment. An orthogonal array L 16 (4 5 ) was adopted in this study, as shown in Table 2. The number of columns in the table was 5, the horizontal number of each column was 4, and the total number of experiments was 16. Each factor was independent, the above factors were placed in the column position of the orthogonal table, and the level of each factor was set in the row position. In addition to the four columns of factors, there was an error column, and the range value (R) obtained by the column was used an experimental error. With such arrangement for formula development, the lowest and highest total concentrations of sizing were 2.5 wt% (Sample No. 1) and 9.8 wt% (Sample No. 16), respectively. Table 2 also summarizes the tensile strength of single and bundle fiber samples with different sizing. For pristine BF, the strength of single fiber was 1258 MPa, and the value for the bundle was 0.14 N/tex. Sample with 10# sizing showed the highest single fiber tensile strength of 1802 MPa among the 17 samples, which was more than 40% higher than that of BF without sizing (Sample No. 0). The strength of bundle fiber for this sample was almost the highest one with a reasonable variation.
The tensile strength of BF with sizing belongs to the larger-the better quality characteristics because one of the main functions of sizing is to enhance the mechanical properties of BF. In other words, the higher strength of fiber means better performance of sizing. Therefore, the larger the k i value (kᵢ is the arithmetic average of single fiber strength at the level i of 1, 2, 3 or 4 for the factors), the more significant improvement of sizing on the tensile strength of BF (Detailed analysis was presented in Table S1 in Supplementary Material). The influence of four controllable factors on the tensile strength of BF monofilament is shown in Figure 1. The strength of a single fiber increased at first and then decreased with an increasing concentration of film former, and the k i of A3 reached the maximum. In contrast, the strength increased with a higher concentration of coupling agent, and the one with B4 level shows the highest value. The trend of lubricant agent was similar to that of film former, where k i of C3 had the maximum value. Interestingly, the part of antistatic agent was irregular in which the biggest k i was 1680 MPa at the level of D1. It can be concluded that the formulation of optimum sizing for improving the tensile strength of BF monofilament was A 3 B 4 C 3 D 1 , where the concentration of film former was taken at level 3 (6.0 wt%), the coupling agent at level 4 (1.0 wt%), the lubricant agent at level 3 (0.5 wt%), and the antistatic agent at level 1 (0.1 wt%) (Verification of optimal formulation was shown in Fig. S1 and Table S2 in Supplementary Material).
Function of film former in sizing
The content of film former in sizing is the highest one except water. Therefore, the effect of film former was studied to illustrate the role of this critical component in sizing. Based on the optimal sizing formula, only the concentration of film former was changed for discussion, and the role of silane coupling agent will be the subject of our future studies. Table 3 shows the experimental scheme for this purpose.
The concentration of film former is expected to affect the property of sizing, thus dominating the formation of sizing formed on fiber surface. With this consideration, the sizing with different concentrations of film former was coated on a glass slide to study morphology and micro-mechanical properties of sizing, which was convenient for direct testing and analysis. For sample preparation, the slide was dipped in the prepared sizing and then taken out. This process was repeated 15 times to ensure that the thickness of sizing was sufficient for characterization. The glass slide with different sizing was dried in an oven at 120 C for 120 h to ensure the complete drying of sizing. Figure 2 shows the surface and cross-section morphologies of sizing with different concentrations of film former. At a low concentration of film former, the surface of sizing was uneven with irregular large size pits ( Figure 2A1). The film thickness was inconsistent, and prominent convex and concave structures were noticed by observing the cross-section of sizing ( Figure 2A2). With increasing concentration of film former, the surface of sizing became smooth with loosely distributed particles ( Figure 2B1), and the number and size of holes on the surface and inside of sizing decreased ( Figure 2B2). When the concentration of film former was 6.0 wt% (F3), the holes were absent in the sizing. Instead, many particles were evenly attached ( Figure 2C1), and the film became more serried inside ( Figure 2C2). In the extreme case, saying the concentration of epoxy was 8.0 wt% (F4), the distribution of particles in sizing became more uniform ( Figure 2D1), and the film became denser and compacted ( Figure 2D2). The reason for these observations was that when the concentration of film former was low, the sizing lacked the driving force for spreading, thus resulting in the formation of an uneven surface with the evaporation of water. When the concentration of epoxy was high, the film former drove the whole solution of sizing to further spread out on the substrate and finally formed a uniform, flat and dense film.
The nano-indentation test quantitatively measured the micro-mechanical properties of sizing. In this test, the indentation depth was fixed at 1000 nm, the loading and unloading speed was set at 0.05 mN/s, and the hardness (H, GPa) and reduced modulus (E r , GPa) of the material were calculated according to the following equations (Burghard et al., 2007;Malzbender et al., 2002): Where P max (mN) is the maximum indenter load, A c (nm 2 ) presents the projected area of elastic contact, h c (nm) stands for the elastic displacement of indenter, h max (nm) means the indenter displacement at the maximum load, S suggests the slope of unloading curve at the maximum load, e is a parameter related to the indenter geometry (For Berkovich indenter used in this study, e ¼ 0.75). Figure 3 shows the load-depth profiles of different sizing samples. A general trend was demonstrated that the sizing with a higher film former concentration was always accompanied by a greater load at the same indentation depth. Specifically, when the indentation depth was 1000 nm, the maximum load of the sample with F1 sizing was about 1.3 mN, and this value increased by more than 100% to 2.7 mN for F4 sizing. During the unloading, the creep occurred due to the viscoelasticity of sizing, resulting in a displacement discontinuity known as the pop-out effect, and this was caused by the relaxation of the molecular chain in the epoxy (de la Rosa-Fox et al., 2007;Liu et al., 2019). The inset in Figure 3 summarizes the obtained H and E r of four sizings. The value of both parameters increased with increasing concentration of film former, indicating the improved ability of sizing to resist mechanical deformation.
The reason behind such observation was that film former promoted the sizing to form a uniform layer on the surface of the glass slide. The higher the concentration of film former, the more uniform and dense film created (As confirmed by the SEM images shown in Figure 2), thus leading to the increased hardness and modulus in the corresponding sizing.
To study the influence of film former concentration on the morphology of BF, fiber samples were characterized by SEM, and the results are presented in Figure 4. The pristine BF exhibited a smooth surface ( Figure 4A). Under higher magnification, it was found that there was no other substance on fiber surface except a few small contaminated particles (inset in Figure 4A). After the application with sizing, the surface of BF changed significantly with the variation on the concentration of film former in sizing. At a low concentration of film former ( Figure 4B), a large number of irregular flakes and granular agglomerates were attached to fiber surface, making the fiber surface rough. It can be seen from the section morphology that the content of sizing on fiber surface was low and the film was not continuous (inset in Figure 4B). As the concentration of film former increased ( Figure 4C), the size of aggregates on fiber surface decreased, some areas became flat. At this time, the continuous film of sizing on fiber surface was thinner (inset in Figure 4C). When the film former concentration was 6.0 wt% ( Figure 4D), the surface and section morphologies of BF showed that the sizing had the best spreading effect on fiber surface with a uniform film, and the particulate material on fiber surface was further refined, making the sizing appear smooth without obvious agglomerates (inset in Figure 4D). This was because, with the assistance of film former, the spreading ability of sizing on fiber surface was improved, and the whole components were more uniformly dispersed on fiber surface to form a thin layer of sizing. However, when the film former concentration continued to increase ( Figure 4E), the fibers were wrapped by a thick layer of sizing, and the bonding phenomenon occurred between the individual filaments. When the single fiber was separated from the bundle, the fiber surface became irregular and even sizing was peeled off from the filament to form a discontinuous film. Definitely, this was not the case that an ideal sizing should be accounted. Table 4 summarizes the properties of sizing and tensile strength of corresponding fiber. With an increasing concentration of film former, the surface tension of sizing was increased. For example, the surface tension of F1 was 24.76 mN/m, and this value was increased by 10% to 27.38 mN/m for F4. This was caused by the higher amount of epoxy, which was a polar component in sizing (Abbasian et al., 2004). The viscosity of sizing with different concentrations of film former changed marginally, and specifically, the viscosity of all sizing was in a low range of less than 2.5 mPaÁs, suggesting the effect of sizing viscosity on the properties of BF could be neglected (Wang et al., 2017, Xing et al., 2021. From this table, it could be observed that the diameter of BF with different sizing changed marginally, about 15 mm, indicating that the variation on the film former concentration had little effect on the diameter of BF. The fiber with F3 sizing exhibited the highest tensile strength of 1967 MPa, with an improvement of 45.60% when compared with that of pristine BF. It seemed that the higher concentration of sizing for BF brought a negative effect on enhancing the strength of the filament. SEM image showed that with excessive content of film former (Figure 4E), the fiber was severely covered by sizing. The whole sample was more like a composite structure, thus limiting the enhancement of tensile strength for the filament.
The relationship between the sizing content on fiber surface and the concentration of the film former is shown in Figure 5A. It demonstrated the content of sizing increased with an increasing concentration of film former. The tensile strength of single fiber with F3 sizing was the highest in the experimental scheme. Whereas the content of sizing was 3.94%, not the case that the higher the sizing applied to the fiber, the better mechanical performance of fiber demonstrates. Therefore, moderate content of sizing on fiber surface played a significant role in promoting the mechanical properties of BF, and these results were in good agreement with our previous findings (Xing et al., 2021). Figure 5B schematically shows the distribution of defects on BF surface with different concentrations of film former. Film former made the mixture of sizing adhered to the fiber surface evenly, and it covered more than 80% content of sizing except for water. Therefore, the higher the concentration of film former was used, the more sizing content on fiber surface was formed. When the concentration of film former was low (Condition I in Figure 5B), the film of sizing was thin, the physical and chemical interactions between BF and sizing were weak, and a small number of the surface defects could be repaired, such as the BF with F1 or F2 sizing. As a result, the mechanical properties of fibers were improved slightly. When the content of the film former was proper (Condition II in Figure 5B), like the BF with F3 sizing, the tensile strength of BF with sizing was enhanced dramatically, which was contributed from the reparation of defects on fiber surface and decreasing stress concentration. When the content of film former was too high (Condition III in Figure 5B), like the fiber with F4 sizing, the tensile strength of BF with sizing also increased, but the improvement effect was limited due to the inherent poor mechanical performance of sizing (a mixture of polymer) on fiber surface. It is known that the mechanical properties of fiber generally outperform that of the matrix, and the introduction of excessive polymer onto fiber in the form of sizing contributes marginally to the strength modification of filament. Hence, it is important to select an appropriate concentration of film former to improve the mechanical properties of fibers.
To reveal the mechanism behind the enhanced strength of fiber with sizing, we examined the cross-section area of fractured fiber using SEM. For a brittle material, the fiber was stretched at a constant speed until the breakage, and the fractured morphology of the fiber was composed of several regions, known as local failure, mirror, mist and hackle (Lund & Yue, 2008). This process is schematically shown in Figure 6A. Under low stress condition, the main crack of the fiber slowly expanded through a single crack to form a flat and smooth semi-circular mirror area. As the applied stress gradually increased, non-dangerous sub-weak points in the material became dominant, and mist region was formed with expended crack. Since the primary and secondary cracks did not usually expand in the same plane, the region was no longer as smooth as a mirror. When entering the stage of rapid propagation, the rough area of the hackle was shaped by simultaneously expanding many secondary cracks on the uneven plane. With the increase of tensile force, the stress concentration appeared at the defect with a large size first, followed by the rapid propagation and the brittle failure when the stress exceeded the critical value. The relationship between fracture strength r (MPa) and crack size a (mm) can be expressed by the following two equations (Lund & Yue, 2008): where A m is the mirror constant related to the material property, it is generally believed that the mirror radius r m (mm) near the crack initiation point and the strength of the fiber r follow the empirical formula (6): E (MPa) is the elastic modulus of the material, c (mN/m) is the surface energy of the material, Y is a geometry parameter of the material (Y ¼ 1.39 for elliptical surface flaw, and Y ¼ 1.13 for circular internal pores). By combining the above two formulas, the following one was expressed, which described the relationship between the crack size a and mirror radius r m : In this study, the elastic modulus of BF was remained at around 75 GPa due to the inherent nature of the material, and the surface energy of pristine BF was 55.45 mN/m (Xing et al., 2021). The surface tension of four sizings was about 26 mN/m, as summarized in Table 4, so the surface energy of pristine filament was much higher than that of BF with different sizing, and this was also reported in a previous study (Chen & Huang, 2021). The fiber strength can be qualitatively expressed in Equation (7), in which E and Y are constant, and c was reduced due to the introduction of sizing on fiber surface. The r would be improved only when the a was reduced. The variation in crack size a on fiber surface can be directly correlated with the mirror radius r m , as expressed in Equation (8). Results arising from the cross-section area of fractured fiber showed that the average r m of pristine BF was 3.07 mm ( Figure 6B), and this parameter was reduced to 1.85 mm with F3 sizing ( Figure 6C). It seems that the content of film former played a leading role in governing the value of r m , and the value of the latter was saturated when the concentration of epoxy was 6.0%, as shown in Figure 6D. The reduced r m led to a smaller a on fiber surface, thus the r of fiber was enhanced according to Equation (8). This can be qualitatively expressed as a healing effect of sizing for fiber samples.
To reveal the mechanism of improving fiber strength caused by sizing, the steady-state stress distribution of the two-dimensional fiber tensile model was simulated by using the solid mechanics module of COMSOL software. One was the partial original BF with a surface crack, another was the model that the crack on BF surface was filled by sizing. The upper end of BF was fixed, and the lower end was applied force to simulate the tensile process of BF. The parameters used in the simulation are summarized in Table 5, and the results are presented in Figure 6E. It can be seen that when pristine BF was subjected to tensile force, the stress concentration occurred at the tip of crack on fiber, and the force dispersed to the periphery like butterfly wings from the tip of the crack. After applying the sizing, the stress concentration at the crack tip was decreased, and both ends of fiber with sizing had the capability to bear more stress. The force for BF was more evenly dispersed, which was the reason that BF with sizing exhibited a higher tensile strength.
Conclusions
In summary, Taguchi method with orthogonal array design was implemented to formulate the optimum sizing for BF, and the effectiveness of this methodology was confirmed by measuring the tensile strength of the individual fiber. The results showed that sizing brought a positive effect on enhancing the tensile strength of BF. Additionally, our comparative data demonstrated that film former in the sizing drove the mixture to spread on fiber surface to form a uniform film. The sizing diffused and formed a film by filling the defects on fiber surface. Under such circumstances, the defects were repaired, resulting in reduced stress concentration and improved mechanical properties of BF. Sizing with a suitable concentration of film former had the best healing effect on rectifying the mechanical properties of BF.
Disclosure statement
No potential conflict of interest was reported by the authors. | 2022-09-25T15:14:27.045Z | 2022-09-23T00:00:00.000 | {
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225910576 | pes2o/s2orc | v3-fos-license | Dynamical symmetry breaking in Abelian geometrodynamics
A new tetrad is introduced within the framework of geometrodynamics for non-null electromagnetic fields. This tetrad diagonalizes the Einstein-Maxwell stress-energy tensor, any stress-energy tensor in a local and covariant way, and allows for maximum simplification of the expression of the electromagnetic field, in any curved four-dimensional Lorentzian spacetime, allowing for the identification of its degrees of freedom in two local scalars. New isomorphisms are proved. A new internal-spacetime mapping is established using these new tetrads. It is possible to map the local group of electromagnetic gauge transformations into the transformation groups of tetrad vectors on two local orthogonal planes. The planes that diagonalize the stress-energy tensor. We will discuss through a first order perturbative formulation the local loss of symmetry when a source of electromagnetic and gravitational field interacts with an agent that perturbs the original geometry associated to the source. The loss of symmetry will be manifested by the tilting of these planes under the influence of an external agent. In this strict sense the original local symmetry will be lost, however a new symmetry will arise. The purpose of this report is to show that the geometrical manifestation of local gauge symmetries is dynamic.
Introduction
A new tetrad is introduced within the framework of geometrodynamics for non-null electromagnetic fields. This tetrad diagonalizes the Einstein-Maxwell stress-energy tensor, any stress-energy tensor in a local and covariant way, and allows for maximum simplification of the expression of the electromagnetic field, in any curved four-dimensional Lorentzian spacetime, allowing for the identification of its degrees of freedom in two local scalars [1][2][3][4]. The Einstein-Maxwell equations will also be simplified. New isomorphisms are proved. The local group of electromagnetic gauge transformations is isomorphic to the new groups LB1 and LB2, independently. A new internal-spacetime mapping is established using these new tetrads [2,3]. It is possible to map the local group of electromagnetic gauge transformations into the transformation groups of tetrad vectors on two local orthogonal planes. The planes that diagonalize the stress-energy tensor. LB1 is the group of local tetrad transformations comprised by SO(1,1) plus two different kinds of discrete transformations. One of these two discrete transformations is the full inversion or minus the identity two by two. The other discrete transformation is not Lorentzian because it is a reflection or flip with zeroes in the diagonal and ones off-diagonal also two by two. The local group of electromagnetic gauge transformations is isomorphic to the local group of tetrad transformations LB2 on the orthogonal local plane 1 Former Professor at Universidad de la República, Av. 18 (2). These group results amount to proving that the no-go theorems of the sixties like the S. Coleman and J. Mandula, the S. Weinberg or L. O Raifeartagh versions are incorrect [5][6][7]. Not because of their internal logic, but because of the assumptions made at the outset of all these versions. The explicit isomorphic link between the Abelian local "internal" electromagnetic gauge transformations and the local tetrad transformations on special orthogonal local planes is manifest evidence of these incorrect assumptions as has been proved. Simply because the Lorentz transformations on a local plane in a four-dimensional curved Lorentzian spacetime do not commute with Lorentz transformations on a different local plane in general, element of contradiction with the no-go theorems assumptions. We will discuss through a first order perturbative formulation the local loss of symmetry when a source of electromagnetic and gravitational field interacts with an agent that perturbs the original geometry associated to the source [8,9]. It has already been proved that the local gauge groups are isomorphic to local groups of transformation of special tetrads. These tetrads define at every point in spacetime two orthogonal planes or blades such that every vector in these local planes is an eigenvector of the Einstein-Maxwell stress-energy tensor. As the local gauge symmetry in Abelian or even non-Abelian field structures in four-dimensional Lorentzian spacetimes is displayed through the existence of local planes of symmetry that we will refer to as blades one and two, the loss of symmetry will be manifested by the tilting of these planes under the influence of an external agent. In this strict sense the original local symmetry will be lost. We will be able to prove in this way that the new planes or blades at the same point will correspond "after the tilting generated by perturbation" to a new symmetry. The purpose of this report is to show that the geometrical manifestation of local gauge symmetries is dynamic. Despite the fact that the local original symmetries will be lost, new symmetries will arise. A dynamic evolution of local symmetries will be manifested. This result will produce a new theorem on dynamic symmetry evolution. This new classical model will be useful in order to better understand anomalies in quantum field theories. These new tetrads are useful in astrophysics spacetime evolution algorithms since they introduce maximum simplification in all relevant objects, specially in stress-energy tensors making the evolution relativistic differential equations simpler [10][11][12].
Diagonalization of the Einstein-Maxwell stress-energy tensor
Throughout the paper we use the conventions of reference [1]. In particular we use a metric with sign conventions -+++. We will call our geometrized electromagnetic potential A µ , where f µν = A ν;µ −A µ;ν is the geometrized electromagnetic field f µν = (G 1/2 /c 2 )F µν . The stress-energy tensor according to equation (14a) in reference [1], can be written as, where * f µν = 1 2 µνστ f στ is the dual tensor of f µν , see section Appendix A for the object µνστ . The local duality rotation given by equation (59) in paper [1] f µν = ξ µν cos α + * ξ µν sin α, allows to express the stress-energy tensor in terms of the extremal field T µν = ξ µλ ξ λ ν + * ξ µλ * ξ λ ν . We can write the extremal field as, Extremal fields are local electromagnetic gauge invariants as it can be noticed from equation (2). Extremal fields satisfy the equation This a condition-equation imposed on extremal fields in order to find the local scalar called the complexion α. The explicit expression for the complexion, which is also a local electromagnetic (2), by the expression tan(2α) = −f µν * f µν /f λρ f λρ . We have also available the general identity, which is valid for every pair of antisymmetric tensors in a four-dimensional Lorentzian spacetime [1]. When applied to the case A µα = ξ µα and B να = * ξ να , it it is straightforward to prove that condition (3) yields the equivalent condition, The extremal field ξ µν and the scalar complexion α have been previously defined through equations (22)- (25) in reference [1]. It is our purpose to find a tetrad that diagonalizes the Einstein-Maxwell stress-energy tensor. This tetrad will simplify the analysis of the geometrical properties of the electromagnetic field as we will see. There are four tetrad vectors that at every point in spacetime diagonalize the stress-energy tensor in geometrodynamics, where Q = ξ µν ξ µν = − T µν T µν according to equations (39) in manuscript [1]. Q is assumed not to be zero, because we are dealing with non-null electromagnetic fields. Non-null we clarify means basically that f µν f µν = 0 and * f µν f µν = 0. In turn and by definitions these last equations imply that ξ µν ξ µν = 0. We are free to choose the vector fields X α and Y α , as long as the four vector fields (6)-(9) do not become trivial. Two equations in the extremal field will be used extensively in this work, in particular, to prove that tetrad (6)-(9) diagonalizes the stress-energy tensor. The first equation is given by (64) in [1], also given in equation (5). When we replace A µα = ξ µα and B να = ξ να in equation (4), the second identity is found, When we make iterative use of (5) and (10) we find, In paper [1] the stress-energy tensor was diagonalized through the use of a Minkowskian frame in which the equation for this tensor was given in equations (34) and (38). In this work, we give the explicit expression for the tetrad in which the stress-energy tensor is diagonal. The freedom we have to choose the vector fields X α and Y α , represents available freedom that we have to (5) and (10), it is straightforward to prove that (6)-(9) is a set of orthogonal vectors. Vectors (6)-(7) define or span the local plane or blade one. Vectors (8)-(9) define or span the local orthogonal plane or blade two. Let us introduce some names. The tetrad vectors have two essential components. For instance in vector V α (1) there are two main structures. First, the skeleton, in this case ξ αλ ξ ρλ which is a local electromagnetic gauge invariant as can be noticed from equation (2), and second, the gauge vector X ρ which is gauge. The gauge vectors it was proved in manuscript [2,4] could be anything that does not make the tetrad vectors trivial.
Electromagnetic potentials in Einstein-Maxwell geometrodynamics
Our goal is to simplify as much as we can the expression of the electromagnetic field through the use of an orthonormal tetrad, so its geometrical properties can be seen in an easier way. As it was mentioned above we would like to show this simplification through an explicit example by making a convenient and particular choice of the gauge vector fields X α and Y α . In geometrodynamics, the Maxwell equations, f µν ;ν = 0 and * f µν ;ν = 0 say that two potential vector fields exist [13], f µν = A ν;µ − A µ;ν and * f µν = * A ν;µ − * A µ;ν . For instance, in the Reissner-Nordström geometry the only non-zero electromagnetic tensor component is f tr = A r;t − A t;r and its dual * f θφ = * A φ;θ − * A θ;φ . The symbol ";" stands for covariant derivative with respect to the metric tensor g µν and the star in * A ν is just a name, not the dual operator, meaning that * A ν;µ = ( * A ν ) ;µ . The vector fields A α and * A α represent a possible choice in geometrodynamics for the vectors X α and Y α . It is not meant that the two vector fields have independence from each other, it is just a convenient choice for a particular example. A further justification for the choice X α = A α and Y α = * A α could be illustrated through the Reissner-Nordström geometry.
In this particular geometry, f tr = ξ tr and * f θφ = * ξ θφ , therefore, A θ = 0 and A φ = 0. Then, for the last two tetrad vectors (8)-(9), the choice Y α = * A α becomes meaningful under the light of this particular extreme case, when basically there is no magnetic field. The choice Y α = A α would make in the Reissner-Nordström geometry the vectors (8)- (9) to become zero and this would be meaningless.
Electromagnetic gauge transformations on blades one and two
Once we made the choice X α = A α and Y α = * A α the question about the geometrical implications of electromagnetic gauge transformations of the tetrad vectors (6)-(9) arises. We first notice that a local electromagnetic gauge transformation of the "gauge vectors" X α = A α and Y α = * A α can be just interpreted as a new choice for the gauge vectors X α = A α + Λ ,α and Y α = * A α + * Λ ,α . When we make the transformation, A α → A α + Λ ,α , f µν remains invariant, and the transformation, * A α → * A α + * Λ ,α , leaves * f µν invariant, as long as the functions Λ and * Λ are local scalars. It is valid to ask how the tetrad vectors (6)-(7) will transform under Schouten defined what he called, a two-bladed structure in a spacetime [14]. These local blades or planes are the planes determined by the pairs ). Given the space constraint in these presentation we will limit ourselves to show a few illustrative results as far as tetrad transformations for gauge vector choice given by electromagnetic gauge transformations. The whole analysis is given in manuscript [2]. In order to simplify the notation we will write Λ ,α = Λ α . First we study the change in (6)- (7) under A α → A α + Λ ,α . Using the following notation, (2) ) and (1) ), several cases arise on blade one. We would like to calculate the norm of the transformed vectorsṼ α (1) andṼ α (2) , where the relation V α α has been used and V α (1) assumed timelike for simplicity. In order for these transformations to keep the timelike or spacelike character of V α (1) and V α the condition [(1 + C) 2 − D 2 ] > 0 must be satisfied. If this condition is fulfilled, then we can normalize the transformed vectorsṼ α (1) andṼ α (2) as follows, The condition [(1+C) 2 −D 2 ] > 0 allows for two possible situations, 1+C > 0 or 1+C < 0, the proper transformations on blade one. For the particular case when 1+C > 0, the transformations (17)- (18) are telling us that an electromagnetic gauge transformation on the vector field A α , that leaves invariant the electromagnetic field f µν , generates a boost transformation on the normalized tetrad vector fields The case 1 + C < 0, represents the composition of two transformations. An inversion of the normalized tetrad vector fields and V α (2) will change their timelike or spacelike character, These are improper transformations on blade one. They have the property of being a composition of boosts and a discrete transformation given by We notice that this discrete transformation is not a Lorentz transformation because it is a reflection. They might also be composed with an inversion, see reference [2] for the whole analysis. On blade or plane two, the choice Y α = * A α + * Λ ,α induces just local spatial rotation tetrad vector transformations. We reiterate that local tetrad electromagnetic gauge transformations can be interpreted as new or different gauge choices X α = A α + Λ ,α and Y α = * A α + * Λ ,α .
Group Isomorphisms
We will just limit ourselves to state these new theorems proved in detail in reference [2].
Theorem 1
The mapping between the local group of electromagnetic gauge transformations and the local group LB1 defined above is isomorphic.
Theorem 2
The mapping between the local group of electromagnetic gauge transformations and the local group LB2 defined above is isomorphic. (6)-(9) is not trivial, we can proceed to normalize, In analogy with the electromagnetic case and without altering anything fundamental we assume for simplicity that See sections Appendix A and Appendix B for more details about this tetrad. The four vectors (21)-(24) have the following algebraic properties, Any other scalar product is zero. It is possible to find expressions for the metric tensor and the stress-energy tensor in the new tetrad (21)- (24). The new expression for the metric tensor is given by, The stress-energy tensor can be written in terms of the new tetrad (21)-(24) as, In order to find the expression for the electromagnetic field in terms of the tetrad (21)- (24), it is necessary to find some previous results. The extremal field tensor and its dual can be written, see reference [2], Equations (28)- (29) are providing the necessary information to express the electromagnetic field f µν = ξ µν cos α + * ξ µν sin α in terms of the new tetrad, We then proceed to apply all these geometrical elements to the Reissner-Nordström case with the choice X ρ = A ρ and Y ρ = * A ρ , where the symbol * in this particular last case is not the Hodge operator but a particular nomenclature [8,9,15]. The line element for this spacetime is given by ds 2 = −(1 − 2m r + q 2 r 2 ) dt 2 + (1 − 2m r + q 2 r 2 ) −1 dr 2 + r 2 (dθ 2 + sin 2 θ dφ 2 ). In the standard spherical coordinates t, r, θ, φ the only non-zero components for the potentials will be A t = −q/r and * A φ = −q cos θ. With these potentials we find that the only non-zero components for the electromagnetic tensor f µν = A ν;µ − A µ;ν and its Hodge dual * f µν = * A ν;µ − * A µ;ν are f tr = −q/r 2 and * f θφ = q sin θ. The symbol ; stands for covariant derivative with respect to the metric tensor g µν , in our case the Reissner-Norström geometry. It is easy to check that the only non-zero components of the extremal field and its dual are ξ tr = f tr and * ξ θφ = * f θφ . We In this particular coordinate system we would have to be careful because both vectors V α and V α (4) before normalizing would be zero at the coordinate value θ = π/2. As the purpose of this section is not to find suitable coordinate coverings but to show that the conserved currents are vectors inside either blade one or two, we proceed to exhibit these currents and conserved charges taken from reference [16]. In reference [16] the energy-momentum currents are defined as T α β ξ β j for j : 1 · · · 4, where the vectors ξ β j are Killing vector fields. These Killing vectors are defined by ξ 1 = (1, 0, 0, 0), ξ 2 = (0, sin θ, cos φ cot θ, 0), ξ 3 = (0, cos φ, − sin φ cot θ, 0) and ξ 4 = (0, 0, 0, 1). The corresponding conserved currents are then, Index a ∈ 2, 3, 4. The main objective of this section is to show that the conserved currents are located or belong to either blade one or blade two. We can now check comparing equations (35)-(36) with equations (31)-(34) that the conserved current J(ξ 1 ) belongs or is in the plane determined by the vectors (U α , V α ), that is blade one, and the other three conserved currents J(ξ a ) for a : 2 · · · 4 lie in the orthogonal plane or blade two determined by (Z α , W α ). The conserved charges are calculated exactly as in reference [16]. The only one that is non-zero corresponds to the current vector inside blade one and it is given in the case where m 2 > q 2 and r + = m + m 2 − q 2 by the value Q 1 = 4 π q 2 /r + . Therefore we have proven our point which states that the four energy-momentum conserved currents belong to either blade one or two. From reference [16] we can easily see that the Bel currents [16][17][18][19] are also inside these blades. This finding should not be surprising, see section Appendix C. A vector inside blade one is invariant under local gauge transformations through the vector Y ρ = * A ρ , and a vector inside blade two is invariant under local gauge transformations through the vector X ρ = A ρ . This is because local Lorentz transformations on blade two do not affect the orthogonal blade one, and viceversa. All geometrical constructions presented in this section will help visualize the ideas that in a general setting we will present in the next section where we will study the dynamics of symmetries under a perturbative scheme.
First order perturbations in geometrodynamics
It is the purpose of this section to demonstrate that the whole geometric analysis in previous sections can still be reproduced step by step in a dynamic situation, see reference [8,9]. We introduce to this end first order perturbations to the relevant objects where ε is an appropriate perturbative parameter,g µν = g µν + ε h µν (37) ξ µν = ξ µν + ε ω µν . The perturbation objects h µν , ω µν and the one we will introduce next for the electromagnetic tensor are of a physical nature caused by an external agent to the source of preexisting fields. It is worth stressing that they are not the result of a local first order coordinate transformation. We will raise indices with the perturbed metricg µν = g µν − ε h µν . We can write the perturbed electromagnetic field through a new local duality transformation as, The perturbed local complexionα will not be explicitly involved in our analysis. As done in references [1,2] we impose the new condition, and through the use of the identity (4), which is valid for every pair of antisymmetric tensors in a four-dimensional Lorentzian spacetime [1], we will show that when applied to the case A µα =ξ µα and B να = * ξ να it results in the equivalent condition, Even though we are developing a first order perturbative scheme, we avoid writing explicitly the first order approximations, specially in this section, in order to create a general framework that allows to understand the ideas with more clarity. The complexion, which is a local scalar, on account of imposing condition (40) can then be expressed as, It will be useful for this matter to write explicitly the stress-energy tensor for the perturbed fields (37)-(38),T µν =ξ µλξ λ ν + * ξ µλ * ξ λ ν .
We next proceed to write the four orthogonal vectors that will become an intermediate step in constructing the tetrad that diagonalizes the first order perturbed stress-energy tensor (43), In order to prove the orthogonality of the tetrad (44)-(47) it is necessary to use the identity (4) for the case A µα =ξ µα and B να =ξ να , that is, whereQ =ξ µνξ µν is assumed not to be zero. We also need the condition (41). We are free to choose the vector fields X α and Y α , as long as the four vector fields (44)-(47) do not become trivial. In this section we have essentially proved that we can build for the perturbed fields a replica of our previous formalisms and constructions used for the unperturbed fields. In particular, we are able to write our new local tetrad keeping the same local extremal skeleton structure as in the unperturbed case and define the new local planes of symmetry associated to the perturbed stress-energy tensor. The new local planes of symmetry will be tilted with respect to the unperturbed planes.
Dynamical symmetry breaking in geometrodynamics
In order to study the notion of symmetry breaking in geometrodynamics we will need the results from sections Appendix D and Appendix E. We proceed next to write the first order perturbed covariant derivative of a first order perturbed local contravariant current vector, We can rewrite equation (49) as follows, where the first order perturbed local current has been written asJ λ = J λ + ε J λ (1) . The objective of this section is to show that between an initial constant time hypersurface and an intermediate constant time hypersurface, just when the perturbation starts taking place, the unperturbed local currents are considered to be conserved, that is ∇ µ J µ = 0. This is because the unperturbed energy-momentum current for instance, lies inside the local blade one or blade two, that is to say, the local planes of gauge symmetry. Between the intermediate constant time hypersurface and a final hypersurface the original local current J µ will be conserved no longer. After the perturbation takes place, the ensuing conservation equation will be∇ µJ µ = 0 for the perturbed local current. This is because the local planes of symmetry, both blade one and two, will be tilted by the perturbation with respect to the planes on the initial setting. There will be new local planes of symmetry at every point in spacetime. We can see through the new perturbed unnormalized vectors (44)-(47) that diagonalize the new perturbed stress-energy tensor (43), that the new local planes or blades of symmetry in spacetime after the perturbation took place, will no longer coincide with the old ones. This is the reason why after the perturbations already took place the equation ∇ µ J µ = 0 is no longer valid and according to equation (50) the following result will be correct, This is exactly what we might call dynamic symmetry breaking. The old currents J λ will be no longer conserved, only the new onesJ λ will be. Using all the elements of analysis developed so far we will proceed to state the following theorem, see reference [8,9], Theorem 3 The local orthogonal planes of symmetry or diagonalization of the Einstein-Maxwell stress-energy tensor and associated local groups of tetrad transformations LB1 and LB2 evolve as the continuous perturbation of an external agent takes place. Symmetries are continuously broken and transformed into new symmetries as the local planes of symmetry evolve.
Conclusions
A new tetrad that diagonalizes the Einstein-Maxwell stress-energy tensor for non-null electromagnetic fields was introduced. However, this tetrad has an inherent freedom in the choice of two vector fields. Geometrodynamics is an arena in which an explicit example or choice can be given for these two vector fields, because Maxwell's equations are providing two vector potentials. The simplicity of the expression for the electromagnetic field in this new tetrad and the associated null tetrad becomes evident [2]. It was also proved that the local gauge group of electromagnetic gauge transformations is related to the group LB1, and also to the group LB2 through isomorphisms. It was also proved that when written in terms of the [2]. We have been able to develop the concept of dynamic symmetry breaking for classical electromagnetic fields in a curved four-dimensional Lorentzian spacetime. The analogous notion developed under the realm of Quantum Field Theories for the Standard Model aimed at the creation of mass through a dynamical interacting mechanism [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. In our work we have produced a dynamical breaking of symmetry through a change in spacetime curvature. The symmetries in the gauge theory of electromagnetic fields are understood through the isomorphisms proved in manuscript [2] as local Lorentz transformations on either plane one or two. These local groups have been named LB1 and LB2. New local tetrad vectors transform inside these blades under the action of these groups. Therefore, symmetry breaking is equivalent to a change in local planes or blades one and two through perturbations. When an external agent to the preexisting geometry perturbes the original system, the local planes of symmetry are tilted with respect to the original ones. Since there are conserved energy-momentum currents represented by vectors that are locally either inside blade one or blade two as we explicitly proved in the Reissner-Norström geometry case, and then in a general case in section Appendix D, symmetry breaking means that these currents will be inside the new local planes of symmetry after the perturbation takes place, which will be the perturbed ones. It is even possible to conceive and reproduce all these ideas in the quantum realm since quantum fluctuations are just perturbations, see reference [35]. We assumed that the energy-momentum currents conservation equations are locally invariant either under LB1 or LB2, evident in the Reissner-Norström geometry case and in a more general aspect for the energy-momentum currents introduced in sections Appendix C and Appendix D. The symmetries will correspond to new local orthogonal planes so that the new currents will also be inside the new planes. The old local conservation laws will no longer hold. There will be new ones associated to the new planes of symmetry. The vectors that locally diagonalize the old stress-energy tensor will no longer diagonalize the new perturbed stress-energy tensor. It is worth reminding ourselves that the Einstein-Maxwell stress-energy tensor is form covariant under perturbations because the perturbed version can be written exactly as the unperturbed version just using the perturbed metric tensor and the perturbed extremal fields. Even though the tetrad that diagonalizes the original stress-energy tensor is not the same as the new one for the perturbed stress-energy tensor, the tetrad vectors in both cases are locally structure invariant because the stress-energy tensors are structure invariant. In conclusion, in this work we have been able to prove that a change in curvature is associated with a local dynamic symmetry breaking process that we might reinterpret as an evolution of local symmetries into new local symmetries. There is a symmetry evolution, and we evaluate this evolution through the local plane symmetry evolution, or the evolution of blades one and two, which are the local planes of gauge symmetry. In the classical settings it is a continuous evolution while in the quantum setting it could be discrete. In other words, the local evolution of the LB1 and LB2 groups.
We can state the following conclusions as a summary.
• New orthonormal tetrad for non-null electromagnetic fields in four-dimensional curved Lorentzian spacetimes. This tetrad diagonalizes locally and covariantly the Einstein-Maxwell stress-energy tensor. Astrophysical applications in spacetime evolution [10][11][12]. • Isomorphisms between the local electromagnetic gauge group and the local groups of tetrad spacetime transformations LB1 and LB2. There is an isomorphism between kinematic states and gauge states of the gravitational fields, locally. These results can be extended to the non-Abelian cases [36][37][38]. • Maximum simplification of relevant tensors and field equations.
• New tetrads encode gravitational and electromagnetic gauge information.
• We are introducing an explicit "link" between the "internal" and the "spacetime", so far detached from each other. • Extension or generalization to non-Abelian theories with gauge groups SU (2) × U (1) and SU (3) × SU (2) × U (1), see references [36][37][38]. • Hypotheses made at the outset of the no-go theorems [5][6][7] proved incorrect. Therefore, the no-go theorems are incorrect. It can be noticed that the signs in e αβµν will be opposite to the standard notation [39]. The reason for this is that we want to keep the compatibility with [1] where the definition e 0123 = [0123] = 1 was adopted. With these definitions we see that in a spacetime with a metric g αβ , are the components of a contravariant tensor [39][40][41]. The covariant components of (A.1) are where g ασ g βρ g µκ g νλ e σρκλ = −g e αβµν , The tetrad vectors (U α , V α , Z α , W α ) have the following expressions in the Minkowski reference frame given by equations (38)-(39) in [1], where | ξ 01 | = (ξ 01 ) 2 .
Appendix C.
In this section we will prove that if the locally conserved energy-momentum current T µν ξ ν satisfies invariance either under the local groups LB1 or LB2, then the vector ξ µ has to lie either on blade two or blade one respectively, see reference [8,9]. The stress-energy tensor can be written [2], We write the vector field ξ µ in a general way using the orthonormal tetrad (21)-(24), where A and B are local scalars as well as φ and ϕ. Equation (C.2) represents the superposition of a general vector on blade one and a general vector on blade two. The equation for conservation of the energy-momentum current will be, Using the orthonormal tetrad vectors (21)- (24) and equation (C.1) we can rewrite (C.3) as, From reference [2] we know that we can produce a full inversion on blade one in expression (C.4), (U µ , V µ ) → (−U µ , −V µ ).
Adding (C.4) and (C.5) we get, Now substracting (C.4) and (C.5) we get, If we now impose current conservation under boosts in expression (C.7) we necesarily get A = 0. Therefore, if we impose on conserved currents local gauge invariance under LB1, then the vector ξ µ must lie on blade two and equation (C.6) will be satisfied. Again in expression (C.4) we can produce a rotation on blade two ϕ → ϕ + π and (cos φ, sin φ) → (− cos φ, − sin φ). If we now impose current conservation under general spatial rotations in expression (C.10) we necesarily get B = 0. Therefore, if we impose local gauge invariance under LB2, then the vector ξ µ must lie on blade one and equation (C.9) will be satisfied. Summarizing the results in this section, from (C.6) and (C.9) we conclude that if we impose local gauge invariance either under LB1 or LB2 on the local energy-momentum current conservation equation (C.3), the vectors ξ µ have to lie either on blade two or blade one.
Appendix D.
The Reissner-Nordström geometry is an exception in the sense that the complexion locally satisfies tan(2 α) = 0. The Killing vector fields lie on either plane one or two. Locally, Killing vectors in principle would not have to be vectors lying on either of the two planes if we are talking about geometries other than the Reissner-Nordström case. Then, the question arises about the existence of locally conserved current vectors lying on either planes in a more general dynamic geometry, for instance where non-null electromagnetic fields are present in a curved four-dimensional spacetime but without the spherical symmetry, see reference [8,9]. That is to say, a spherically symmetric source under the dynamic perturbative action of an external agent as stated at the beginning of this work or specifically in section 7. Electromagnetic and gravitational fields would have to satisfy the Einstein-Maxwell equations even under perturbative interaction. The bottom line is that we are assuming that the perturbed fieldsg µν ,ξ µν and α will also satisfy the Einstein-Maxwell equations. In this section we will analyze conserved currents for the unperturbed case in general, not necessarily the Reissner-Nordström geometry case. For the perturbed situation the analysis would be similar by replacing in the Einstein-Maxwell equations for the perturbed fieldsg µν ,ξ µν andα . One simple way to see that there are always locally conserved currents lying on both planes or blades is the following. If we replace the electromagnetic field in terms of the complexion and the extremal field, see expression f µν = ξ µν cos α + * ξ µν sin α, inside the Maxwell equations and following reference [1] equations (62)-(63), we can see that the extremal field and the complexion must satisfy, in accordance to the Maxwell equations which are a subset of the Einstein-Maxwell equations, where α ν represents the derivative ∂α/∂x ν . Therefore we can try and explore the vectors ξ µν α ν and * ξ µν α ν , and see if they are conserved on one hand and if they belong to the planes one and two on the other hand. First we can see that due to the antisymmetry of the extremal field ξ µν and its dual * ξ µν and to the scalar nature of the complexion α the following equations are satisfied, ξ µν ;νµ = −( * ξ µν α ν ) ;µ = 0 (D.3) * ξ µν ;νµ = (ξ µν α ν ) ;µ = 0 . 4). If the geometry is such that the complexion gradient α ν is not trivial, then we have two conserved local vector | 2020-06-25T09:10:16.255Z | 2020-05-01T00:00:00.000 | {
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234440893 | pes2o/s2orc | v3-fos-license | Evaluation of shielded metal arc welding learning in vocational high school
This study aims to determine the level of planning, implementation, and assessment of learning outcomes in the XI class of shielded metal arc welding (SMAW) techniques at the state vocational high school 1 Sedayu, Bantul Regency. This type of research is a descriptive study with a survey method. Data collection using a questionnaire. The study population was 90 students of class XI state vocational high school 1 Sedayu. The sample was taken the same as the population. Data collection using a questionnaire. The data analysis technique used a quantitative descriptive analysis. Based on the data analysis results, the planning category is medium, with a mean of 29.48. The implementation is a very high category with a mean of 49.60, the assessment of learning outcomes categorized as high with a mean of 10.68. The results showed that the evaluation SMAW technical learning was in the high category. Results also showed that the level of evaluation of the SMAW technique learning on planning is in the medium category and the evaluation of the implementation of the SMAW technique learning was in the very high category.
Introduction
Learning is a teaching effort carried out to students to learn and understand through the existing environment, and the results are reflected in behavioral changes that are cognitive, affective, and psychomotor. Learning is an organizing activity to regulate the environment and connect it with students so that the teaching and learning process can be carried out [1]. Learning evaluation is related to accountability; assessing learning outcomes has become an essential, albeit frightening, responsibility for many educators [2]. The evaluation aims to determine whether implementing the learning system that has carried out is effective and efficient or not, seen from the factors directly related to the learning process. The purpose of learning evaluation is to determine the learning system's effectiveness and efficiency regarding the objectives, materials, methods, media, learning resources, environment, and the research system itself [3].
The learning success factor is very significant based on the context and strategy used [4]. Learning is essential, but this is not according to the reality in the field, so it is not applied to learning. During the School Field Practice (SFP) at Vocational High School 1 Sedayu, several things were found to be obstacles in learning electric welding practices, namely in planning, implementing, and assessing learning outcomes learning evaluation was necessary. Evaluation is the process of planning, obtaining, and providing the information needed to find new sources. Measurement is the measurement, while assessment is evaluation [5]. The word evaluation obtained that the Indonesian word evaluation means assessing, but it is done by measuring it first. Evaluation is planned and carried out regularly [6]. Evaluation is an activity carried out at the beginning, during the program, and at the end of the program. Evaluations in practical learning usually include observation, oral and written communication, selfevaluation, and simulation techniques [7]. Shielded metal arc welding (SMAW) involves joining two or more metals that use heat energy to melt the work piece and electrodes (filler).
During the SMAW process, heat energy is generated due to a jump of electric ions (cathode and anode) at the electrode's tip and the material's surface. The liquid metal from the electrode and the objects joined then freezes, and the two or more metals are connected [8]. During the welding process, the protective type used is the flux membrane found on the electrodes. The flux on the electric arc welding electrode protects the weld metal from melting during the ongoing welding process. This flux will become a slag when it is reliable. A learning strategy can be defined as a plan that will contain a series of activities designed to achieve specific educational goals. The learning strategy is a plan of action or a series of activities, including methods and various learning resources compiled to achieve a specific goal.
There is a research on visual inspection on shielded metal arc welding products of welding contest whose participants are from vocational high school student. The results showed that the aspects that become the weakness of contestants were a lack of knowledge and a lack of understanding in determining the welding requirements. Selecting the welding current, recognizing the type of material, identifying the welding code and selecting the SMAW electrode were the difficult factors [9]. The other study also showed that students need the other media in order to improve their understanding on SMAW subject. The conventional job sheets have not been able to solve some of the problems that exist in the learning activities of SMAW subject [10].
Determine the success of the quality of student learning outcomes, it is necessary to evaluate, which aims to measure the extent to which students understand [11]. Implementation of learning is the implementation of strategies that have been designed to achieve learning objectives and are supported by the existence of facilities and infrastructure. The implementation of learning is an essential component in realizing the quality of education. Therefore, the implementation of learning must be carried out appropriately, ideally, and prospectively. Thus, teachers must be able to implement theories related to learning theory into actual learning realities.
Assessment is a systematic implementation of the collection, analysis, and interpretation of information or data to determine the extent to which learners have achieved learning objectives. Assessment is the implementation of giving or determining the value to particular objects based on specific criteria. In the implementation of learning, assessment plays an essential role in determining whether the learning implementation has been done. This study aims to determine how high the level of evaluation of the XI class of SMAW technique learning in Vocational High School 1 Sedayu, Bantul Regency.
Methods
This type of research is a descriptive study using a survey method. This study aims to describe the evaluation of the XI class of SMAW technical learning at the State Vocational High School 1 Sedayu. This study's subjects were all class XI students with 90 students consisting of 3 classes, namely XI TPA, XI TPB, and XI TPC.
The research procedure for the evaluation of learning techniques for SMAW begins by making a questionnaire by dividing three indicators, namely, planning, implementing, and assessing learning outcomes. The questionnaire will be distributed to all students-this research was conducted by distributing questionnaires through google form after the data was collected and analyzed. The data used in this study used a questionnaire instrument. Instrument testing used a one-shot case study research design. The score used in this study is based on a Likert scale. According to Hadi, the Likert scale has four answers [12], namely: strongly agree (4), agree (3), disagree (2), and strongly disagree (1).
The instrument's validity is the content validity estimated by testing the test content's appropriateness or relevance through expert judgment. Content validity is carried out to ensure that the
Results and Discussion
Levels of learning evaluation techniques SMAW depicted in figure 1. The results showed that the evaluation of the XI class of SMAW technical learning at the State Vocational High School 1 Sedayu Bantul Regency was in the high category, the mean obtained was 89.76, which was in the high category, namely 84.25 <89.76 <91.75. In detail, it can be explained as follows: very high 29 students or 32.3%, high 54 students or 60%, moderate five students or 5.5%, low one person or 1.1%, and very low one person or 1.1 % with high results obtained, namely 54 students or 60.0%.
Figure 1. Levels of learning evaluation techniques SMAW
The picture above shows that the level of evaluation of the SMAW technique learning has an average of 89.76, which is in the high category. This means that students following learning theory and applying it in SMAW practice are in the high category. This indicates that the learning activities are of good quality and are carried out well, starting from planning, implementing, and assessing learning outcomes. These results describe the evaluation function categorized into four functions: determining students' progress, development and success after experiencing or carrying out teaching and learning activities for a certain period.
Planning Indicators
Levels of planning indicator result is shown in figure 2. Based on the results of the analysis, it shows that the level of evaluation of the SMAW technique learning on planning is in the medium category. The mean obtained is 29.48, which is in the medium category, namely 28.25 <29.48 <30.75. In detail, it can be explained as follows: very high category nine students or 9.5%, high 23 students or 25.7%, medium 28 students or 31.5%, low 25 students or 27.8%, and very low five students or 5.5%. These results can be seen from planning and learning the SMAW technique in the medium category. This shows that in the teaching and learning process, the teacher designs learning activities that will be carried out systematically so that in delivering the material it can be conveyed in language that is easily understood by students and with limited infrastructure, the teacher can balance it by modifying it with other teaching materials. Teachers and students were balanced in teaching and learning process, and quality will emerge in the learning process if it is well planned and conceptualized by the teacher.
Implementation Indicators
The analysis results show that the level of implementation of the SMAW technique learning towards implementation is in the very high category, the average obtained is 49.60, including the very high category, namely 49.60> 48.9. In detail, it can be explained as follows: very high category 62 students or 68.9%, high 26 students or 28.9%, moderate one person or 1.1%, low 0 students or 0% and very low one person or 1, 1%. The results are illustrated in figure 3. From these results, it can be seen from the evaluation of the implementation of the SMAW technique learning in the very high category. This shows that the implementation of teacher learning always prioritizes student safety by always prioritizing Occupational Safety and Health, and students always obeying the teacher's orders. Teachers also respond to students according to their characteristics to complement each other between teachers and students. Reciprocity between students and teachers is
Learning Outcomes Assessment Indicators
The analysis results show that the SMAW technique's level of learning on assessing learning outcomes is in the high category, the mean obtained is 10.68 and is included in the high category, namely 10.65 <10.68 <11.95. In detail, it can be explained as follows: very high category 22 students or 24.5%, high 33 students or 36.7%, medium 18 students or 20.0%, low 12 students or 13.3%, and very low four students or 5.5%. The results are illustrated in figure 4. Indicators assessing the learning evaluation construct SMAW technique has a high category with 36.7% as 33 learners. These results can be seen from evaluating learning outcomes in learning SMAW techniques, including in the high category. This shows that the teacher prioritizes discipline so that students always work on assignments on time and do performance results well following the teacher's orders.
Conclusion
Based on the discussion that has been described, it can be concluded that the quality of learning in planning indicators in SMAW technique learning is included in the medium category, which indicates that it is good enough in planning to start from the program structure and needs to be improved again to make it better. The quality of learning on the implementation indicators in learning the SMAW technique is included in the very high category, which indicates that it is excellent in implementing learning starting from the methods, facilities, and infrastructure. The quality of learning on the indicators of assessment of learning outcomes in SMAW technique learning is included in the high category, which indicates that the teacher conveys to students coherently in assessing learning outcomes. | 2021-01-07T09:06:21.374Z | 2020-12-01T00:00:00.000 | {
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233837113 | pes2o/s2orc | v3-fos-license | Determination of the moment of resistance to turning of a forestry articulated machine
This article considers the calculation of the moment of resistance to turn off a wood articulated machine. The total moment is determined from the sum of the moment of resistance to turning the tire in place, the moment that occurs when the ground is cut off by the side surface of the tire, the moment of resistance to rolling the wheel.
Introduction
The initial parameter for calculating the articulated frame is the moment of resistance to mutual turn of the links. For the calculation, it is necessary to consider the turn from a static position, since in this case resistance to turn is maximum. The calculation scheme is shown in figure 1. Figure 1: ܤ -track of the designed machine, m; ܮ б -distance between the wheels of the balancing cart, m; ܽ, ܾ -respectively, the longitudinal and transverse axis of the tire contact patch, reduced to an equal rectangle, m; ݈ 1 , ݈ 2 -distances, respectively, of the first and second links from the axis of the vertical hinge of the articulated module assembly to the axis of the tandem bridge.
Moment for the implementation of the relative turn of the links, ܰ • ݉ [1][2][3][4]: where ܶ сопр1 , ܶ сопр2 -moments of resistance to turn of the first and second modules, respectively, ܰ • ݉ . When the module is turned, the wheel performs a complex movement, which can be represented as the sum of two movements: turning the wheel in place and rolling the wheel. Thus, the moment of resistance to turn of the n-module is equal to, ܰ • ݉ : where ܯ тр -is the moment of resistance to turn the tire in place, ܰ • ݉; ܯ ср -the moment arising when cutting off the soil by the side surface of the tire, if the module is in the track, ܰ • ݉; ܯ к -is the moment of resistance to wheel rolling, ܰ • ݉ .
To determine the moment of resistance to turning the tire on the spot, let us assume that the coefficient of adhesion φ and specific pressures at each point of the tire print with the supporting surface are the same. We will take into account that the wheel axis is located parallel to the reference surface perpendicular to the longitudinal axis of the module.
The moment of resistance to turn the tire in place
The car is equipped with diagonal low-profile tires, so the contact spot of the tire with the support surface will have the shape of a rectangle. To determine the size of the contact spot, we use the calculation scheme ( Figure 2).
where -ܭ is the empirical coefficient; ݓ -is the tire pressure, kPa.
We determine the value of the coefficient ,ܭ knowing the load carrying capacity ܩ ш and the radius under static load rG: Calculation of the length of the longitudinal axis of the contact patch of the n-module tire, m: Let's determine the pole of the tire turn. Let's select an elementary area with sides ݔ݀ and ݕ݀ on the contact spot ab (Fig. 3).
The projection of the elementary friction force on the AX axis is equal to: The projection of the elementary friction force on the axis will take the form AX: dFтрх will tend to rotate the wheel about its axis. Thus, when turning the wheel in place, the turning pole will be at the intersection of the projection of the wheel axis on the support surface and the direction of action of the projection of the resultant friction force in the contact spot when turning the tandem bridge. Formula for determining the Y turn pole coordinate: The center of the tire contact spot is determined by the coordinates ܮ( б /2; .)2/ܤ As a consequence, the limits of integration of ݕ 1 , ݕ 2 , ݔ 1 , ݔ 2 can be represented as: Let's determine the moment of resistance to turning the tire in place. The calculation scheme is shown in (Fig. 4), where O is the pole of turning the tire in place. Contact tire boundary coordinates ݑ 1 , ݑ 2 , ݒ 1 , ݒ 2 are defined as: Resistance to turning the tire in place: ܯ݀ тр = ܨ݀ тр • ;ݓ Module frictional moment: Let's determine the moment that occurs when the soil is cut by the side surface of the tire (Fig. 5). The depth on the drag can be equal to the clearance. However, on the drag, the turn is made in motion. Turning in place occurs when loading assortments, where the track size is determined by the deformation of the soil under the tire of the machine during a single pass and depends on the tire pressure on the soil and the strength properties of the soil. On drained soils with a single pass, the depth of the track does not exceed 5 cm [4][5][6][7][8]. Т сопр ݊ = ܯ тр ݊ + М ср ݊ + М к ݊ .
Let's determine the end formula:
Conclusion
A formula for determining the moment of resistance to turn of the module is derived and calculation schemes and formulas for determining the depth of the track depending on the type of surface are proposed. | 2021-05-07T00:04:33.041Z | 2021-03-01T00:00:00.000 | {
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254332518 | pes2o/s2orc | v3-fos-license | H∞ State-Feedback Control of Multi-Agent Systems with Data Packet Dropout in the Communication Channels: A Markovian Approach
The paper presents an H∞ type control procedure for multi-agent systems taking into account possible data dropout in the communication network. The data dropout is modelled using a standard homogeneous Markov chain leading to an H∞ type control problem for stochastic multi-agent systems with Markovian jumps. The considered H∞ type criterion includes, besides the components corresponding to the attenuation condition of exogenous disturbance inputs, quadratic terms aiming to acquire the consensus between the agents. It is shown that in the case of identical agents, a state-feedback controller with Markov parameters may be determined solving two specific systems of Riccati equations whose dimension does not depend on the number of agents. Iterative procedures to solve such systems are also presented together with an illustrative numerical example.
Introduction
Multi-agent systems received a considerable interest in control engineering over the last decades due to their wide area of applications including terrestrial, maritime, aerial and space surveillance and monitoring missions. Some early developments and comprehensive surveys in this field may be found, for instance, in [1][2][3][4]. The design requirements for the control of multi-agent systems may be formulated from different perspectives, a lot of literature treating these topics being available these days. Although the review of the control design methodologies for multi-agent systems is beyond the purpose of this paper, one will mention however some monographs as [5][6][7][8], presenting different multi-agent control problems. An important aspect related the multi-agent systems is their distributed control which is characterised, in contrast with the centralised case, by the absence of a control decision-maker. Such formulations, primarily considered for agents with single and double integrators models (see, e.g., [9]) have been then investigated for more general linear systems, as in [10]. Consensus of nonlinear agents using output feedback has been analysed, for instance, in [11] and an algorithm based on feedback linearisation of nonlinear agents with output measurements may be found in [12]. In [13], a stability analysis from the perspective of a hybrid modelling is proposed using invariant sets and the Lyapunov stability theory. Consensus problems for Bernoulli networks have been considered for instance in [14] and for high-order multi-agent systems under deterministic and Markovian switching network topologies, in [15]. Other consensus problems in stochastic sense have been investigated, for instance, in [16] and in [17] under Markov switching networks for agents with first and second order dynamics. Many formulations of the control problems for multi-agent systems include optimisation criteria. As shown for instance, in [18], the complexity associated with the computation of distributed optimal controller significantly increases. In [19], the authors study the structural properties of optimal control problems with infinite-horizon linear-quadratic criteria by analysing the spatial structure of the solution to corresponding Lyapunov operator and Riccati equations. A problem of synthesising a distributed dynamic output feedback achieving H ∞ performance is presented in [20]. In [21], a decentralised Markovian H ∞ control problem is considered for first-order dynamics of the agents in presence of time-delay conditions; the optimal control is expressed in terms of the solutions of a system of linear matrix inequalities depending on the dynamics of the multi-agent system. An H ∞ state-feedback consensus controller for undirected multi-agent systems is derived in [22] for a more general class of N identical agents; the complexity of the H ∞ consensus problem is reduced by representing the problem by N number linear systems. In fact, optimal control problems of multi-agent systems remain a domain of interest due to the design and implementation complexity of the control laws, even in the case of identical agents (see, for instance, [22][23][24][25]). In [26], a linear-quadratic control problem for identical agents is considered, for which it is proved that the optimal solution depends on the number of agents and on the stabilising solutions of two Riccati equations having the same order as the agents dynamics. This conclusion shows that the computational requirements may be significantly reduced, especially for large-scale multiagent systems. The research has been continued in [27] where the robustness properties of the decentralised linear-quadratic optimal controller are analysed.
More recently, an H ∞ optimisation problem for identical stochastic linear models corrupted with multiplicative noises was formulated and solved in [28], aiming to provide disturbance attenuation performance together with robust stability with respect to parametric uncertainties in the agents models. Based on the state-feedback gains of the centralised state-feedback controller, a distributed controller depending on the adjacency matrix associated with the undirected graph of the communication network was obtained using the spectra of Lyapunov operators.
In this paper, the loss of links between agents is modelled by linear stochastic systems with Markovian jumps. The proposed methodology allows to consider different configurations of the network, each of them corresponding to a state of the Markov chain. Markov switching network models may be found in many papers between which one mentions [29][30][31]. The formulation and the developments presented in this paper considers stochastic models both for the agents and for the network. The H ∞ cost function for the optimal control design includes, besides the expression for the attenuation of exogenous disturbance inputs, quadratic terms aiming to acquire the state consensus between the agents. The optimal H ∞ state-feedback gains are expressed in terms of the stabilising solutions of two systems of coupled game-theoretic Riccati equations having the same order as the dynamics of a single agent. This coupling between the Riccati equations is typical in the optimal control of stochastic Markovian systems, depending on the elements of the stationary transition rate matrix.
The paper is organised as follows: the H ∞ control problem for stochastic system with Markovian jumps is formulated and solved in Section 2. The optimal gains of the control law are expressed in terms of the stabilising solution of a system of coupled algebraic Riccati equations with indefinite sign. A convergent iterative algorithm to determine the stabilising solution of this system of coupled Riccati equations is also presented. The third section analyses the case of multi-agent H ∞ control. The main result of this section allows to determine the optimal control law for multi-agent systems solving two specific systems of coupled game-theoretic Riccati equations corresponding to the dimension of a single agent. In Section 4, the case of dropout data packages in the communication networks is discussed and illustrated by a numerical example for a large-scale multi-agent system with two states of the Markov chain. The paper ends with some concluding remarks.
H ∞ Type Control for Stochastic Systems with Markovian Jumps; The Case of a Single Agent
Consider the linear stochastic systeṁ where x ∈ R n denotes the state vector, w ∈ R m 1 is an exogenous input, u ∈ R m 2 stands for the control input, y 1 ∈ R p 1 is the quality output and y 2 ∈ R n denotes the measured output. Throughout the paper η(t), t ≥ 0 denotes a continuous Markov chain with the state space D = {1, . . . , d} and with the probability transition matrix P(t) = p ij (t) = e Πt , i, j ∈ D, t ≥ 0 in which the stationary transition rate matrix of η is The triple {Ω, F , P } denotes a given probability space, E[x] stands for the expectation of the random variable x, E[x|H] represents the conditional expectation of x with respect to the σ-algebra H ⊂ F and E[x|η(t) = i] is the conditional expectation with respect to the event η(t) = i. In the following developments it will be assumed that C (i)D(i) = 0 and D (i)D(i) = I m 2 , ∀i ∈ D. For invertible D(i) D(i), i = 1, . . . , d, if these assumptions are not accomplished one may perform the following change of the control variable u for which one can easily check that with the new control variableũ the orthogonality condition C(i) D(i) = 0 holds and D(i) D(i) = I m 2 , i = 1, . . . , d.
For a multi-agent system with N agents, the indexes k, = 1, . . . , N will be used to define the connection between the agents k and .
Some known definitions and results used in the following developments will be briefly reminded (more details and proofs may be found, for instance, in [32,33]). Definition 1. The stochastic system with Markov parameterṡ is called exponentially stable in mean square (ESMS) if there exists β ≥ 1 and α > 0 such that E |Φ(t)| 2 |n(0) = i ≤ βe −αt , ∀t ≥ 0, i ∈ D, where Φ(t) denotes the fundamental (random) solution of the differential system (2). (2) is ESMS if and only if there exist the matrices X(i) > 0, i = 1, . . . , d verifying the system of Lyapunov-type inequalities
Proposition 1. The stochastic system
Throughout the paper it is assumed that the systeṁ is stochastically detectable, namely there exist a set of matrices H(i), i = 1, ..., d such that the systemẋ(t) = (A(η(t)) + H(η(t))C(η(t)))x(t) is ESMS. The proof of the next result may be found, for instance, in [33] (Theorem 7 of Chapter 3).
Proposition 2. If the system (4) is stochastically detectable and if the system of Lyapunov-type equations
has a symmetric solution with X(i) ≥ 0, ∀i ∈ D, then it is ESMS.
where x(t) is the solution of the system (2) with the initial condition x 0 .
The main result of this section is the following theorem.
Theorem 1. If the system of coupled Riccati equations
has a stabilizing solution (X(1), ..., X(d)) with X(i) ≥ 0, ∀i ∈ D for a certain γ > 0, namely if the stochastic system with Markov jumpṡ then the state-feedback control law u(t) = F(η(t))x(t) stabilises the system (1) and , where the quality output y 1 (t) is determined with the initial condition x(0) = 0 of the system (1) .
Proof.
In order to prove that the state feedback gain (6) stabilises (1) one may firstly rewrite the Riccati system (5) as On the other hand, since the system (4) is assumed stochastically detectable, it follows that the systeṁ is also stochastically detectable and then, based on Proposition 2, one concludes that the above stochastic system with the state matrix A(η(t)) + B 2 (η(t))F(η(t)) is ESMS. Since the system (1) with the control law u(t) = F(η(t))x(t) has the same state matrix it follows that the state-feedback (6) is stabilising. In order to prove the last part of the theorem one introduces the function V(x(t), η(t)) = x (t)X(η(t))x(t). Using Proposition 3 for a certain initial condition x 0 , it follows that Taking into account (5), one obtains For t → ∞ and u(t) = F(η(t))x(t), from the above equation it follows that Then, for x 0 = 0, the inequality (7) directly follows and it becomes equality for w
Remark 1.
Matrix Riccati equations with indefinite sign as in the system (5) appear in H ∞ control ( [34]) and in mixed H 2 /H ∞ control problems ( [35]) in the deterministic framework.
The next result proved in [36] gives a numerical procedure to compute the stabilising solution (X(1), . . . , X(d)) of the system of game theoretic Riccati-type equations (5) assuming that such a solution exists.
Proposition 4. Assume that the system (1) is stochastically detectable and that the system of Riccati Equation (5) has a stabilising solution. Then the sequences {X k (i)} k≥0 , {Z k (i)} k≥0 defined by X 0 (i) = 0 and X k+1 (i) = X k (i) + Z k (i), i = 1, . . . , d, where Z 0 (i) are the stabilising solutions of the system of Riccati type equations Z k (i), k ≥ 1 are the stabilising solutions of the un-coupled Riccati equations are convergent and the limit of X k (i), i = 1, . . . , d when k → ∞ is the stabilising solution of the system (5).
An iterative algorithm to solve the system of coupled Riccati equations with definite sign is given for completeness in Appendix A. The proof of the algorithm convergence may be found in [33] (Theorem 21 of Chapter 4).
Remark 2.
Although in (9) one considered the same standard homogeneous Markov chain for all agents one may also treat the case when each agent is modelled with its own stochastic process η k (t). Indeed, if each agent dynamics is modelled with a standard homogenous Markov chain η k (t), k = 1, ..., N with d states then one may consider for the multi-agent system (9) an extended Markov chain with d N states. Since in the present paper the Markov parameters are used to characterise the availability or the link failure between agents it follows that the maximal number of states of the Markov chain in these applications is 2 N .
The main result of this section is given by the following theorem.
Proof. Part (i) of the statement is a direct consequence of Theorem 1.
The Data Packet Dropout Case
From (28) and (29) it follows that the control of each agent is determined as a combination of its own states and the states of all other network agents. The case when the states of all agents are available for every agent represents the nominal case and it will be denoted as the state i = 1 of the set D. In the case when the state of a certain agent is not available, the corresponding terms in the control expression of the other agents will be zero which is equivalent with the fact thatF 2 (2) = 0. This case will be associated with the state i = 2 of D. Supposing that the communication network fails at a certain moment of time, it follows that F(2) = diag (F 1 (2), . . . , F 1 (2)) and X 2 (2) = 0. The condition X 2 (2) = 0 is accomplished if the weights corresponding to the coupling terms in (11) are taken to be zero, namely if P(2) = 0 and if the corresponding row in the stationary transition rate matrix Π has null elements. To conclude, in the above considered scenario in which either the communication network properly works or it completely fails leads to a Markovian model with d = 2 states of the set D. If the connection with a single agent, let say k is lost then the gainF will have all extra diagonal elements of the k-th column equal to zero. Similarly, in the case when the connections with more agents fail, the columns ofF corresponding to these agents will have zero extra diagonal elements; in this situation the Markov chain will have maximum 2 N states.
In order to illustrate these ideas one considers a networked system with N = 100 agents whose planar motions are described by the kinematic equations where x k and y k denote the Cartesian coordinates and u i and v i their commanded accelerations, respectively. Two states of the Markov chain have been considered in this example. The first one corresponds to the case when the communication properly works and the states of all agents are available for all others; the second state of the Markov chain characterizes the situation in which each agent can only access its own state vector. Assuming that the quality outputs (denoted in Section 3 by y 1k ) are x k y k u k v k it follows that the matrix coefficients in the representation (1) are identical for both states of the Markov chain, namely only the weights Q k (i) in the cost function (11) being different for the two states. Thus for i = 1 one chose Q k = 100 · I 4 (namely P(1) = 10 2 · I 4 ) and for i = 2, Q k (2) = 0 4 , for all k = . It was assumed that stationary transition rate matrix of η is The elements of the second row of the stationary transition matrix were taken zero in order to accomplish the condition X 2 (2) = 0. Indeed, for P(2) = 0 and for π 21 = π 22 = 0, the unique stabilising solution of the Riccati system (21) has the form (X 2 (1), 0) since (X 1 (1), X 1 (2)) is the stabilising solution of (20). The elements of the first row of Π have been chosen such that the transition from the nominal first state to the second one corresponding to the network failure takes place in about 5 s as illustrated in Figure 1. Solving the Riccati systems (20) and (21) for γ = 100 one obtains Using the aforementioned gains one determined the agents planar trajectories for both cases when the network communication properly works and the case when it fails, respectively. Figure 2 presents two snapshots at t = 0.5 s and at t = 2 s obtained for random initial positions of the agents in both cases. One can see from the two snapshots in the upper half of the figure, that the matrix gain P giving the coupling between agents is important in determining a short settling time. By contrast, numerical simulations show that in the case when the communication network is not available the settling time is about 6.5 s.
Concluding Remarks
An optimal H ∞ type control method for large-scale multi-agent systems with identical dynamics and independently actuated is presented. It is shown that regardless the number of agents, the optimal solution may be obtained solving two systems of algebraic Riccati equations whose dimension correspond to a single agent. Convergent iterative numerical procedures are presented and used for a case study revealing the benefits of the coupling between agents. The proposed design methodology may be also used in applications in which only some of the links between agents fail. The dimension of the Riccati equations remains the same but their number increases due to the larger number of states of the Markov chain. Future research will be devoted to applications in which a sensitivity analysis with respect to the transition probabilities will be included.
Conflicts of Interest:
The authors declare no conflict of interest. | 2022-12-07T20:22:58.797Z | 2022-11-28T00:00:00.000 | {
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12830364 | pes2o/s2orc | v3-fos-license | Annokey: an annotation tool based on key term search of the NCBI Entrez Gene database
Background The NCBI Entrez Gene and PubMed databases contain a wealth of high-quality information about genes for many different organisms. The NCBI Entrez online web-search interface is convenient for simple manual search for a small number of genes but impractical for the kinds of outputs seen in typical genomics projects. Results We have developed an efficient open source tool implemented in Python called Annokey, which annotates gene lists with the results of a keyword search of the NCBI Entrez Gene database and linked Pubmed article information. The user steers the search by specifying a ranked list of keywords (including multi-word phrases and regular expressions) that are correlated with their topic of interest. Rank information of matched terms allows the user to guide further investigation. We applied Annokey to the entire human Entrez Gene database using the key-term “DNA repair” and assessed its performance in identifying the 176 members of a published “gold standard” list of genes established to be involved in this pathway. For this test case we observed a sensitivity and specificity of 97% and 96%, respectively. Conclusions Annokey facilitates the identification of genes related to an area of interest, a task which can be onerous if performed manually on a large number of genes. Annokey provides a way to capitalize on the high quality information provided by the Entrez Gene database allowing both scalability and compatibility with automated analysis pipelines, thus offering the potential to significantly enhance research productivity.
Background
Recent advances in high-throughput DNA sequencing have allowed large regions of a sample genome (or genomes) to be sequenced rapidly at high coverage [1]. The detection of rare variant associations with disease is one of many applications enabled by this new technology [2]. However, current analysis pipelines tend to produce large numbers of genetic variants, only a small subset of which are likely to be relevant to the phenotype under study [3,4]. This necessitates filtering and annotation to obtain manageable lists of candidates for further investigation. A typical workflow includes the identification of a list of variants in a sample, annotation of those variants with metadata drawn from a variety of sources, and filtering using research-specific criteria. Existing annotation tools, such as Annovar [5], are good at associating variants with basic biological information, such as which genes are affected. However, they are less helpful in deciding whether those genes are relevant to a given domain of interest. For a single DNA sample it is not uncommon for an experiment to produce thousands of variants, which may be collectively associated with hundreds of genes. It is therefore infeasible to manually inspect every candidate gene to determine if it is known to be associated with a particular domain of investigation. In order to address this problem we have implemented the Annokey tool described in this paper. We discuss its main features, how it is used and implemented, and analyse its performance on a simple test case relative to a published "gold standard" of DNA repair pathway genes [6].
Annokey's inputs are a list of genes and a list of key terms (e.g. "DNA repair pathways") that are considered by the user to be relevant to a research question. For each gene, Annokey searches for instances of those terms within the NCBI Entrez Gene database [7] and PubMed article abstracts and records their frequency and occurrence contexts. This provides an indication of each gene's relevance as a candidate for follow-up study. The key terms are listed by the user in descending order of significance, and the rankings of matched terms are included in the output. This offers an additional level of information for prioritisation, which becomes more important as the number of key terms increases. The tool automates what would otherwise be a labour intensive task and can greatly increase research productivity. Whilst Annokey was originally developed in the context of rare variant detection, it is applicable to a much wider range of genomic studies; any situation where a researcher wants to know which of a set of genes are related to particular topics, e.g. RNA-Seq, ChIP-Seq, siRNA screens.
The Entrez Gene database is provided by the National Center for Biotechnology Information (NCBI) and contains detailed information about genomes from a variety of organisms. Gene records for a particular organism are curated by NCBI's Reference Sequence project, drawing data from other databases within NCBI and other sources such as the Gene Ontology Database [8]. These represent individual genes and each is labelled by a unique identifier called a GeneID. Each entry contains information about (amongst other things) nomenclature, gene sequences, gene product sequences, pathways, interactions, markers and phenotypes. Entries also indicate other resources within and outside the NCBI, including literature citations in the form of PubMed article references. The database can be accessed in two main ways. The first access method is via a web interface [9] which is suitable for interactive browsing and querying. The second access method is via a programming interface (which is used by Annokey). Whilst the web interface provides an expressive search engine, it is tedious to use for large numbers of genes or large numbers of search terms, and it is difficult to incorporate into an automated analysis pipeline. The programming interface is also not straightforward to work with for non-technical researchers, as also noted by Mrozek et al. who aim to provide a more userfriendly interface for complex exploration of the NCBI resources [10]. Annokey is a targeted gene annotation tool that allows researchers to capitalize on the high quality information provided by the Entrez Gene database in a way that scales to large numbers of genes and queries, is tailored to a specified user context, and is compatible with automated analysis pipelines.
Annokey operates from the command line and has the following four main features: 1. Online and offline search capability. 2. Flexible search terms, allowing use of regular expressions. Search terms are ranked in importance by the user according to the order in which they are listed in the input. 3. Summary search results are provided as annotations on the input gene list for quick inspection, prioritisation and integration with workflows based on spreadsheets. 4. Detailed search results are provided in an HTML report with hyperlinks back to the Entrez Gene web interface. The HTML report also shows each matching instance of a search term and the context in which it was found.
Annokey's online search retrieves data from the NCBI databases directly over the Internet. This provides access to the most recent version of the data but, due to network latencies, is typically only applicable to queries involving a small number of genes (<100), although the number of search terms is not a limiting factor. Offline search utilises a locally cached copy of the Entrez Gene database, which can be populated using a snapshot of the whole database for a given organism. Offline search is appropriate for queries involving hundreds or thousands of genes and search terms, or in cases where a large number of different searches are required. The Annokey distribution provides an additional tool that automates the process of downloading and preparing the latest database snapshot.
Search terms are provided by the user in a text file, one per line, and are written as regular expressions [11]. This provides an easy interface for exact-match literal terms but also allows sophisticated search patterns to be constructed. For example, the search term "tumor cell" matches exactly (and only) that text, whereas "([Cc]ancer|[Tt]umou?r) [Cc] ell" generalises the search to allow for both "cancer cell" or "tumor cell", an optional spelling of "tumour" (with a "u"), and variation in capitalisation of the first letter of each word. The use of regular expressions provides a good compromise between the needs and abilities of novice and advanced users. The terms are ranked by importance according to the relative order of the lines in the input file. In its summary output, Annokey annotates each gene with information about the search results. One of the annotations is the highest rank of any matching search term, which provides a simple way for the user to prioritise the results.
For each gene in the input, Annokey retrieves its corresponding entry from the Entrez Gene database (if it exists), and then searches for each keyword in the fields of that entry. The location and frequency of each search term is recorded. The search optionally extends to the PubMed article summaries referenced in the gene record. The results of the search are presented in two ways: 1) as summary annotations to the input gene file; 2) as a more detailed HTML report. The summary output provides information that is easy to understand at a glance and practical to use in a typical bioinformatics workflow using spreadsheets (or CSV files). The detailed output provides a more fine-grained breakdown of the search results and includes hyperlinks back to the Entrez Gene web interface. We anticipate that most users will first look at the summary results to select a set of candidate genes, and then use the detailed report to investigate the candidates in more depth.
Implementation
Annokey is implemented in Python 2.7, is intended to be used as a command-line application, and can be used on any POSIX-compatible operating system (e.g. Linux, Mac OSX).
The algorithm is realised by the ANNOKEY_SEARCH procedure, which takes four parameters: 1) a list of gene names; 2) a list of search terms; 3) an organism name (which defaults to human); and 4) a Boolean flag indicating whether the search should include PubMed articles referenced from the gene entries. To avoid ambiguities such as those caused by synonyms, Annokey matches input gene names against the "Official Symbol" as specified by NCBI.
Each gene is processed separately by the loop spanning lines 4 to 26. The database record for a gene is retrieved (line 6) as an XML document, and parsed into its constituent fields (line 7). The precise set of fields that are searched are presented in the "NCBI Entrez Gene database fields" subsection below. Depending on how Annokey is used, the gene record might be found in the local file cache, or it might be fetched by an online query to the NCBI Entrez database. Each field of the gene record is processed separately by the loop spanning lines 9 to 13, and for a given field, each search term is processed separately by the loop spanning lines 11 to 13. All the matches of the search term are collected (line 12) and added to the set of hits (line 13). Each hit records the gene name, the search term, the field in which the matches were found, and a list of match locations. If the parameter "include_pubmed" is true then Annokey will also search through the titles and summaries of PubMed articles that are referred to in each gene record (lines 15 to 26). The set of PubMed articles referenced by a gene are collected (line 17) and the corresponding PubMed database entries are retrieved (line 18). Annokey first looks for PubMed database entries in a local file cache, and if that is unsuccessful, it then tries to fetch them from the online PubMed database (saving the downloaded entry into the local cache to optimise future requests). The XML PubMed entries are parsed into fields and searched in much the same way as the fields of the gene database. The total set of search hits is returned on line 28. In practice, to save memory, Annokey incrementally generates outputs after each gene is processed. It further reduces memory requirements by employing a streaming XML parser [12], which avoids the need to store the entire entry in memory for any given gene. Multiple different genes in a search could refer to the same PubMed article. Annokey avoids repeating the same search by saving the results of previous searches in a table indexed by PubMed ID.
Annokey uses the BioPython [13] library to request data from the online Entrez Gene and PubMed databases, and optimises the retrieval database entries by keeping a local file cache of database records. Cached retrieval of database entries avoids the latency of network access, which significantly reduces the running time of the program, and also reduces the load on the NCBI servers. Snapshots of the Entrez Gene database are provided periodically on the NCBI FTP server [14]. Annokey provides an additional tool to automate the process of populating its local file cache from a database snapshot. At the time of writing the uncompressed snapshot for human genes was roughly twelve gigabytes, with similar sizes for other organisms. Annokey's local cache stores each gene entry in a separate XML file, using a directory structure indexed by organism and then by gene name. To reduce the load on the file system, the gene names are further distributed evenly over 256 sub-directories by hashing the gene name (with a stable hash function) modulo 256. During the development of Annokey we experimented with storing the file cache in a relational database but found a slight degradation in performance and so we opted for a simpler solution based on the filesystem. The PubMed database is much larger than Entrez Gene and also more tightly restricted in its terms of use. This means that Annokey cannot populate a local cache of PubMed from a snapshot in the same way that it does for Entrez Gene. However, Annokey does keep a cache of previously downloaded PubMed entries to avoid requesting the same entry more than once from the online servers. In future work we will consider using the downloadable snapshots of MEDLINE [15] the bibliographic database underlying the PubMed retrieval interface, to populate the PubMed cache.
The detailed HTML report is facilitated by the use of the "html" Python library [16] using version 5 of the HTML syntax, which is supported by all modern web browsers. The "hide/show details" facility in the report is implemented using embedded Javascript. We have validated the HTML report using the W3C validator [17].
Example usage
The following example illustrates how Annokey can be used to perform a simple search. More complex scenarios are described in the user documentation [18]. Annokey requires two input files: a list of genes, and a list of search terms. The input gene list is a comma or tab separated file with at least one column headed "Gene". Other columns are allowed; they will be ignored by the search but preserved in the output. For illustrative purposes, suppose we have supplied the following gene list of three genes (although typically the list would be much longer, and contain many more columns): The input search term list is a text file with one term per line. Search terms can be literal text or regular expressions. The example below illustrates the contents of a file with two terms (again, in a real experiment, the list would be much longer):
[Bb]reast [Cc]ancer DNA [Rr]epair
If, for example, the gene list and the search term list are stored in files called "genes.csv" and "terms.txt" respectively, then an offline (cached) search can be performed by executing the following on the command line: annokey -terms terms.txt -genes genes.csv The summary output of the search is a new CSV file (printed to the standard output device) with three additional columns added, as follows: The output contains the original gene column (and any other columns in the input gene file) plus three additional columns of annotations, based on matches to the provided search terms: 1. Highest Rank: the numerical rank of the highest ranked matching search term. 2. Highest Rank Term: the highest ranked matching search term.
Total Matched Entries: for each search term
Annokey counts the number of database fields where that term is found at least once. This column contains the sum of all those counts over all the search terms.
The first two annotation columns show the highest ranked matching term (its numerical rank, and the term itself). The numerical rank allows the user to sort the search results in terms of the relative importance of matched terms. In the example above, we have ranked "breast cancer" as having higher importance than "DNA repair". The third annotation column shows how many matching fields were found over all the search terms. This allows the user to sort the results in terms of the weight of evidence. In the example above, there were no matches for the CANT1 gene, so its annotations are empty. It is important to note that the annotated output provides a heavily summarised view of the search results. It is intended to provide an overview that is relatively easy to understand and prioritise. Brief, sortable summaries are particularly useful when dealing with large numbers (>100) genes, as is typical in many genomics projects. Annokey also provides more detailed search results in the form of a HTML report, which we describe below.
The detailed output of the search is an HTML file containing an entry for each input gene describing the location and frequency of each matching term. Figure 1 shows the output for the example search above, as rendered by a web browser. The report begins with metainformation about the search, such as the version of Annokey that was used, the command line arguments and so forth. Then the search results are broken down by gene, and within a gene, by search term. The report only shows genes having at least one match, which explains the absence of CANT1 in the example. Each gene entry contains a table showing, for each matching search term, its rank and the locations and frequencies of its matches in the various fields of the gene database. In the figure we can see that "DNA [Rr]epair" was ranked second and matched once in the Process and Pathways fields and eight times in the GeneRIF (Gene Reference into Function) fields. Figure 1 Screen-shot of an example detailed search report, showing matches for two genes RPA1 and BRCA1 against the search terms "[Bb]reast [Cc]ancer" and "DNA [Rr]epair". The top part of the report shows meta information about the search. This is followed by an entry for each of the genes. Each gene entry contains hyperlinks back to the online Entrez Gene database entry for the gene, its list of GeneRIFs and PubMed references. The green coloured table summarises the matches for each search term, including the rank of the term and the fields where matches were found and their frequency. Following the table is a list of each search term showing all the contexts where the term was matched. This section can be quite long, so it is hidden by default, and can be expanded by clicking on the "hide/show details" button. In the picture above, the details are shown for RPA1 but hidden for BRCA1.
Following the table is a more detailed view showing the context of each search term match, where the term is highlighted within the field in which it was found. This section can become quite large, so it is hidden by default, and can be displayed by clicking on the "hide/show details" button for the corresponding gene.
Analysis of performance
In order to gauge the effectiveness of Annokey, we measured its sensitivity and specificity against a published "gold standard" list of human DNA repair genes [6]. This is by no means an exhaustive experiment but it gives us an idea about its behaviour in a reasonably well-controlled scenario. We ran Annokey with an input list of all the human genes contained in the Entrez Gene database, searched for the key term "DNA [Rr]epair", and included linked PubMed articles in the search. We counted every gene with at least one search-term match as a positive result (and any other gene as a negative result), and compared the list of positives against the gold standard. Any positive result that was also in the gold standard list was considered a "true positive", whereas the remaining ones were considered "false positives". Any gene in the gold standard list that was not also within the positive results was considered a "false negative", whereas the remaining ones were considered "true negatives". The results were as follows: We also examined "true negatives" and "false positives" by mixing a random sample of 100 genes from each set, and compared them to a blinded classification by a human expert. For each gene the expert manually looked up its entry on the Entrez Gene database and scored its relevance to "DNA repair" on a scale of 0 to 2, where 0 means not related, 1 means moderately related, and 2 means highly related, based on the evidence in the database (and not prior knowledge). The rules for scoring were defined before the experiment, and are listed in Table 1. The human expert agreed with Annokey on all 100 of the "true negatives" (all the "true negatives" were scored 0 by the human expert). In Annokey's "false positive" set, the human expert classified 2 genes as moderately related and 16 genes as highly related, which is to say that the expert agreed with Annokey that 18/100 of the "false positives" with respect to the gold standard were in fact correctly classified. We expect a relatively high false positive rate with keyword search, especially in the situation where even a single match is considered to be a positive result. In practice a user is likely to apply more stringent thresholds and thus reduce the false positive rate (though perhaps at the expense of an increased false negative rate). We also believe that the high false positive rate of keyword search can be further mitigated by the ability of the user to rank the importance of the results in the summary output. Of course the question of "truth" is a moving target in genomics and our tests are limited to the information contained in the Entrez Gene database. However, we believe the experiment shows that keyword search of Entrez Gene is a simple and effective way to classify genes.
Related work
Myriad tools exist to support functional analysis of gene lists. A 2009 survey identified nearly 70 tools that highlight "interesting" genes derived from high-throughput studies [19]. Many such tools take advantage of structured information in gene databases, including Gene Ontology annotations [8]. PubMed is used by some tools as a source of functional information about genes. However, these tools generally rely on statistical tests to establish the relevance of specific genes and their annotations with respect to a set of reference or control genes. In contrast, Annokey applies a user-directed ranking algorithm that relies on curated associations between genes and an area of interest.
There are also a number of tools that provide search functionality for the biomedical literature. Web-based, general search tools comparable to the PubMed system are reviewed in [20]. A few such tools offer the ability to limit results to particular biological entity types, such as genes, which can help to identify the literature most relevant to a given gene. A small number of literature search tools are also specifically designed to support the analysis of lists of genes. The most directly comparable in aims to Annokey are GoGene [21] and GeneValorization [22]. Table 1 Rules employed by a human expert to score the relevance of genes against the topic "DNA repair" using manual inspection of evidence from the Entrez Gene database GoGene is a tool that draws on associations between genes and functionally-related terms extracted from the literature, in combination with structured information in Entrez Gene and UniProt, to organise genes according to particular functional categories (e.g. Gene Ontology terms). Genes related to specific functional categories or diseases can be identified by browsing the relevant ontology structures. Like Annokey (when the PubMed search is activated), it performs a search against both PubMed and Entrez Gene. Unlike Annokey, the direct literature search is based only on matches to the gene names in the abstracts. Associations between genes and specific diseases or functional concepts are based on identification of those concepts in the abstracts mentioning the genes. The tools are therefore somewhat complementary; while Annokey relies on direct links to the literature to identify relevant abstracts rather than the occurrence of a gene name, it can support more flexible search for relevant terms (including in the Gene database itself, not restricted to terms found in the literature alone) and users have direct control over the relative importance of those terms.
GeneValorization is a web-based tool that shows the relationship between genes and contexts of study (keywords) using frequency of co-location of the gene name and the keyword in the literature. It employs a graphical format to display its results. Whilst having similar goals to Annokey, it differs functionally in a number of ways. Annokey is a command-line tool that is designed to work with existing analysis pipelines, whereas GeneValorization is designed for interactive use. Annokey allows more flexible search using regular expressions, whereas GeneValorization uses literal terms only. Annokey searches within various fields of the Entrez Gene databases plus linked PubMed articles, whereas GeneValorization focuses on literature search only. Annokey caches database entries, which allows it to support very large numbers of genes and terms, whereas GeneValorization performs all queries online.
Conclusions
Annokey is a freely available open-source tool that allows users to annotate a list of genes using a keyword search of the Entrez Gene database and linked PubMed article summaries. It produces two types of search results: 1) a summarised output of annotated genes, which is useful for filtering and prioritisation; and 2) a detailed search report which shows how each search term was matched within the various fields of a gene record. We believe that Annokey will be a useful addition to many bioinformatics workflows in which lists of candidate genes need to be prioritised with respect to a domain of interest. | 2016-05-12T22:15:10.714Z | 2014-06-26T00:00:00.000 | {
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252896499 | pes2o/s2orc | v3-fos-license | Postoperative analgesic effect of dexmedetomidine combined with TPVB applied to open gastrectomy for gastric cancer
Abstract Objectives This study is aimed to investigate the analgesic effect of combined dexmedetomidine and thoracic paravertebral nerve block (TPVB) on gastric cancer (GC) patients undergoing open gastrectomy. Methods From May 2019 to Nov 2020, a total of 80 GC patients preparing for open gastrectomy were enrolled in our hospital and were divided into the ropivacaine (RO) group and the ropivacaine + dexmedetomidine (RD) group ad libitum. All of the patients underwent TPVB. The characteristics, usage of patient-controlled analgesia (PCA), adverse events, visual analogue scale (VAS) scores, inflammatory cytokines, and T cell subgroups between the two groups were compared. Results Patients in the RD group showed the decreased occurrence rate of postoperative adverse events and VAS scores and improved anti-inflammation and immune function. These findings implied that the application of dexmedetomidine in combination with RO in TPVB has a good postoperative analgesic effect, as well as anti-inflammatory and immune-enhancing effects. Conclusions Dexmedetomidine as an adjunct analgesic may be potentially applied in clinical practice for GC patients undergoing open gastrectomy.
Introduction
Gastric cancer (GC) is the most common malignant tumor of the digestive system in the world, and its morbidity and mortality are among the top three of all malignant tumors in our country [1]. Surgical operation is the most common and efficient strategy for GC therapy at the early stage, and the treatment outcomes are usually satisfactory [2]. It is easy to cause severe hemodynamic fluctuations during the operation and the postoperative pain of patients is obvious [3]. Postoperative adverse effects include insomnia, hypotension, and bradycardia, which may increase the risk of cardiovascular and cerebrovascular and is not conducive to the rapid recovery of patients [4]. Therefore, postoperative analgesia is a necessary application in perioperative nurse.
Currently, patient-controlled analgesia (PCA) system is a commonly used postoperative analgesia mode, which administration is more accurate and follows the principle of individualization [5]. However, the postoperative analgesic effect of PCA alone is often not satisfactory in traumatic operations such as gastrectomy [5]. Epidural analgesia is regarded as the gold standard for the analgesia of abdominal and thoracic surgeries [6]. The local anesthetics act upon the spinal nerve roots in epidural space directly, block the introduction of pain stimulation, and thus provide good analgesic effect for postoperative operation [7]. Although epidural analgesia has many advantages, some limitations such as spinal nerve injury and epidural hematoma infection are also possible [8].
In recent years, epidural analgesia has been gradually replaced by other analgesia methods [9]. Yeung et al. indicated that thoracic paravertebral nerve block (TPVB) has the same analgesic effect as epidural block, but reduced the risks of developing minor complications [10]. Additionally, compared with epidural block, TPVB has a wider scope of application and can maintain hemodynamic stability, which is conducive to rapid postoperative recovery [11,12]. It has been used in some upper abdominal surgeries, such as hepatectomy [13] and nephrectomy [14]. However, there are relatively rare researches on TPVB for GC surgery.
Dexmedetomidine is a highly selective a2-adrenoreceptor agonist recently introduced to anesthesia, which can not only inhibit the excitability of sympathetic nerves and maintain hemodynamic stability, but also plays an important role in sedation, anxiolysis, and analgesia [15][16][17]. Recently, dexmedetomidine in combination with local anesthetics in TPVB for postoperative analgesia has attached much attention [18][19][20]. Wang et al. conducted a meta-analysis on the analgesic effects of dexmedetomidine combined with local anesthetics in TPVB and revealed that this combination improved postoperative pain scores, prolonged the duration of analgesia, and reduced postoperative analgesic consumption [18]. Xu et al. believed that dexmedetomidine combined with ropivacaine (RO) prolongs the duration of postoperative analgesia and improves recovery rate of patients undergoing video-assisted thoracoscopic surgery [19]. Liu and Liu demonstrated that in esophageal cancer patients received radical surgeries, dexmedetomidine combined with RO in TPVB can significantly improve the quality of anesthesia, promote the postoperative analgesic effect, and reduce the incidence of postoperative adverse events [20]. However, the postoperative analgesia effect of dexmedetomidine combined with TPVB applied to open gastrectomy in clinic is still unclear.
This study is aimed to investigate the effects of RO alone and combined with dexmedetomidine in TPVB on postoperative analgesia, inflammation, and immune function in patients undergoing gastrectomy. Our findings may deepen the understanding of the impact of dexmedetomidine combined with TPVB applied to open gastrectomy in clinic.
Patients
A total of 80 GC patients preparation for open gastrectomy were enrolled in our hospital from May 2019 to November 2020. The inclusion criteria were: (i) body mass index (BMI) was 18-27 kg/m 2 , (ii) American Society of Anesthetists (ASA) grade I-II, (iii) no obvious abnormalities of heart and lung function, and (iv) no communication disorders. The exclusion criteria were: (i) patients with puncture contraindications such as infections, scars, and tumors at the puncture sites, (ii) patients with allergic history of RO and opioids, (iii) pregnant and lactating women, (iv) patients with long-term drinking, chronic pain, and history of taking psychotropic drugs. Based on our preliminary study, we calculated the sample size with an online tool 'Power and Sample Size.com'.
Preparation before anesthesia
Written informed consent was provided by each patient. This study was performed in line with the principles of the Declaration of Helsinki. This study obtained the approval of the Ethics Committee in Huizhou Municipal Central Hospital. The included patients were divided into two groups ad libitum using a computer-generated random number table with sealed opaque envelope technique for allocation concealment: the RO group and the ropivacaine þ dexmedetomidine (RD) group. Preoperative lower limb vascular color Doppler ultrasound was performed to exclude deep venous thrombosis. All patients were fasting and not given medication 8 h before operation. The routine peripheral vein channel was established after entering the operating room and ringer lactate was instilled at a rate of 8 mL/kg/h to supplement the fluid loss caused by fasting. Meanwhile, the patients received routine electrocardiogram (ECG), heart rate (HR), pulse oxygen saturation (SpO 2 ), blood pressure (BP), and bispectral index (BIS) monitoring.
Afterwards, ultrasound-guided TPVB was performed by an experienced anesthesiologist that was blind to the group allocation. In brief, the patient was in lateral position, and the 8-9 spinous processes of thoracic vertebrae were determined first. Before puncture, 1% lidocaine was used for local infiltration anesthesia. The puncture point was 3 cm beside the upper edge of spinous process. In-plane puncture technique was used to puncture into thoracic paravertebral space. No blood and cerebrospinal fluid were drawn back. The catheter was implanted 2.5 cm and fixed. Fifteen milliliters 0.5% RO with 2 mL dexmedetomidine (1 lg/kg) was injected paravertebrally to the patients in RD group under ultrasound guidance. Patients in the RO group received 15 mL RO (0.5%) and 2 mL normal saline. The anesthetic effect was tested to determine that nerve block was successful.
Anesthesia induction
After the intravenous injection of 0.2-0.4 lg/kg sufentanil, 0.2-0.3 mg/kg etomidate, and 0.2 mg/kg cisatracurium, tracheal intubation was used for mechanical ventilation. Partial pressure of end-tidal carbon dioxide (P ET CO 2 ) was maintained at the level of 30-35 mmHg.
Maintenance of anesthesia
The flow of fresh oxygen during the operation was 1 L/min and the inhaled oxygen concentration was 80%. Inhalation of 1.5% sevoflurane and continuous infusion of remifentanil (25 lg/mL) was used for intraoperative maintenance. Besides, the value of BIS needed to maintain at 40-50. After the operation, the endotracheal tube was pulled out when the patients were conscious.
Postoperative analgesia and care
All patients received intravenous 0.02 lg/kg/h sufentanil via a PCA pump (100 mL), with a lockout time of 15 min. The anesthetist conducted postoperative monitoring, pain assessment, and management was blinded to the patient groups. Nurses cared for patients 24 h a day. After anesthesia for 6 h, lower limb massage combined with ankle pump exercise (passive during anesthesia and active after awakening) was performed for 15 min each time and 4 times a day, and timely ambulation was recommended. According to the Caprini score, prophylactic dose of low molecular weight heparin (Dasaichang) was subcutaneously injected if necessary.
Variables recorded
The visual analogue scale (VAS) pain score (0-10) was used to evaluate the pain of patients at rest or coughing in the two groups at 6, 12, 24, and 48 h after awakening (restored mental activity). Score 0: no pain; score 1-3: mild pain; score 4-6: moderate pain; score 7-10: severe pain. In addition, the frequency of PCA pressed, sufentanil consumption, and the adverse events after operation such as nausea, vomiting, cardiac arrhythmia, bradycardia, and hypotension were also recorded.
Measurement for cytokines and T cell subgroups
Blood samples (5 mL) were collected on the day before operation (T0) and on postoperative days 1 (T1), 3 (T2), and 7 (T3). The plasma levels of IFN-c, IL-6, and TNF-a were measured using the corresponding commercial ELISA kits. The percentages of CD4 þ and CD8 þ cells in peripheral blood were measured by flow cytometric analysis.
Statistical analysis SPSS 20.0 software (SPSS; Chicago, IL) was used in analysis. Continuous variables conformed to normal distribution were analyzed using t-test and presented as means ± SD, while Chi-square test was used for categorical variables and shown as absolute number or percentage. p Value less than 0.05 indicated a statistically significant difference.
Analysis of patient characteristics
We analyzed the clinicopathologic characteristics of the patients in the RO and RD groups first. As shown in Table 1, there were no significant differences between RO patients and RD patients in age, sex, BMI, ASA, tumor stage, and operation time.
Comparison for the usage of PCA in the patients Afterwards, the usage of PCA in the patients of these two groups was assessed. We found that PCA duration, sufentanil consumption in PCA 48 h after operation, and PCA pressing times 48 h after anesthetic recovery in RD patients were significantly reduced compared to the RO patients (4.7 ± 1.5 vs. 6.3 ± 2.4, p¼ 0.0004; 32.5 ± 5.4 vs. 45.4 ± 8.3, p< 0.0001; 11.4 ± 1.7 vs. 14.3 ± 2.5, p< 0.0001; Table 2). However, the postoperative wake-up time of RD patients (the time from the end time point of surgery to the time when the patient fully restored mental activity) (12.8 ± 1.3) was relatively longer than that of RO patients (9.5 ± 1.1) (p< 0.0001).
Comparison for the adverse events in the patients
Regarding adverse events noted in the first 48 h postoperatively, on the whole, fewer adverse effects were observed in RD patients (4/40) compared to the RO patients (9/40) ( Table 3). For details, two patients in RD group and five in RO group had postoperative nausea and vomiting. Respiratory depression occurred in one patient in the RO group, while no cases were found in the RD group. Meanwhile, one RO patient and one RD patient presented the bradycardia symptom, and two RO patients and one RD patient had postoperative hypotension. No cardiac arrhythmia was recorded.
Comparison for VAS scores in the patients
As shown in Figure 1(A), VAS scores in RD patients were lower than those of RO patients at 12 and 24 h after anesthetic recovery when in a rest state (p< 0.05). Interestingly, we found that when coughing, VAS scores of RD patients were significantly decreased compared to the RO patients within one day (p< 0.05, Figure 1(B)).
Comparison for cytokines and T cell subgroups at different time points in the patients
Before operation (T0), the levels of IFN-c, IL-6, and TNF-a showed no significant difference between RO and RD groups ( Figure 2(A-C)). On postoperative day 1 (T1) and day 3 (T2), their levels in both two groups were increased compared with those at T0. On postoperative day 7, the levels of these factors were decreased to baseline except for TNF-a, whose level was higher than the baseline. Besides, IFN-c level was significantly higher but the levels of IL-6 and TNF-a were lower in RD groups compared with those in RO group at T1 and T2. In terms of T cell subgroups, the percentage of CD8 þ cells in both groups showed no significant changes from T0 to T3. Both CD4þ percentage and CD4þ/CD8þ ratio were decreased in two groups at T1 and T2 compared to those of pre-operation (p< 0.05). Meanwhile, the percentages of CD4 þ cells and the ratio of CD4 þ /CD8 þ cells in RD group were higher than those in RO group at T0 and T3 (Figure 2(D-F)) (p< 0.05).
Discussion
GC is a kind of malignant tumor of the digestive system and it is currently believed that open gastrectomy-based surgical methods can improve the long-term survival rate of GC patients to a certain extent [21]. However, postoperative pain generally leads to a series of adverse effects and increases the risk in perioperative period [4]. Therefore, effective anesthesia modes are important for improving the recovery rate of GC patients undergoing open gastrectomy. This study focused on the postoperative analgesia effect of 0.5 mg/kg dexmedetomidine combined with 0.5% RO in TPVB applied to open gastrectomy, suggesting that addition of dexmedetomidine potentiated the analgesic properties of RO, reduced requirement for postoperative sufentanil consumption, and decreased pressing frequency of PCA. In addition, adverse events, inflammation reactions, and immunosuppression in patients of anesthetics combination were also improved. Currently, many studies have confirmed that the analgesic effect of TPVB is better than that of epidural block and the occurrence rate of complications and contraindications in TPVB such as hypotension and bradycardia are significantly less than those in epidural block [10][11][12]22,23]. We speculated that the block plane generated by epidural block is wider than that of TPVB and the inhibitory effect on sympathetic nerve is more obvious, which was consistent with the previous results [24]. On the other hand, most of the current studies on TPVB are unilateral block [25,26] and we believed that the inhibitory effect on sympathetic nerve is slighter compared with the bilateral block in epidural block. In this study, a bilateral TPVB in patient undergoing open gastrectomy was performed. We found that there are relatively few Table 3. Comparison of the adverse events between the RD and RO groups. adverse effects in patients of the RO group (9/40) and RD group (5/40), suggesting that application of TPVB in open gastrectomy may be safe and effective.
Recently, dexmedetomidine as an adjuvant has been widely used in epidural analgesia and anesthesia [27][28][29]. Zhang et al. found that dexmedetomidine as an adjuvant can act synergistically and provided an improved sedation and analgesic [27]. Li et al. reported that the usage of adjunct drug dexmedetomidine (>5 lg) in epidural block makes great contributions on the decrease of post-anesthetic shivering compared to that of 5 lg [28]. A study by Cheng et al. revealed that the hemodynamics are more stable and VAS scores are lower in 0.5 lg/mL dexmedetomidine þ 0.08% RO group than that of the 0.5 lg/mL dexmedetomidine in combination with high concentration of RO (0.125%) group [29]. Given that the occurrence rate of complications and contraindications in TPVB are less than those in epidural block [10][11][12]22,23], we speculated that dexmedetomidine as an adjunct anesthetic in TPVB may be more efficient for analgesia. In addition, numerous studies demonstrated that the application of dexmedetomidine in patients undergoing GC surgery can improve the postoperative cognitive function [30][31][32]. We further speculated dexmedetomidine combined with RO in TPVB may prolong the duration of analgesia, reduce the dosages of anesthetic, and promote the recovery of GC patients undergoing open gastrectomy. In the current study, we found that the duration of PCA, drug dosages in PCA, pressing frequency of PCA, VAS scores, and postoperative sufentanil consumption in RD group were all decreased compared to those of RO group, which confirmed our assumption. However, the postoperative wake-up time was longer in RD group than RO group. Although dexmedetomidine prolonged the anesthetic time, it did not influence the outcomes, including early ambulation. Therefore, RD is benefit for the recovery of patients through enhancing analgesia and reducing anesthetic dose.
Additionally, adjunct analgesic (such as RO) combined with epidural block is also reported to repress inflammation and improve immune functions in GC surgery [33,34]. Interestingly, the effects of dexmedetomidine on inflammatory cytokines and T lymphocytes are confirmed in patients with kidney calculi [35] or GC surgery [36] without any block. In this study, to our knowledge, we for the first time investigated the influences of dexmedetomidine in combination with RO in TPVB on inflammation and immune functions for GC patients undergoing open gastrectomy. Immune function during the early postoperative period was clearly reduced. We found that both in RO and RD groups, the levels of IFNc, IL-6, and TNF-a at T1 and T2 were significantly increased in comparison to those at T0. IFN-c increased more in the RD group than in the RO group, while the elevated amplitudes of IL-6 and TNF-a were lower in the RD group compared to the RO group. Based on these results, we believed that dexmedetomidine combined RO in TPVB can inhibit the augmentation of inflammatory factors in post-operation. Tlymphocytes act crucial roles in anti-tumor immune activity, in which the reduced CD4 þ percentage and the ratio of CD4 þ /CD8 þ suggested the declined immune functions [37,38]. In our study, we demonstrated that although CD4 þ and CD4 þ /CD8 þ were decreased in two groups at T1 and T2 compared with T0, the amplitudes of reduction in the RO group were significantly larger than those in the RD group. These findings revealed that dexmedetomidine administration helps relieve immunosuppression. The combination of dexmedetomidine and TPVB protected the immune function against a large decline in our study. This finding again confirms that dexmedetomidine combined with TPVB contributes to cellular immune functioning. The detailed mechanisms remain to be verified by in-depth researches.
There were also some other limitations of this study. First, the epidural block groups were not set as the current medical environment does not allow serious complications presented. Second, the immune system is a complex system and other immune cytokines needed to be studied. We will elucidate these issues in future studies.
For GC patients undergoing open gastrectomy, combination use of dexmedetomidine and RO in TPVB not only decreases the occurrence rate of postoperative adverse events, reduces postoperative anesthetic consumption and prolongs the duration of analgesia, but also has the functions of anti-inflammation and improving immune activity.
Ethical approval
This study was performed in line with the principles of the Declaration of Helsinki. This study obtained the approval of the Ethics Committee in Huizhou Municipal Central Hospital (approval no. Kyll20210124).
Consent form
Informed consent was obtained from all individual participants included in the study.
Author contributions
WLW, ZQH, and QYQ made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; WLW took part in drafting the article and revising it critically for important intellectual content. All authors read and approved the final manuscript.
Disclosure statement
No potential competing interest was reported by the author(s).
Funding
The author(s) reported there is no funding associated with the work featured in this article.
Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. | 2022-10-15T06:17:44.777Z | 2022-10-14T00:00:00.000 | {
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11569374 | pes2o/s2orc | v3-fos-license | Neuropilin-1 Modulates p53/Caspases Axis to Promote Endothelial Cell Survival
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets.
INTRODUCTION
Apoptosis of the endothelial cell (EC) has been suggested to play an important role in a number of common and life-threatening vascular diseases, such as atherosclerosis, hypertension, and restenosis [1][2][3][4][5]. EC apoptotic death-induced loss of EC number and EC dysfunction may constitute an initial causative step in, and have a critical role in, the progress of many vascular pathological situations by compromising vascular wall permeability to cytokines, growth factors, lipids and immune cells, increasing smooth muscle cell proliferation and enhancing blood coagulation [6][7][8]. However, the cascade of molecular events that precede these final outcomes is largely unknown. The intracellular signaling that regulates the onset and execution of apoptosis has only been partially elucidated [9,10].
One possible reason for EC apoptosis may be the inappropriate function of growth factors and their receptors [11], which are critically involved in controlling cellular differentiation, growth, and function. One functionally relevant vascular growth factor is vascular permeability factor/vascular endothelial growth factor (VPF/ VEGF) [12,13] that has been cited as one of the most important pro-angiogenic factors. Neuropilin-1 (NRP-1) was recently found to be one of the VPF/VEGF receptors, which is expressed in EC and functions as an isoform-specific receptor for VPF/VEGF [14,15].
Although the original evidence suggests that the function of NRP-1 in VPF/VEGF signaling in EC occurs via the formation of complexes involving VEGFR-2 and NRP-1 [14,15], our findings and those of others have identified that NRP-1 regulates EC migration and adhesion to extracellular matrix proteins independently of VEGFR-2 [16,17]. Moreover, recently published data by Pan et al. indicated that NRP-1 is crucial to modulating EC motility, thus demonstrating that it plays roles beyond acting as an enhancer of VEGFR2 signaling [18]. These observations suggest the possibility that NRP-1 may either interact with other signaling receptors or independently promote cell signaling. The latter is supported by our further studies in which we revealed that the C-terminal three amino acids of NRP-1 (SEA-COOH) can interact with neuropilin-1 interacting protein (NIP, also called RGS-GAIP-interacting protein ,GIPC.), which mediates EC migration and angiogenesis [19]. Nevertheless, the molecular mechanisms of the downstream signaling of GIPC and its related function towards NRP-1 are not well understood.
Early in 1995, Alon et al. reported that VPF/VEGF could function as a potent inhibitor of apoptosis of ECs comprising newly formed retinal vessels in neonatal rats exposed to hypoxia [20]. Subsequently, Jain et al. reported VPF/VEGF could protect EC of newly formed immature tumor vessels [21]. Considerable progress has also been made towards delineating the VPF/VEGF survival signaling distal to the activation of VEGFR-2 [22][23][24][25]. However, a recent study found that the embryonic stem cell death pathway is overruled by a survival pathway during prolonged hypoxia (48 h) in a process involving HIF-1a-dependent up-regulation of VPF/VEGF which, in an autocrine action involving VEGFR-2 and NRP-1, protects against apoptosis [26]. Barr and co-workers [27] showed that NRP-1 plays an essential role in autocrine antiapoptotic signaling by VPF/ VEGF in tumor cells and that an NRP-1-blockade with an anti-NRP-1 peptide corresponding to exon 7 [28] of VEGF165 induces tumor cell and EC apoptosis. However, a VEGFR-2-blockade only induces EC apoptosis [27], which suggests that both VEGFR-2 and NRP-1 play an important role in VPF/VEGF-mediated EC survival signaling. It also suggests that they may function together as a receptor complex. More interestingly, Bachelder's study found that NRP-1 supported VPF/VEGF autocrine function and further cell survival and chemotaxis in tumor cells lacking expression of VEGFR-1 and VEGFR-2 [29,30]. To date, it is unknown whether NRP-1 can mediate VPF/VEGF-induced survival in EC independent of VEGFR-2 and the involved molecular signaling events.
By selectively activating NRP-1 in cell culture and by using a reverse genetic strategy in a zebrafish model, we explored the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1. We show here a novel mechanism for a VEGFR-2-independent, NRP-1-specific control of the antiapoptotic pathway in EC which most likely involves GIPC and then is mediated by the activation, respectively, of PI3K/Akt, the p53/caspases and FoxOs axis, and p21.
Results NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2, and its C-terminal three amino acids are essential for this function We first established a system to efficiently and reproducibly induce apoptosis in porcine aortic endothelial cell (PAEC) and human umbilical vein endothelial cell (HUVEC). Subconfluent cells were serum-starved (0.1%FBS) for 24 hours. Using TUNEL and Annexin V-FITC/PI apoptosis detection assays, we found that serum-starved cells showed a much higher prevalence of apoptosis and typical apoptotic nuclei ( Figure S1, a, b) as compared to the cells grown in full medium.
As VEGFR-1, VEGFR-2, and NRP-1 are all expressed on EC, it is difficult to distinguish their biological functions and signaling pathways induced in EC by VPF/VEGF individually. Therefore, to elucidate the distinct roles of NRP-1, we first used PAEC that express none of the VPF/VEGF receptors. Wild-type PAEC and PAEC transfected with NRP-1 cDNA (PAEC/NRP-1) were analyzed for their survival when treated with VPF/VEGF. As shown in Supp. Fig. 1a, apoptotic cells of parental PAEC treated with VPF/VEGF were observed by TUNEL assay. However, VPF/VEGF induced a significant decrease in apoptosis in PAEC/ NRP-1 relative to parental PAE cells, about five-and six-fold less than VPF/VEGF-stimulated parental PAEC or serum-starved PAEC/NRP-1 (Figure 1a). The percentage of apoptotic cells, which was determined using an Annexin V-FITC Apoptosis Detection Kit (Roche, Nutley, N.J.) with flow cytometry, demonstrated that the stimulation of PAEC/NRP-1 with VPF/ Figure 1. NRP-1 mediates VPF/VEGF-induced EC survival; the C-terminal three amino acids of NRP-1 are essential for this function. a. Quantitative determination of apoptotic nuclei in PAEC (n = 8). TUNEL assay was performed in apoptosis-induced PAEC and PAEC/NRP-1 with or with out 10ng/ml VPF/VEGF stimulation for 48 hrs to assess whether NRP-1 mediates PAEC survival. b. FACS apoptosis analysis to determine the percentage of apoptotic PAEC. c. Quantitative determination of apoptotic nuclei in HUVEC (n = 8. n: The number of counted field. All are same in this study). EGNP-1 mediates HUVEC survival; the C-terminal three amino acids of NRP-1 are essential for this function. TUNEL assay was performed in apoptosis-induced HUVEC transfected with EGNP-1 or EGNP-1DSEA and stimulated with or without 10ng/ml EGF. d. FACS apoptosis analysis to determine the percentage of apoptotic HUVEC. All the experiments at least were repeated three times and duplicated-wells were used for each experiment. *p,0.001, **p,0.005, ***p,0.01, ****p,0.05 in a Student's t test. e. NRP-1 mediates EC survival in zebrafish; the C-terminal three amino acids (SEA-COOH) are essential for the function. Fluorescent image of embryos injected with indicated morpholinos and mRNA, then subjected to the antibody stain with anti-GFP antibody and TUNEL assay to detect apoptosis. Insets show a magnification of apoptotic foci in the vessel. The arrows indicate apoptotic EC. i. Control; ii. Mismatch MO (4.5 ng); iii. zNRP-1a/1b MOs (4.5 ng+4.5 ng); iv. zNRP-1a/1b MOs (4.5 ng+4.5 ng)+hNRP-1 mRNA (0.3ng); v. zNRP-1a/1b MOs (4.5ng+4.5ng)+hNRP-1DSEA mRNA (0.3 ng). f. The dynamics of NRP-1-induced EC apoptosis in the zebrafish embryo. Fluorescent images of different stage embryos injected with indicated morpholinos and subjected to the antibody stain and TUNEL assay to detect apoptosis. Insets show a magnification of apoptotic foci in the vessel. The arrows indicate apoptotic EC. i. Control; ii. zNRP-1a/1b MOs (3.0 ng+3.0 ng). doi:10.1371/journal.pone.0001161.g001 VEGF significantly reduced apoptotic cell number, especially necrotic/late-apoptotic cells which were about three-fold lower than in VPF/VEGF-stimulated PAEC ( Figure. 1b). This result is consistent with our findings using the TUNEL assay.
Furthermore, we engineered a chimeric receptor, EGNP-1, by replacing the extracellular domains of NRP-1 with the N-terminal domain of the epidermal growth factor receptor (EGFR), and another chimeric mutant receptor, EGNP-1?SEA (created by deleting the C-terminal three amino acids of NRP-1 (SEA-COOH)) [16].These receptors were used to further elucidate the role of NRP-1 in VPF/VEGF-induced EC survival and the importance of the Cterminal three amino acids of NRP-1 (SEA-COOH). The concomitant stimulation of serum-deprived EGNP-1-transduced HUVEC with epidermal growth factor (EGF, 10 ng/ml) led to a decrease in apoptosis more than five-fold compared to the nonstimulated control and the LacZ-transduced HUVEC. However, EGF stimulation in EGNP-1?SEA-transduced HUVEC did not yield a significant difference in apoptosis when compared to the control and to the LacZ-transduced HUVEC ( Figure S1b, Figure 1c). Flow cytometry confirmed that EGF induced a significant decrease of apoptotic cells in EGNP-1-transduced HUVEC, but not in EGNP-1?SEA-transduced HUVEC, which, by contrast, showed more necrotic/late-apoptotic cells (Figure 1d). From these data, we concluded that NRP-1/EGNP-1 mediates ligand-induced EC survival independent of VEGFR-2, and that its C-terminal three amino acids (SEA-COOH) are essential for this function.
We employed a reverse genetic strategy to explore the function of NRP-1 in EC survival in a zebrafish model. Previously, we cloned zebrafish NRP-1 genes (zNRP-1a/1b) and analyzed their expression patterns by in situ hybridization [19]. In the current study, to examine functions of NRP-1 in zebrafish EC apoptosis, morpholinos are injected into a Fli-GFP transgenic fish line (TG(fli1:egfp)y1), where all of the ECs are labeled with GFP. Because of the transparency of zebrafish embryos, ECs can be observed in living embryos in fluorescent green. After that, the embryos were subjected to the TUNEL assay to detect apoptosis. The identity of the apoptotic cells is confirmed by using the red channel where EC should be labeled by Fli-GFP signal. The evaluation of apoptotic EC should consider the overlay of red signal and Fli-GFP green signal, and only red signal in EC location. As shown in Figure 1e and Figure S1, c, d, following knockdown of zebrafish NRP-1 by co-injecting NRP-1a/-1b splicing donor morpholinos (MO) into single-cell staged embryos, we found elevated levels of EC apoptosis compared with non MO injected or nucleotide exchange MO injection embryos. TUNELpositive cells showed orange masses and were either rounded or shrunken (pycnotic). Furthermore, the specific EC apoptotic phenotype was suppressed by coinjection of human NRP-1 mRNA (Figure 1e, Figure S1, c, d). The ectopic expression of human NRP-1 in zebrafish efficiently blocked zebrafish NRP-1 knockdown-induced EC apoptosis, and the zebrafish showed normal vessels with established blood circulation (data not shown), and a normal EC phenotype (Figure 1e, Figure S1, c, d). In contrast, the human NRP-1 mRNA lacking its three C-terminal amino acids (SEA-COOH) failed to block zebrafish NRP-1 knockdown-induced vessel defects, nor did it abrogate EC apoptosis ( Figure 1e, Figure S1, c, d). This suggests the functional importance of SEA-COOH in NRP-1-mediated EC survival.
We also investigated the dynamics of apoptosis in the zebrafish embryo. As shown in Figure 1f and Figure S1e, knockdown of NRP-1 revealed EC apoptosis as early as 24hpf embryos, which continued until 48 hpf. The early stage embryo had more apoptotic cells than the late stage embryo. Taken together, these results indicate that NRP-1 mediates EC apoptosis in zebrafish angiogenesis, and that its C-terminal three amino acids (SEA-COOH) are required for the function.
GIPC conveys NRP-1-mediated EC survival signaling
Based upon the above findings, we set out to further determine downstream pathways and the role of the involved key signaling molecules in NRP-1-mediated EC survival. Because our previous data showed that the C terminus of the RGS-GAIP (regulators of G-protein signaling-Ga interacting protein)-interacting protein, GIPC (also called NRP-1 interacting protein, NIP) can function as an adaptor protein to interact with the C-terminal three amino acids of NRP-1 (SEA-COOH) through its PDZ domain, and can then convey the NRP-1 signal to regulate EC migration and zebrafish angiogenesis [19], we first sought to address the role of GIPC in NRP-1-mediated EC survival. For this purpose, we knocked down GIPC in HUVEC using RNA interferencemediated silencing. After identifying that GIPC siRNA specifically and effectively silenced the expression of GIPC, but not that of NRP-1 or VEGFR-2 (Figure 2a), we then carried out apoptosis assays in HUVEC transduced with EGNP-1. We found that silencing of GIPC resulted in an increase in the percentage of apoptotic cells in HUVEC and also had marked morphological effects on the EC, including cytoplasmic shrinkage and cell retraction ( Figure 2b, Figure S2a). To examine this result in more detail, we analyzed the percentage of apoptotic cells with flow cytometry. Indeed, we observed a notable difference in apoptosis between GIPC knock-down HUVECs and without GIPC knockdown HUVECs (Figure 2c). Necrotic/late-apoptotic and early apoptotic cells were significantly increased by increasing the amount of GIPC siRNA. These results were subsequently confirmed in vivo in our zebrafish model. Zebrafish GIPC morpholinos (1.5ng and 4.5ng) were injected into TG(fli1:egfp) embryos at the 1-4-cell stage. This not only disturbed the formation of the dorsal longitudinal anastomotic vessel (DLAV) and subintestinal vein (SIV) as described in our previous study [19], but also resulted in a significant increase in EC apoptosis, as revealed by TUNEL staining ( Figure S2, b, c; Figure 2d). Additionally, circulation in the DLAV and SIV was diminished or blocked (data not shown). The body size was reduced as well ( Figure 2d). These phenotypes resemble those from zNRP-1a/1b knockdown, which suggests that zGIPC is involved in the survival signaling pathway of NRP-1. Furthermore, we found that the GIPC-mediated EC apoptotic phenotype could be rescued with human GIPC mRNA ( Figure S2, b, c; Figure 2d), whereas coinjection of hGIPCDPDZ mRNA with the zGIPC morpholino did not rescue the zebrafish GIPC knockdown-induced EC apoptotic phenotype. In summary, these results suggest that GIPC is involved in NRP-1-mediated EC apoptosis, and that the PDZ domain of GIPC plays an important role in this function, probably by interacting with the C-terminal domain of NRP-1 (SEA-COOH).
NRP-1-mediated EC survival requires PI3K/Akt activation
Since the survival signal mediated by various growth factors and cytokines may be dependent upon the phosphatidylinositol 3kinase (PI3K)/Akt signaling pathway [31,30], we investigated the possible regulatory role of PI3K/Akt in NRP-1-mediated survival.
We first determined the role of PI3K in NRP-1-mediated EC survival signaling. As assessed by the number of apoptotic cells, after treatment of EGNP-1-transduced HUVEC with Ly294002 (a PI3K inhibitor, 25 mM) we observed a significant increase in HUVEC apoptosis (Figure 3a, Figure S3a), which suggests that blockage of PI3K almost completely impedes the survival effect of NRP-1. It is known that PI3K contains a kinase subunit (p110) and a regulatory subunit (p85) and functions in tyrosine kinase receptor signaling pathways [32]. The p85 adaptor subunit is known to form a constitutive heterodimer with the p110 catalytic subunit. Ligand-activated receptors interact with the p85 subunit, releasing the p110 subunit in an active form. In this study, we further used the dominant negative mutants of the p85 subunit (p85(DN)) and the constitutively activated mutant of PI3K (p110CAAX) to determine whether PI3K was required for EGNP-1-mediated EC survival. Our results showed that the co-transduced HUVEC expressed similar levels of EGNP-1 (data not shown), but the cotransduction of HUVEC with EGNP-1 and p85(DN) increased EC apoptosis compared to HUVEC co-transduced with EGNP-1 and LacZ (Figure 3a, Figure 3Sa). However, co-transfection of p110CAAX with EGNP-1 resulted in a slightly decreased level of EGF-induced, EGNP-1-mediated EC apoptosis. Flow cytometry also showed a similar result to that found with TUNEL: both Ly294002 and p85(DN) induced more EC apoptosis, especially late apoptosis (Figure 3b). These results indicate that the antiapoptotic activity of NRP-1 on EC is mediated through PI3K.
It is known that the stimulation of growth factor receptorinduced PI3K activation mediates an increase in 3-poly-phosphoinositides, which in turn activates downstream effectors, such as the serine/threonine kinase Akt/protein kinase B (Akt/PKB) [32][33][34]. Therefore, we tested whether the PI3K effector protein Akt is involved in the survival response. We first co-transfected HUVEC with EGNP-1 and wild-type Akt or its two mutant forms, Akt(DN) (a dominant negative Akt mutant) and Akt(Active) (a constitutively activated Akt mutant), respectively. As revealed in Figure 3c and Figure 3Sb, Akt(Active) promoted EC survival to levels similar to those seen in EGNP-1 transduced HUVEC grown in the presence of EGF or 10% serum, whereas transfection of HUVEC with the dominant-negative Akt(DN) completely inhibited NRP-1-mediated EC survival activity. Flow cytometry confirmed the result showing significant high levels of late-and early-apoptotic cells ( Figure 3d) in Akt(DN) co-infected HUVEC compared to Akt(Active) or LacZ co-infected HUVEC.
PI3k-dependent activation of Akt involves its phosphorylation at serine 473 [34]. To further verify that Akt is involved in NRP-1- Figure 2. GIPC is involved in NRP-1-mediated EC survival. a. Knockdown of GIPC by siRNA in HUVEC. HUVEC were transfected with siRNA against GIPC in different concentrations. Immunoblotting was performed to quantify the expression levels of GIPC in these cells. GIPC and VEGFR-2 were quantified as a ratio to the amount of NRP-1. b. GIPC is involved in NRP-1-mediated HUVEC survival. Apoptosis assays were performed in apoptosisinduced HUVEC transfected with EGNP-1 and then transfected with siRNA, and followed by 10 ng/ml EGF stimulation for 48 hours. Quantitative determination of apoptotic nuclei (n = 8). Knock down of GIPC inhibits EGF-stimulated HUVEC apoptosis in a dosage-dependent manner. c. FACS apoptosis analysis to assess the percentage of apoptotic HUVEC. All the experiments at least were repeated three times and duplicated-wells were used for each experiment. *p,0.001, **p,0.005, ***p,0.01, ****p,0.05 in a Student's t test. d. GIPC is involved in NRP-1-mediated EC survival in zebrafish. Fluorescent image of embryos injected with indicated morpholinos and mRNA, then subjected to the antibody stain with anti-GFP antibody and TUNEL assay to detect apoptosis. Insets show a magnification of apoptotic foci in the vessel. The arrows indicate apoptotic EC. i. Control; ii. zGIPC MO (4.5 ng); iii. zGIPC (4.5 ng)+hGIPC mRNA (0.3 ng); iv. zGIPC MO (4.5 ng)+hGIPCDPDZ mRNA (0.3 ng). doi:10.1371/journal.pone.0001161.g002 mediated EC survival response, we analyzed whole-cell lysates of EGNP-1-transduced HUVEC treated with 10 ng/ml EGF for Akt activation, as indicated by phosphorylation at serine 473, using a phospho-specific antibody. In initial time course experiments, we found significant phospho-Akt upregulation as early as 2 minutes with maximal activation at 10-15 minutes after exposure of EGNP-1-transduced HUVEC to EGF (Figure 3e). The response gradually decreased after prolonged incubation (data not shown). As in HUVEC, the level of phospho-Akt was also upregulated in PAEC/NRP-1 stimulated by VPF/VEGF (Figure 3e).
Finally, we evaluated the effect of PI3K depletion on the activation of Akt. As shown by Western blot analysis, the PI3K inhibitor Ly294002 (25 mM) completely abolished Akt activation in response to EGF in EGNP-1-transduced HUVEC (Figure 3f), but the VEGFR-2 kinase inhibitor IV (100 nM), which can inhibit VEGFR-2 phosphorylation, did not influence EGNP-1-induced Akt phosphorylation (Figure 3f). These results suggest that Akt is a downstream effector in NRP-1-mediated EC survival signaling and exclude the possibility of involving VEGFR-2-mediated Akt activation in this study. In light of the above results, we conclude that PI3K/Akt is critically involved in NRP-1-mediated EC survival effects.
p53 and the mitochondrial apoptotic pathway axis inactivation are involved in NRP-1-mediated EC survival About 13-18% of MOs used in zebrafish result in off-target effects [35], which are represented by a signature neural death peaking at the end of segmentation (1dpf, day post-fertilization). The embryos display small heads and eyes, somite and notochord defects and craniofacial defects. Collaborative information from Dr. Stephen C. Ekker (Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis) has shown that the off-target effects of MOs are mediated through p53induced apoptosis. In this study, we found that knockdown of NRP-1a/-1b in zebrafish resulted in neural death phenotype and vessel defects, including EC apoptosis, suggesting that NRP-1 Figure 3. PI-3K/Akt is involved in NRP-1-mediated HUVEC survival. a. PI3K is involved in NRP-1-mediated EC survival signaling. Apoptosis assay was performed in apoptosis-induced HUVEC transfected with EGNP-1 or co-transfected with EGNP-1 and p85(DN) and stimulated with or without 10ng/ ml EGF for 48hours, and then treated with LY294002 for 30 minutes, accordingly. Apoptotic nuclei were determined quantitatively (n = 8). b. FACS apoptosis analysis to quantify the percentage of apoptotic HUVEC. c. Akt is involved in PI3K-mediated NRP-1 survival signaling in EC. Apoptosis assays were performed in apoptosis-induced HUVEC transfected with EGNP-1 or co-transfected with EGNP-1 and Akt(DN) or Akt(Active) and stimulated with or without 10ng/ml EGF. Apoptotic nuclei were determined quantitatively (n = 8). d. FACS apoptosis analysis to determine the percentage of apoptotic HUVEC. All the experiments at least were repeated three times and duplicated-wells were used for each experiment. *p,0.001, **p,0.005, ***p,0.01 in a Student's t test. e. Akt phosphorylation by NRP-1/EGNP-1 activation. HUVEC were transfected with EGNP-1 and then stimulated with EGF. Immunoblotting with anti-phospho-Akt was carried out to quantify the expression levels of phospho-Akt in these cells. b-Actin was used as a loading control f. The influence of PI3K depletion and VEGFR-2 phosphorylation inhibition on EGNP-1-mediated Akt activation. Immunoblotting was performed in HUVEC transfected with EGNP-1 and then treated with Ly294002 or VEGFR-2 Kinase inhibitor IV. Phospho-Akt and Akt were quantified as a ratio to the amount of b-Actin doi:10.1371/journal.pone.0001161.g003 MOs may result in p53 activation. Alternatively, this may also suggest p53 is involved in NRP-1-mediated antiapoptotic effects. The latter seems more convincing in this study because we also observed that the morphant phenotypes from NRP-1 knockdown were rescued by human NRP-1 mRNA. It is quite likely that NRP-1 is specifically involved in cell death. For this purpose, Mdm2 MO and p53 MO co-injection embryos were used as controls. As we have shown previously, NRP-1 MOs induced vessel defects and EC apoptosis. In addition, we also observed tail and body axis, and neuron apoptosis (data not shown), that are very similar to the neural death phenotype induced by off-target effects of MOs. Interestingly, after co-injecting p53 MO with NRP-1a/-1b MOs, we found that knockdown of p53 mostly alleviated off-target neural death compared to the positive control, while the vessel defects by knockdown NRP-1 were also mostly rescued (Figure 4a, Figure S4, a, b). These results suggest that p53 is involved in NRP-1-mediated EC survival.
This finding was further confirmed by investigating the effect of NRP-1 activation on p53 expression. By stimulating EGNP-1transduced HUVEC with 10 ng/ml EGF, p53 protein expression levels decreased significantly at 30 minutes to 24 hours when compared to the control group without EGF stimulation ( Figure 4b). This indicates that the EGNP-1-mediated EC antiapoptotic effect is induced by inhibition of p53 activation. Similar results for p53 expression level were also observed in PAEC/ NRP-1 (Figure 4b). These results support our in vivo findings.
A previous study reported that effective recruitment of Akt by appropriate survival signals may lead to activation of Mdm2, inactivation of p53, and eventually inhibition of p53-dependent apoptosis [36]. We, therefore, further investigated whether p53 is downstream of PI3K/Akt in NRP-1 signal. As shown in Figure 4c, the inhibition of PI3K with Ly294002 or blockage of Akt with the dominant negative mutant of Akt (Akt(DN)) reversed the expression of p53 to a higher level in EGNP-1-transduced HUVEC stimulated with EGF, which suggests that p53 is, in fact, downstream of PI3K/Akt in NRP-1-mediated EC survival.
Caspases are involved in both the mitochondria-independent (extrinsic) and the mitochondria-dependent (intrinsic) pathways [37,38], the two main pathways of cell death signaling in biological systems. p53 can regulate the secretion of cytochrome c to initiate the mitochondria-dependent pathway, leading to the activation of caspase-9 then caspase-3 followed by apoptosis [39]. p53 also regulates a series of genes that initiate the mitochondria-independent pathway, resulting in caspase-8 and -3 activities and apoptosis [40]. For these reasons, we sought to determine whether NRP-1's antiapoptosis is associated with inactivation of caspases. We examined the cleavage of the key apical caspases in the extrinsic pathway (caspase-8) and in the intrinsic pathway (caspase-9) as well as the cleavage and activity of caspase-3, which is the major executioner protease for both pathways. Cleavage of caspase zymogens is instrumental to the activation of caspase-3 and directly follows proximity-induced activation of apical caspases, thus contributing to their sustained activity [37,41]. Pro-caspase-9 is the apical caspase of the intrinsic pathway and is efficiently processed within the apoptosome [37]. In this study, cleavage of pro-caspase-9 was induced by serum starvation for 24 hours in EGNP-1-transduced HUVEC. Western blot analysis showed the full length caspase-9 (47 kDa) and a cleaved fragment (37 kDa) (Figure 4d). Following 30 minutes to 24 hours of stimulation with EGF in these cells, we found that the levels of cleaved caspase-9 decreased, as indicated by the concomitant increase of the pro-caspase band and the decreased formation of the p35 fragment (Figure 4d). Activated or cleaved caspase-3 (17 and 19 kDa) is crucial for the induction of apoptosis. After 24 hours serum starvation, pro-caspase-3 is proteolytically cleaved to generate a p19 signature fragment in EGNP-1-transduced HUVEC, but the cleavage level decreased following EGF stimulation (as shown in Figure 4d). In this study, cleavage of caspase-8 was not detected (data not shown). These results indicate that sustained activation of NRP-1 inhibits caspase-9, and consequently directly suppresses the cleavage of its preferred substrate, caspase-3, therefore may reduced EC apoptosis. Taken together, the above results confirm that the NRP-1-mediated EC anti-apoptotic affects occur through the axis inactivation of p53 and mitochondrial apoptotic pathway.
Induction of p21 is involved in NRP-1-mediated EC survival p53 is a transcription factor, and its loss results in changes in the expression of downstream target genes. One such target gene is p21 Cip1/WAF1 (p21), which we examined in this study. As shown in Figure 5a, we observed a significant increase in p21 protein levels from 30 minutes to 24 hours after EGF stimulation in EGNP-1transduced HUVEC or VPF/VEGF stimulation in PAEC/NRP-1. Obviously, this increased p21 expression level is independent of p53 inhibition. The cyclin kinase inhibitor p21 was initially believed to be an inhibitor of cell cycle progression, but has since been shown to have additional functions other than CDK inhibition. A recent study reported that AKT/PKB phosphorylation of p21 enhances the protein stability of p21 and promotes cell survival [42]. Based on this evidence, we first examined whether NRP-1 activation can induce p21 phosphorylation. We used two antibodies against the potential AKT/PKB phosphorylation sites of p21, Thr 145 and Ser 146 . Figure 5b demonstrates that p21 is phosphorylated by EGNP-1 in the two positions. Furthermore, we tested whether the increased expression level of p21 results from NRP-1-mediated PI3K/AKT activation. Indeed, our results showed that there was a reduced expression level of p21 in LY294002 treated or Akt(DN) co-infected HUVEC, compared to the control HUVEC infected only with EGNP-1. Meanwhile, we found that the phosphorylation levels of p21 at sites Thr 145 and Ser 146 were also reduced ( Figure 5c). Together, these data suggest that NRP-1-mediated EC survival occurs through Akt/PKB activation-induced phosphorylation and up-regulation of p21.
Modulation of FoxO transcription factor activation in NRP-1-mediated EC survival signaling
Studies have suggested that PI3K-mediated Akt activation in mammals negatively regulates the Forkhead box class O (FoxO) subfamily of Forkhead transcription factors [43,44]. Akt mediates phosphorylation of FoxO proteins [44,45], which prevents their nuclear translocation, thereby inhibiting their transcriptional competence. Studies in mammalian cells have shown that the overproduction of FoxO1 (FKHR), FoxO3a (FKHRL1), and FoxO4 (AFX) induces either cell cycle arrest or apoptosis [44,46]. Based upon these findings, we decided to address whether PI3K/ Akt-mediated NRP-1 survival signaling occurs through inhibition of FoxO transcription factors. HUVEC are known to express FoxO1 and FoxO3a, whereas FoxO4 could not be detected [47]. We therefore focused on these two FoxO transcription factors. As we expected, Western blot analysis showed that 10ng/ml EGF or VPF/VEGF stimulation resulted in a significant increase in the phosphorylated forms of FoxO1a and FoxO3a in the EGNP-1transduced HUVEC or PAEC/NRP-1 (Figure 6a). Furthermore, depletion of PI3K or blockage of Akt resulted in a significant decrease in the phosphorylated forms of FoxO1 and FoxO3a proteins in EGNP-1-transduced HUVEC (Figure 6b). Our results suggest that the survival signaling of NRP-1 in EC is mediated by PI3K/Akt through the inhibition of FoxO1 and FoxO3a.
DISCUSSION
In this study, we show a novel function for NRP-1, which can mediate VPF/VEGF-induced EC survival independent of VEGFR-2. We used PAEC transfected with NRP-1 cDNA to examine the specific function of NRP-1. PAEC/NRP-1 was a generous gift of by Dr. Shay Soker (Molecular and Cell Biology, Wake Forest Institute for Regenerative medicine). Previously, Soker et al. established stable cell lines synthesizing neuropilin-1 (PAEC/NRP-1) and characterized VEGF165 binding to NRP-1 in the cell [48]. PAEC were chosen for this study due to their lack of expression of any VPF/VEGF receptors [49]. To explore the possibility that NRP-1 might transduce VPF/VEGF signaling alone, NRP-1 must be selectively activated, since VPF/VEGF receptors VEGFR-1 and VEGFR-2 are also expressed in EC. To do so, we used the chimeric receptor EGNP-1 by replacing the extracellular domains of NRP-1 with the N-terminal domain of EGFR. At #80% confluence, early passage HUVEC doesn't express EGFR, and therefore, the fusion receptor can be used to distinguish the signaling pathway that is mediated by NRP-1 [16]. This might seem to be scant evidence because NRP-1 might form a complex with VEGFR-2 through its intracellular domain. However, previously using EGNP-1, together with co-transducing HUVEC with EGDR mutants or blocking VEGFR-2 with an anti-VEGFR-2 antibody, we demonstrated that NRP-1 mediates HUVEC migration independent of VEGFR-2 [16]. This suggests that NRP-1 can indeed function independently of VEGFR-2 in VPF/VEGF signaling and supports our findings in this study. The current understanding of NRP-1 function is primarily based upon studies of its extracellular domain. However, the chimeric receptor EGNP-1, though it lacks the extracellular domain of NRP-1, has been shown to be involved in EC survival. This hints there may be a distinguishing pathway whereby NRP-1 mediates EC survival ii. zNRP-1a/1b MOs (4.5 ng+4.5 ng); iii. zNRP-1a/1b MOs (4.5 ng+4.5 ng)+zp53 MO (9.0 ng); iv. zMdm2 MO (4.5ng); v. zMdm2 MO (4.5 ng)+zp53 MO (9.0 ng). b. NRP-1/EGNP-1-mediated p53 inactivation. EGNP-1 transduced HUVEC or PAEC/NRP-1 were subjected to EGF or VEGF for the indicated times and their lysates were immunoblotted for p53 with b-actin as a loading control. c. p53 is the downstream signaling molecule of PI3K/Akt in EGNP-1-mediated EC survival. HUVEC were transfected with EGNP-1 then treated with Ly294002 or co-transfected with EGNP-1 and Akt(DN). Immunoblotting was performed to determine p53 expression level. d. Inhibition of mitochondrial apoptotic pathway in EGNP-1-mediated EC survival. Immunoblotting was carried out in EGNP-1 transduced HUVEC stimulated with EGF at indicated times to assess the activation of caspase-9 and caspase-3. p53 and caspases were quantified as a ratio to the amount of b-Actin *p,0.001, **p,0.005, ***p,0.01, ****p,0.05 in a Student's t test. doi:10.1371/journal.pone.0001161.g004 through its intracellular domain and is independent of VEGFR-2 signaling. Indeed, our published data have shown that the Cterminal three amino acids of NRP-1 (SEA-COOH) can interact with GIPC and are involved with NRP-1-mediated EC migration [19], whereas we failed to identify VEGFR-2 in the complex of NRP-1 and GIPC (data not shown). These facts, together with the Figure 5. Induction of p21 through PI3K/Akt is involved in the survival signaling of NRP-1. a. The activation of NRP-1/EGNP-1 increased p21 expression. EGNP-1 transduced HUVEC or PAEC/NRP-1 were subjected to EGF or VEGF for the indicated times and their lysates were immunoblotted for p21 with PLC-c as a loading control. b. The activation of EGNP-1 induced p21 phosphorylation at its Tyr 145 and Ser 146 sites. Immunoblotting was performed with anti-p21Tyr and anti-p21Ser in EGNP-1 transduced HUVEC stimulated by EGF for the indicated times. c. Akt mediates the phosphorylation of p21 in EGNP-1-mediated signaling. HUVEC were transfected with EGNP-1 then treated with Ly294002 or co-transfected with EGNP-1 and Akt(DN). Immunoblotting was performed to quantify the expression and activation of p21. p21 and phospho-p21 were quantified as a ratio to the amount of PLC-c. *p,0.001, **p,0.005, ***p,0.01, ****p,0.05 in a Student's t test. doi:10.1371/journal.pone.0001161.g005 observation in the present study that the C-terminal three amino acids and GIPC are essential for NRP-1-mediated EC survival, support the existence of an independent signaling pathway for NRP-1-mediated VPF/VEGF function. Overall, these results indicate that NRP-1 can mediate VPF/VEGF-dependent EC survival as an independent receptor.
Our previous study demonstrated that GIPC functions as a key adaptor protein that is involved in NRP-1-mediated EC migration and angiogenesis [19]. Similarly, we found that knockdown of GIPC in this study suppresses NRP-1-mediated anti-apoptotic effects in HUVEC and increases EC apoptosis in the zebrafish model, suggesting that GIPC addresses the same signaling pathway as NRP-1 in NRP-1-mediated EC survival. Consequently, how GIPC conveys NRP-1 survival signaling in EC is obviously an important issue that should be targeted.
In this study, we found that the PI3K inhibitors Ly294002 and p85(DN), a dominant negative mutant of the p85 subunit, reduced levels of NRP-1/EGNP-1-dependent survival. These observations indicate that PI3K is involved in NRP-1-mediated EC survival signaling, which is consistent with the findings by Bachelder et al. that NRP-1 supported VPF/VEGF autocrine function and promoted breast carcinoma cell survival by stimulating the PI3K pathway [29]. Activation of PI3K phosphatidylinositol-3, 4, 5triphosphate positively regulates Akt by binding to the pleckstrin homology domain of Akt [50]. In our experiments we found that Akt phosphorylation levels increased after EGF stimulation of EGNP-1-transduced HUVEC, and that such activation could be inhibited by the PI3K-specific inhibitor Ly294002 but not influenced by inhibiting VEGFR-2 phosphorylation. This confirms that NRP-1 survival signaling induces PI3K-dependent Akt phosphorylation. The ability of activated Akt to promote survival has been found in fibroblasts [51] and heochromocytoma cells [52] and neuronal cells [32]. In accordance with these findings, we found that the kinase-inactive Akt mutant (Akt(DN)), that has been suggested to act in a dominant-negative manner [53], blocks NRP-1-mediated survival activity on EC. This finding constitutes the further demonstration of a role played by the PI3-kinase/Akt pathway in mediating a NRP-1 survival activity. Despite the additional information provided by the above results, how PI3K is activated in NRP-1 survival signaling still remains unknown. p85, the regulatory subunit of PI3K, contains two SH2 domains and a SH3 domain. The SH2 domain of p85 is responsible for interaction with phosphorylated tyrosine in the context of motif YXXM [54,55]. However, unlike VEGFR-2, the intracellular domain of NRP-1 lacks the YXXM motif. Since GIPC is a central adapter protein for NRP-1 signaling, it is tempting to speculate that GIPC may function as an adaptor molecule, mediating the activation of p85 by NRP-1, which is currently under investigation.
Given that a transformed approach might alter signaling pathways (Dr. Stephen C. Ekker, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis), especially those that regulate cell survival, the involvement of p53 in regulating the apoptotic pathway suppressed by survival signals from NRP-1 was addressed more comprehensively by both in vivo and in vitro studies in this work. In the zebrafish model, EC apoptosis and vessel defects resulting from the loss of NRP-1 are recovered when p53 expression is downregulated. This suggests that p53 acts upon downstream components of a NRP-1 activation-induced survival cascade. The p53 pathway is composed of hundreds of genes and their products that respond to a wide variety of stress signals, including apoptosis and cell cycle arrest. The former involves caspase cascade while the latter is mediated by p21. Decreased levels of p53 might suppress caspase cleavage and therefore downregulate apoptosis. Indeed, our in vitro data found that activating NRP-1 decreases the expression of p53, caspase-9 and caspase-3, which confirms our in vivo observations. Our findings that NRP-1-induced cell survival more directly affects caspase-9, then caspase-3, but not caspase-8 cleavage, also supports the concept that phospholipases may be primarily involved in the intrinsic (mitochondria-dependent) arm of the bifurcate pathways of apoptosis mediated by death receptors. In addition, the data shown here that p53 is the downstream signaling molecule of PI3K/Akt hints that Mdm2 may be another considerable signaling component in NRP-1 survival signaling in EC, thereby connecting connects PI3K to p53. p21 was originally isolated as a p53-inducible protein and identified as an inhibitor of cyclin-dependent kinases (CDKs), and thus thought to function as a critical regulator of the cell cycle [56][57][58]. However, a number of previous studies suggested that p21 levels were alternatively regulated by p53-independent mechanisms. Serum and other growth factors might be involved with the up-regulation of p21 levels in various cell types [59,60]. The activator of the PI3K pathway, EGF, increased p21 levels by increasing the half-lives of both transcript as well as protein [61]. Additionally, the PI3K pathway was involved with the modulation of p21 levels [62,63]. Inhibitors of the PI3K pathway reduced the level of p21 in both cases. Moreover, recent investigations have clearly demonstrated that p21 also plays a role in allowing cell cycle transit as well as preventing apoptosis. Many late stage, rapidly proliferating tumors show a profound elevation in the levels of p21, which is counterintuitive in light of the proposed inhibition of cell cycle progression mediated by p21 [64][65][66]. The pro-survival activity of p21 in cells treated with chemotherapeutic agents such as taxol also argues for a role for this protein in the protection of tumors in vivo [67,68]. The similar pro-survival effect is also observed in terminally differentiated cells such as myoblasts [69], hematopoietic stem cells [70], and macrophages [71]. An exciting finding shown by Li et al., indicated that Akt phosphorylation of p21 at sites 145 and Ser 146 resulted in increased p21 stability as well as enhanced glioblastoma cell survival [42]. Notably, the results from our current study are consistent with these observations, confirming a crucial role for p21 as an antiapoptotic protein, and also providing evidence that p21 levels can be regulated directly by PI3K/Akt. In addition, the elevated levels of p21 may also partly result from activated PI3K/Akt-induced caspase-3 inactivation, which reduces caspase-3-mediated p21 cleavage. This is consistent with a previous study that caspase-3mediated cleavage of p21 during the apoptotic process sends growth-arrested cells to apoptosis [72]. While this novel role of p21 is evolving, a recent study revealed that p21 blocks irradiationinduced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation [73]. Since the mechanisms by which p21 interferes with the death machinery are still largely unknown, however, this remains our current focus.
FoxO transcription factors are key regulators of cell fate. The mammalian FoxO family of transcription factors FoxO1, FoxO3a, and FoXO4 are major substrates of the protein kinases PKB (Akt). Activation of the PI3K pathway blocks the function of all 3 FoxO members by Akt-dependent phosphorylation of three conserved residues, which relay PI3K signals to target genes [74,75]. A recent study has shown that FoxO1 and FoxO3a are the predominant FoxO isoforms expressed in mature EC [47]. In this study, we found that NRP-1 induced the rapid phosphorylation of FoxO1 and FoxO3a and was mediated by activated PI3K/Akt. These data suggest a requirement for FoxO1 and FoxO3a inactivation during NRP-1-induced EC survival and are consistent with previous studies into the roles of FoxO1 and FoxO3 in the induction of cell apoptosis [44,76]. The mechanism whereby FOXO proteins promote apoptosis in different cell types appears surprisingly diverse and includes induction of death-receptor ligands (e.g. FasL) and proapoptotic factors (e.g. Bim) as well as repression of cytoplasmic (e.g. c-FLIP) and mitochondrial antiapoptotic proteins (e.g. Bcl-2 and Bcl-xL) [77][78][79][80]. How FoxO1 and FoxO3a function in PI3k/Akt-dependent NRP-1 survival signaling in EC has not been fully defined in this study and remains to be elucidated.
In summary, after finding that NRP-1 can mediate VPF/ VEGF-induced EC survival independent of VEGFR-2, our results also highlight, for the first time, the distinct signaling pathways of NRP-1 in VPF/VEGF-induced survival effects in EC by connecting NRP-1 and GIPC to activation of PI3k/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. Based upon these findings, we proposed the working model in detailed in Figure 7.
What is emerging from this study, also in the light of reports from our previous studies and others, is that NRP-1 can function alone to mediate VPF/VEGF signaling. The consistency of our results supports the two current hypotheses for NRP-1 function in VPF/VEGF signaling in EC: either that NRP-1 is involved in VPF/VEGF-induced EC function by acting as a VEGFR-2 coreceptor, or alternatively, as a functional receptor alone. Not only this, recognizing the mechanistic aspects of coreceptor-based or single receptor-based signaling ligand guidance cues may have implications for other ligand guidance and complicated receptormediated events which form the basis of key biological functions. In addition, the multiple pathways involved in NRP-1-mediated VEGF survival signaling indicate a degree of complexity that is paralleled by the emerging complexity of interaction between VEGF ligands and their receptors, and the downstream signaling pathways they mediate.
To the best of our knowledge, both the VEGFR-2 signal alone, as well as NRP-1 acting in concert with VEGFR-2, are involved in VPF/VEGF-induced EC survival in vivo. As a consequence, NRP-1 signaling alone only partly determines EC fate. This implies that an understanding of the role of NRP-1 only provides part of the picture of VPF/VEGF survival signaling. However, given our increasing understanding of NRP-1, we can generate a more complete scenario of how the signaling molecule determines cell behavior.
Cell culture and treatment
Parental PAEC and PAEC expressing NRP-1 (PAEC/NRP-1) [were kindly provided by Dr. Shay Soker (Molecular and Cell Biology, Wake Forest Institute for Regenerative medicine) and were grown in F12 medium containing 10% FCS and GPS [48]. HUVEC (CLonza, San Diego, CA) were cultured as described previously. HUVEC were grown on 30 mg/ml vitrogen-coated dishes using the EGM-MV Bullet Kit (5% fetal bovine serum [FBS] in EBM with 12 mg/ml bovine brain extract, 1 mg/ml hydrocortisone, and 1 mg/ml GA-1000). HUVEC (passage 3 or 4) that were ,80% confluent were used for most experiments. Apoptosis was induced by deprivation using EBM supplemented with 0.1% BSA for 24 h. After that, cells were exposed to 10ng/ml VPF/VEGF or EGF in the same medium for 48 hours and then used for apoptosis testing.
Zebrafish lines and maintenance
Zebrafish were maintained and bred as described previously [81] (All experiments were performed using the TG(fli1:egfp)y1 l transgenic line that has been described previously [82] (Embryos were staged according to Kimmel et al [83].
TUNEL assay
Detection and quantification of EC apoptosis was performed using an In Situ Cell Death Detection Kit, TMR red Roche, Nutley, NJ). The assay was performed following the manufacturer's protocol. Samples were dechorionated and fixed in 4% paraformaldehyde for 1 hour at room temperature. Then they were washed with PBS twice and permeabilized with 0.1% sodium citrate, 0.1% TritonX for 2 minutes on ice. After washing twice in PBS, samples were incubated with the reaction mixture containing the terminal deoxynucleotidyl transferase and TMR labeled nucleotides for 1 hour in the dark at 37uC. The reaction was stopped by washing with PBS three times. Terminal deoxynucleotidyl transferase catalyzed the incorporation of labeled nucleotides to 3-OH DNA and ends in a template-independent reaction. The fluorescent signal was visualized and imaged using a Zeiss Axioplan 2 microscope coupled to an Apotome, using AxioVision 4.2 software (Zeiss). Z-stacks were superimposed using Extended Focus feature of the software.
Flow cytometry analysis
Cells (5610 4 ) were seeded on the slide cover in six-well culture plates and treated for 48 h with VPF/VEGF (10 ng/ml) in medium containing 0.1% FBS. Cells were washed with PBS and apoptosis was assessed using the Annexin V-FITC/PI apoptosis detection kit (BioVision, Inc., Mountain View, CA) according to the manufacturer's instructions. The respective proportions of apoptotic cells and necrotic/late-apoptotic cells were measured on 20,000 cells with a FACS Calibur analysis system (Becton Dickinson, CA, USA).
RNA interference
HUVECs at 70% confluency will be transfected in optimem medium with the siRNA duplexes using lipofectamine 2000 (Invitrogen). After 4 hours the transfection medium will be removed, the cells will be washed twice with PBS and then maintained in complete HUVEC medium for 48 hours before performing experiments with VPF/VEGF stimulation as above. Morpholino gene knock-down in zebrafish Morpholinos were designed targeting splice donors of the specific genes to efficiently block pre-mRNA splicing. Zebrafish NRP-1a/ NRP-1b MOs, p53 MO, and MDm2 MO were designed as described. We purchased Morpholino antisense oligonucleotides from Gene Tools, Inc, (Philomath, OR). The sequences are available upon request. The injection solution was prepared as described. Morpholino solutions with 3.0 and 6.0 ng MOs were injected into zebrafish embryos at the 1-4 cell stage, respectively. Embryos were examined for EC apoptosis and vascular defects at different time points. Quantitative RT-PCR (Ambion, Austin, TX) was used to monitor the splicing events. All experiments were repeated at least three times mRNA microinjection The human full-length NRP-1 and a construct with the last three amino acids deleted (S-E-A), the human full-length GIPC and its PDZ domain deletion mutant were each cloned into the pCS2 vector respectively. The transcribed mRNAs were injected together with the NRP-1 morpholino or alone into 1-4 cell staged embryos. By using a splicing donor morpholino, it is possible to reduce the endogenous expression of the gene, without disturbing the expression of ectopic mRNAs. All experiments were repeated at least three times.
Antibody staining
4% paraformaldehyde-fixed zebrafish embryos were permeabilized with Proteinase K, then incubated with anti-GFP antibody at 4uC overnight. After washing with PBST, the zebrafish embryos were used for TUNEL staining.
Detection of apoptotic EC in zebrafish
To examine the functions of NRP-1 in zebrafish EC apoptosis, morpholinos were injected into a Fli-GFP transgenic fish line, where all of the EC are labeled with GFP. Then, zebrafish embryos were fixed at different time points after injection (24, 30, 36, 42 and 48 hpf) with a freshly prepared fixation solution (4% PFA) overnight at 4uC. Antibody-stain with anti-GFP antibody was performed to increase the fluorescence green signal in vessel that has been reduced due to fluorescence diffusion by fixation. Apoptosis was characterized with similar steps as described above using the In Situ Cell Death Detection Kit, TMR red (Roche). Antibody-stained embryos were incubated with the reaction mixture directly. Imaging of blood vessels in TG(fli1:egfp)y1 embryos was performed using a Zeiss confocal laser microscope. Transmitted light images were obtained with a Leica MZ FLIII microscope equipped with a Nikon COOLPIX8700 digital camera. The identity of the apoptotic cells was confirmed by using the red channel where EC would be labeled by Fli-GFP signal. | 2014-10-01T00:00:00.000Z | 2007-11-14T00:00:00.000 | {
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2236504 | pes2o/s2orc | v3-fos-license | Clean and fast cross-coupling of aryl halides in one-pot
Summary Unsymmetrically coupled biaryls are synthesized in high yield starting from different aryl bromides and bis(pinacolato)diboron by carrying out the Miyaura borylation reaction followed by the Suzuki–Miyaura reaction in the same reaction pot over 1–2 mol % SiliaCat DPP-Pd. The SiliaCat DPP-Pd catalyst is air-stable and the method does not require the use of inert conditions. The use of non-toxic isopropanol or 2-butanol as reaction solvent further adds to the environmental benefits of this new green synthetic methodology.
Findings
Affording valued biaryls and heterobiaryls, namely ubiquitous chemical moieties in pharmaceuticals, natural products, photoactive species and many other functional molecules, the Suzuki-Miyaura cross-coupling reaction is widely employed by the fine chemicals and pharmaceutical industries [1]. The reaction involves a boron-containing nucleophile (a variety of aryl and heteroaryl boronic acids, esters, Ar-BBN, trifluoroborate and other boron species) and vinyl-or aryl halides as the electrophilic species. The use of the Suzuki-Miyaura reaction became routine both in industry and in research laboratories following Miyaura's discovery from 1995. He demonstrated a direct route to boronic esters [2], namely the cross-coupling of bis(pinacolato)diboron (B 2 Pin 2 ) with aryl or vinyl halides catalyzed by PdCl 2 (dppf) ([1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride) in the presence of excess KOAc at 80 °C in dioxane or in DMSO.
In contrast to a number of Pd catalysts that are air sensitive, PdCl 2 (dppf) is air stable. This made the method more versatile and provided a long awaited easy way to synthesize a broad range of boronic esters that are conveniently used in place of boronic acids in the Suzuki-Miyaura reaction when reactive Scheme 1: Heterogeneously catalyzed synthesis of boronic acid pinacol esters over SiliaCat DPP-Pd.
Scheme 2:
Heterogeneously catalyzed cross-coupling of two aryl bromides by consecutive borylation and Suzuki-Miyaura reactions, both of which are mediated by SiliaCat DPP-Pd. functional groups are present in the electrophilic aryl halides [3,4].
Several homogeneous palladium catalysts of enhanced versatility for the cross-coupling of pinacolborane with aryl halides have since been introduced, for example by Buchwald and co-workers [5,6]. Of direct relevance to this research report, Giroux and co-workers reported the homogeneously palladiumcatalyzed one-pot borylation/Suzuki coupling reactions already in 1997 [7].
Yet, it remains desirable to efficiently heterogenize palladium catalytic species used in cross-coupling reactions with the aim to prevent product contamination with palladium, reuse the expensive catalyst and streamline the whole synthetic process [8]. Furthermore, the replacement of DMSO or dioxane with a less toxic solvents is of obvious environmental and health relevance.
Sol-gel-entrapped catalysts are rapidly emerging as a promising tool for synthetic organic chemistry [9]. In this research context, we have recently described the heterogeneously catalyzed synthesis of boronic acid pinacol esters using a wide range of aryl chlorides, bromides and iodides and bis(pinacolato)diboron as borylating agent over the sol-gel-entrapped SiliaCat DPP-Pd catalyst (Scheme 1) [10]: SiliaCat DPP-Pd is a commercially available catalyst [11] made of an organosilica matrix functionalized with diphenylphosphine ligands bound to Pd 2+ ( Figure 1). The catalyst, which is highly active in C-C coupling reactions [12], has typical 0.2-0.3 mmol/g palladium loading, a high surface area and large accessible mesoporosity (300-650 m 2 /g, depending on the applied parameters of the sol-gel synthesis).
The leach-proof and truly heterogeneous nature of SiliaCat DPP-Pd in the synthesis of boronic acid pinacol esters has been recently shown elsewhere [10], wherein the issue of catalyst recycling is also addressed in detail. Now we report a new method that establishes a direct route, in one-pot, to unsymmetrically coupled biaryls starting from two different aryl bromides and bis(pinacolato)diboron without the need to isolate the intermediate boronic acid pinacol ester. The overall process is described by Scheme 2. The SiliaCat DPP-Pd catalyst mediates the borylation and the subsequent Suzuki-Miyaura reaction in an elegant one-pot sequential synthesis. Hence, an aryl bromide is first converted into the boronic acid pinacol ester (step 1 in Scheme 2). A different aryl bromide is then added along with aqueous base (step 2).
The reaction is carried out in iPrOH or in 2-BuOH. No work-up is performed after the borylation in the first step, nor is any catalyst added prior to conducting the second step of the sequence, the Suzuki-Miyaura reaction. Results in Table 1 show that, following aqueous work-up, different unsymmetrically coupled compounds are obtained in good to excellent yields by coupling numerous different aryl bromides with different aryl halides, including heteroatom-containing aryls.
In detail, entries 1 and 2 in Table 1 show that 3-bromophenol and 4-bromoanisole are entirely converted into the boronic acid pinacol ester in 2-3 h over 2 mol % SiliaCat DPP-Pd. The same aryl halides are coupled with 4-bromotoluene or 2-bromotoluene to form the Suzuki-Miyaura coupling product in, respectively, 85% and 92% yield in 3 h. In conclusion, unsymmetrically coupled biaryls can be synthesized in high yield starting from different aryl bromides and bis(pinacolato)diboron by carrying out the Miyaura borylation reaction followed by the Suzuki-Miyaura reaction over 1-2 mol % catalytic amount of the sol-gel entrapped catalyst SiliaCat DPP-Pd in the same reaction pot. There is no need to isolate the intermediate boronic acid pinacol ester, while the air stable sol-gel entrapped palladium catalyst does not require the use of inert conditions.
Finally, the use of isopropanol or 2-butanol as reaction solvents further points out the environmental benefits of the method. As the fine chemicals and pharmaceutical industries are eventually adopting green chemistry synthetic methodologies [13], this method provides both industries with a clean route to valued compounds that are widely used in many industrial sectors. | 2017-06-16T20:30:50.306Z | 2014-04-22T00:00:00.000 | {
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245133788 | pes2o/s2orc | v3-fos-license | The Impact of Lifetime Work and Non-work Physical Activity on Physical Fitness Among White – and Blue – Collar Retirees: A Cross-Sectional Study
Objective: The literature offers significant amount of data on the effects of occupational activity on health, with a distinct link between retirement and health among the most frequently tackled topics. Studies on the relationship between past occupational activity and physical fitness among older retirees remain scarce. The aim of the study was to assess the effects of physical activity on physical fitness in white- and blue-collar retirees. Methods: A total of 200 participants (aged ≥60) were included in the study. Lifetime physical activity was assessed using the Lifetime Physical Activity Questionnaire. Mean MET/week/year values of total Physical Activity and for each domain separately (occupational, sports, household) were calculated. Participants were stratified to blue- or white- collar group. Physical performance, hand-grip strength (HGS) and pulmonary function were assessed. Results: Mean total MET/week/year values for the blue- and the white-collar workers were 140.48 ± 55.13 and 100.75 ± 35.98, respectively. No statistically significant differences in physical performance scores were found between the white- and blue- collar groups. Adjustment for age, sex weight and height revealed a statistically significant association between work-related PA FEV*1 in the blue-collar group. White – collar workers presented higher odds ratio for membership in highest quartile in regard to short physical performance battery test score. Conclusion: Only minimal association of type of occupation on physical fitness were found despite statistically significant differences between mean intensity and duration of sports- and work-related lifetime physical activity. These findings may indicate that the type of past work is not an independent factor influencing the state of a person in old age. Large-scale investigations with physically fit and unfit participants, are necessary.
BACKGROUND
In the European Union (EU), the number of individuals reporting work-related health problems has been steadily decreasing, from <13% in 2007 to <8% in 2013, for the entire workforce. Regardless, the numbers continue to be high in some EU countries, e.g., Sweden and Finland (20%) (1). The differences stem not only from workload differences for various positions, but also health systems, prevention, and reporting as well as treating work-related health problems (1). An average person devotes up to one-third of their life to work (2,3). The working environment, physical and emotional strain, or forced positions are only a few of the factors which affect the health of the workers (4)(5)(6). The indirect effect of the occupational activity on health, mediated by the impact on the socioeconomic status, has also been reported in the literature (7).
Both, blue-and white-collar jobs can contribute to adverse health outcomes. White-collar workers typically perform sedentary jobs which, due to lack of movement and improper body positioning, may contribute to developing back and neck pain (8). White-collar jobs are frequently associated with markedly elevated stress levels, emotional strain (9), and risk of obesity (10). These factors increase the risk for developing chronic diseases (9). In contrast, blue-collar jobs tend to be more physically demanding and are often correlated with stress, lack of satisfaction, and low socioeconomic status. Blue-collar work has been demonstrated to be a risk factor for developing chronic diseases, heart failure and depression (9). Blue-collar workers are at high-risk for respiratory hazards due to their working conditions and the presence of air pollutants, e.g., dust related to construction or farming jobs (11).
The literature offers an accumulating body of data on the effects of occupational activity on health, with a distinct association between retirement and health among the most frequently tackled topics (12). Physical fitness can be considered as one of the health indicator and is defined, according to The American College of Sports Medicine, as a combination of features and attributes that allow to perform physical activity (13). Studies on the effect of past occupational activity and physical fitness among older retirees remain scarce (14,15). The analysis of such links continues to present a challenge as health in older age is the sum of a multitude of factors accumulating throughout the course of one's life. Physical activity (PA), both related and unrelated to occupational activity, is an example of one such crucial factor.
According to the World Health Organization (WHO) (16), physical activity (for older people: 150 min./week of moderateintensity or 75 min./week of vigorous-intensity aerobic) is the key element of "healthy ageing, " as it lowers the risk for developing severe illness and physical disability (17,18).
PA measuring methods frequently pose a challenge. Self-reported physical activity questionnaires such as IPAQ (International Physical Activity Questionnaire) (19), although uncomplicated, fast and easily applicable, have received a fair share of criticism due to the respondent-related risk of under-or over-estimation. (14,20) Additionally, they will typically cover the current time or the last few weeks or months before the study. More objective methods such as the use of accelerometers are infinitely more demanding in terms of the cost, not to mention time. Also, the more objective methods do not allow for a retrospective analysis, which is necessary to achieve precise evaluation of the effects of lifetime work-related PA on physical fitness in retirees. Furthermore, it is necessary to take into account other types of PA to avoid selection bias.
Searching for a relationship between the type of work performed in the past and the fitness of retired people may enable the implementation of appropriate activities related to health promotion in groups particularly exposed to a decline in fitness level. The aim of the study was to assess the effects of physical activity on physical fitness in white-and blue-collar retirees.
Participants
The participants were recruited from the general population of the Wielkopolska Region using radio, Internet, and print (leaflets/flyers) media and in cooperation with various organizations for seniors. A total of 200 participants (out of 384 who volunteered to participate) were included in the study (Figure 1). The inclusion criteria were as follows: age ≥60 years and cessation of the occupational activity. The volunteers were interviewed to identify possible exclusion criteria such as diseases and injuries which might negatively affect the upper limb performance and/or respiratory tract function (e. g., stroke, asthma, Chronic Obstructive Pulmonary disease (COPD), history of thoracic surgery, upper limb fracture, brachial plexus injury, brachialgia, history of pulmonary surgery, smoking and impaired cognitive function).
Cognitive functioning was assessed with the Mini-Cog test (21). Mini-Cog consists of two components: short-term recall of three words to test short-term memory and the clock drawing test, with 5 points as the maximum score. The score of <3 points may be indicative of cognitive impairment, so it constituted one of the exclusion criteria in the present study. Permission to use the Mini-Cog test was obtained.
The study was conducted between March 2019 and October 2019. The requirements of the Declaration of Helsinki were followed.
Measurements
Lifetime physical activity was assessed using the Lifetime Physical Activity Questionnaire (LTPAQ) (22). Lifetime physical activity in the present study was defined as average PA calculated from lifetime physical activity patterns reported in LTPAQ. The questionnaire consists of 4 domains which measure occupational activity (i.e., work), sports, household and transportation-related physical activity. In the present study, the transportation domain was not analyzed separately but some information collected from that domain were included in the presentation of Sports data. Before the study, all participants received a recall calendar, which facilitated their recall of various kinds of lifetime physical activity, according to the guidelines specified in the LTPAQ COMPREHENSIVE USERS' GUIDE (23). All types of occupational activity or volunteering performed for the minimum of 8 hours/week, for 4 months (min. 128 hours/year or 2.5 hours/week for a year) were treated as occupational activities. Each respondent submitted data on the age at which s/he started and finished working at each position, including the number of months/year, days/week, hours/day. Next, the intensity of physical exertion was described on a scale from 1 to 4 ( Table 1). If the intensity of different tasks varied significantly, the total working time was divided and different intensities were assigned to individual tasks, as per respondent instruction.
Household Activities included housework, yard work, home repair and childcare, which took the minimum of 7 hours/week for 4 months (or 112 hours total/year or 2.15 hours/week per year). Sedentary household activities such as sewing were not registered (23). The level of intensity of physical exertion for Household Activities was described on a scale from 2 to 4 ( Table 1).
As far as Exercise and Sports Activities were concerned, sports activities which were performed for at least 32 hours total/year (or 40 minutes/week per year, or 2 hours/week for 4 months) were included in that category. Next, the Metabolic Equivalent of Task (MET) was assigned to each activity, in accordance with the 2011 Physical Activity Compendium. (24) If a respondent walked/rollerbladed/biked/ran to work, that type of physical activity was also included in the Exercise and Sports Activities.
In order to determine the Total PA MET value, the combined number of the hours spent performing a particular activity was calculated using the following an original formula created for the purpose of this study, based on the LIFETIME TOTAL PHYSICAL ACTIVITY QUESTIONNAIRE COMPREHENSIVE USERS' GUIDE (23) and other publications: (25,26).
YearsxMonthsxDaysxHoursx 4 = TotalLiftimeActivityTime
The obtained number of the hours was divided by the number of the years of life and the result was again divided by 48 to determine the duration of lifetime activity/week. That result was then multiplied by the MET value of a given task to achieve the lifetime MET/week/year value for that task. The following formula was applied: The obtained values for specific activities from the different domains were summed up to determine the following: (27) was used to stratify the participants into the white-or blue-collar groups, depending on the type of occupational activity reported during the LTPAQ interview. Similar to the methodology proposed by Fukushima et al. (27) the blue-collar group included participants performing physical tasks who had worked as mechanics, farmers, builders, production workers and electricians, whereas the whitecollar group comprised office workers, accountants, physicians, managers, engineers, and academics. Participants who could not be classified into the white-or blue-collar group due to the mixed nature of their past work were removed from the analysis.
There is no consensus over physical performance definition, however for the purpose of this study definition proposed by other authors was adopted. According to Beaudart et al. (28) physical performance can be defined as "an objectively measured whole body function related with mobility" which involved not only muscle function measures but also other organs and systems (including cardiorespiratory system). Therefore, in the present study hand-grip strength (HGS), spirometry and Short Physical Performance Battery test (SPPB) results constituted the indicators of the physical performance.
Physical performance, was assessed using the SPPB (29). The test consists of three tasks: rising from a chair and sitting down with five repetitions, a hierarchical test of balance, and a short walk at the usual pace. Each SPPB component is scored from 0 to 4, with 0 representing the inability to perform the test and 4 representing the highest category of performance. The highest possible score is 12, indicating the best body function (29,30).
HGS of the dominant hand was tested using the JAMAR hydraulic dynamometer. The measurements were performed in accordance with the recommendations of the American Society of Hand Therapists (ASHT) (31). During the measurements, the subject was sitting in a chair without a backrest and armrests, with feet resting on the floor, parallel to each other, hip and knee joints set at right angles, arms adducted and touching the torso, elbow joint bent to 90 • , forearm in a neutral position, wrist straightened in the range of 0 • to 30 • . The subject performed a maximum handgrip and held it for 6 s. The dominant hand was tested three times, with a 1-min break between each measurement. The best result was used for further analysis. Pulmonary function was assessed using spirometry. Spirometry was performed using non-turbine Piston PDD 301 spirometer, in accordance with the American Thoracic Society and European Respiratory Society recommendations (32). Spirometer software indicated whether the measurement was correct and the test was valid, or if repeat test was required. Single-use disposable filter mouthpieces were used. FVC (Forced Vital Capacity), FEV * 1 (Forced Expiratory Volume in the 1st second) and IVC (Inspiratory Vital Capacity) parameters were tested. During the tests, the participants were vigorously encouraged by the technician to give it their best effort while inhaling and exhaling. The test was repeated up to 8 times (with 1-min break between the manoeuvers) until three correct measurements were achieved. Both, the technician who performed the test and the software indicated whether the measurement was correct, and the test was valid, or if repeat test was required. Mean score was used for further analysis. This study is part of a project "Type of work performed in the past and physical fitness at retirement."
Statistical Analysis
The statistical analysis was conducted using Statistica 13 (StatSoft, Polska). Mean, median and standard deviation were used to describe group characteristics. In the absence of normal distribution of the investigated variables, U-Mann-Whitney test was used to compare the white-and blue-collar groups. HGS and spirometry results were adjusted for age, sex, weight and height. In order to investigate the effects of PA on HGS and spirometry results for every variable, statistical analysis was conducted using the General Linear Regression (GLR): GLR -without control bias, Model 1 -controlled for age and sex, and Model 2controlled for age, sex, weight and height. The minimal study sample of 96 persons was calculated with assumption of z = 1.96 and error = 10%. The p-value of <0.05 was considered as statistically significant.
RESULTS
A total of 200 people (127 blue-and 73 white -collar retirees) were included in the study. Out of them, 107 were female. Mean patient age was 70.11 ± 6.55 years (69.93 ± 6.49 and 71.36 ± 6.51 for the blue-and white-collar groups, respectively). No statistically significant differences were found between blue-and white-collar workers as far as retirement duration was concerned (p=0. 18 Table 2. Statistically significant differences were found for age, MET/week/year for Work and Sports, and Total as well as h/week/year for Work and Sports. The odds ratio for the membership in the 1st and 4th quartile of measured variables in association with the type of occupation were presented in Table 3. There were no statistically significant associations between type of occupation and measured variables except SPPB. Table 4 presents the effects of PA on the investigated parameters. Before adjustments, statistically significant associations were found between Total MET/week/year and FEV and FEV * 1. Model 1 also revealed statistical significance between these parameters, whereas Model 2 only between MET/week/year and FEV * 1. In the white-collar group, statistically significant associations between PA and SPPB values were found both, before and after adjustments. Work-related physical activity proved to be correlated with HGS, IVC, FEV, and FEV * 1 in the blue-collar group and with HGS and IVC in the white-collar group. Model 1 revealed a statistically significant association between work-related PA and FEV as well as FEV * 1, while Model 2 only in case of FEV * 1 in the blue-collar group. In the white-collar group, neither Model 1 nor Model 2 found a statistically significant association ( Table 4).
Total PA time demonstrated an association only with FEV and FEV * 1 in the blue-collar group in Model 1 and 2 ( Table 5). The number of lifetime work-related hours of PA showed a statistically significant association with all investigated parameters in the blue-collar group before the adjustments. After the adjustments, the effect was detected only for FEV and FEV * 1 both, in Model 1 (age and sex) and Model 2 (Model 1 + height and weight). An association between the number of Total work-related PA hours with IVC pre-adjustment and HGS pre-adjustments as well as in Model 1 and 2 was found in the white-collar group ( Table 5).
DISCUSSION
In the present study, we evaluated the effects of work-related and work-unrelated physical activity on physical performance among retirees, who were either white-collar or blue-collar workers during their professional activity years. HGS, spirometry and SPPB results constituted the indicators of the physical performance. No statistically significant differences in physical performance were found between the white-and blue-collar groups. The results of this cross-sectional study demonstrated an association between lifetime work-related and work-unrelated physical activity and FEV * 1 among blue-collar retired workers. The relationship between work-related PA was detected both, before and after adjusting for age, sex, height and weight. Notably, even though the correlation after the adjustments was weak (β < 0.2), a similar correlation was not observed in the white-collar group.
Hunter et al., (33) demonstrated a positive role of highintensity physical activity in muscle function. Taylor et al. (34) also reported high-intensity physical activity to be more beneficial for health than low-intensity PA. That association may explain why in our study higher physical activity (expressed in MET as well as in hours) seemed to be a factor which improved the FEV * 1 score in the blue-collar group. Rawashdeh and Alnawaiseh (35), reported a statistically significantly higher FEV * 1 in their study group of 72 males undergoing a three-week long high-intensity aerobic training.
These authors demonstrated that physical activity improved the airflow in the respiratory tract, which they attributed to the fact that during the training a larger volume of air was introduced into the airways, resulting in a widening of the respiratory tract. Their findings might help and explain our results. In our study, in the blue-collar group, higher work-related MET/week/year resulted in higher FEV * 1, which indicates higher-intensity PA. Similarly, higher number of work-related h/week/year, i.e., more hours spent at work, contributed to higher FEV * 1. Bertoni et al. (15) showed that blue-collar workers have a tendency to limit their non-work related PA and are more reluctant to engage in sports activities as compared to the white-collar workers, which is consistent with our observations. Statistically significant association between type of occupation and membership in SPPB score quartiles shows, that being retired white -collar worker doubles the odds for membership in highest quartile regarding SPPB score and lowering odds for membership in lowest quartile (OR = 0.36). However, the clinical importance of association presented in studied group could be reduced because participants SPPB scores were relatively high and most participants were considered as independent (SPPB score ≥ 10). For this regard more studies, involving less independent participants should be conducted in future. The difference between blue -and white collar group can also be explained in the ground of abovementioned patterns of participation in sport activities (15). In the white-collar group, total exertion score (MET week/year) correlated with the SPPB results, indicating that higher PA corresponded to higher SPPB scores, both before and after the adjustments (Model 1 and 2).In the blue-collar group, statistically significant relationships were found between all of the investigated parameters and the number of hours spent at work before the adjustments. However, the relationship was no longer statistically significant after adjusting for age and sex. The effect of age and sex on the physical function decline as well as the negative impact of age on lung function have been described by different authors (36,37). Navarro et al. (38) reported that lung function declines 1% per year after the age of 25 years. Various mechanisms of lung function decline have been proposed, especially disturbed respiratory mechanics caused by altered thoracic shape resulting from a progressing spinal kyphosis, impaired diaphragm function, restricted chest and pulmonary alveoli expansion due to loss of elasticity (39,40). Importantly, a statistically significant relationship between the number of hours spent at work and HGS in the white-collar group became negative after the adjustments, indicating that longer time spent at work might have a detrimental effect on the health of white-collar workers. Saidj et al. (41) in their study of 2,544 working adults (aged 18-69), evaluated the relationship between sitting position at work and during leisure time and found a negative correlation between these variables and HGS in subjects aged <50 years. However, no such relationship between sitting time at work and HGS was observed, which led them to the conclusion that sedentary behaviors during leisure time are more harmful than at work. The differences between their results and our findings might stem from the fact that we did not evaluate sitting time but only MET or the number of hours dedicated to different types of work.
Duong et al. (42) in their study conducted in a group of 126 359 adults from 17 countries demonstrated a relationship between mortality and FEV * 1 decline. Impaired FEV * 1 contributed to one-fourth of the deaths and one-sixth of cardiovascular incidents. According to these authors, impaired FEV * 1 contributed to the cardiovascular incidents significantly more than other factors such as hypertension, smoking, or previous cardiovascular diseases. Some authors also mentioned that pulmonary function decline is associated with higher risk for renal failure (43), diabetes (44), and neurocognitive diseases (45). In light of the abovementioned and our findings, it seems probable to conclude that higher work-related PA results in better health during the retirement among blue-collar workers.
Our study is not without limitations. First of all, the "healthy user bias" might have occurred due to the restrictive selection criteria of the retirees (exclusion criteria). Individuals with respiratory system diseases were excluded to avoid bias in spirometric measurements. In consequence, the influence of occupation type on developing such diseases cannot be assessed. Next, as the group of retirees participating in the study was relatively young, it is possible that the results of this study may not reflect the condition of oldest-old retired people. Another limitation is that, lifetime physical activity was evaluated in a subjective manner. Despite having made every effort to help the subjects recall as many details as possible and having conducted a detailed interview, the activities may have been inaccurately reported by the respondents. Regardless, due to the nature of this cross -sectional study assessing past physical activity, it was not possible to apply more objective tools to evaluate the activity or physical exertion of the subjects. Smoking was not analyzed in two occupational groups in this study but was one of the exclusion criteria., the data on the distribution of smokers among occupational groups were not available. Other studies (46)(47)(48) indicates that blue -collar workers presented higher smoking prevalence than white -collar workers. As indicated by de Castro et al. (46) blue -collar work may increase the risk of being a smoker up to 2.5 times, so it can also be assumed that it may increase the risk of smoking-related respiratory diseases, which may reduce the physical fitness of the older adults. However, due to lack of the data, in present study it is not possible to assess the abovementioned relationships of smoking.
In conclusion, in our study, we found no statistically significant relationship between the retired white-and bluecollar workers in terms of physical performance scores. No differences in the HGS, SPPB, FEV, FEV * 1, and IVC scores were found, despite statistically significant differences between mean intensity (MET/week/year) and duration (h/week/year) of sports-and work-related lifetime physical activity. Only association between type of occupation and membership in SPPB score quartiles in favor of white -collar workers was found. The results of present study are important for healthcare practitioners and workers, and are indicating that both blue-and white-collar workers should maintain high non-work related PA, despite different occupational PA levels. These findings may indicate that the type of past work is not an independent factor influencing the state of a person in old age. Blue-collar workers who had worked with higher intensity presented higher FEV * 1 scores after retiring, while white-collar workers who had spent less time at work presented higher HGS after retiring.
As the number of the literature reports about the effects of occupational activity on the health of older people after retiring is limited, it seems prudent to conduct further, large-scale investigations in large sample-size groups, with physically fit and unfit subjects, not just healthy individuals.
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 Bioethics Committee at Poznan University of Medical Sciences. The patients/participants provided their written informed consent to participate in this study.
AUTHOR CONTRIBUTIONS
TT, EZ, and MP developed a study design. TT and AP performed measurements and data collection. AP performed statistical analysis of the data. TT was a major contributor in writing the manuscript. All authors performed interpretation of the data, read and approved the final manuscript. | 2021-12-15T14:22:35.137Z | 2021-12-15T00:00:00.000 | {
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246997786 | pes2o/s2orc | v3-fos-license | Case Report: Superior Mesenteric Artery Syndrome in an Adolescent With Cannabinoid Hyperemesis
Background Superior mesenteric artery syndrome (SMAS) occurs when the third portion of the duodenum is compressed between the superior mesenteric artery (SMA) and the aorta, causing duodenal obstruction. This condition most commonly arises from marked weight loss that reduces the size of the fat pad between these vessels, causing greater acuity of angulation. We present an unusual case of SMAS occurring in an adolescent due to precipitous weight loss resulting from cannabinoid hyperemesis syndrome (CHS). Case Presentation A 17-year-old adolescent presented emergently with voluminous bilious emesis. She endorsed a history of recent weight loss and a longstanding history of chronic heavy cannabis use associated with recurrent nausea and vomiting. Her chronic symptoms satisfied the Rome IV criteria for cannabinoid hyperemesis syndrome, but her acute vomiting symptoms were more extreme. Evaluation was significant for mild abdominal tenderness and fullness of the epigastrium. Contrast abdominal CT demonstrated moderate gastric and proximal duodenal distention with tapering of the lumen between the SMA and the aorta, consistent with SMAS. Conclusions To our knowledge, this is the first reported case of SMAS occurring as the result of CHS. Clinicians should be aware of this possible juxtaposition, when a patient with a history of chronic excessive cannabis use, stereotypical vomiting resembling cyclic vomiting syndrome, and considerable rapid weight loss presents with a sudden exacerbation of symptoms, even when a normal BMI is maintained.
Background: Superior mesenteric artery syndrome (SMAS) occurs when the third portion of the duodenum is compressed between the superior mesenteric artery (SMA) and the aorta, causing duodenal obstruction. This condition most commonly arises from marked weight loss that reduces the size of the fat pad between these vessels, causing greater acuity of angulation. We present an unusual case of SMAS occurring in an adolescent due to precipitous weight loss resulting from cannabinoid hyperemesis syndrome (CHS).
Case Presentation: A 17-year-old adolescent presented emergently with voluminous bilious emesis. She endorsed a history of recent weight loss and a longstanding history of chronic heavy cannabis use associated with recurrent nausea and vomiting. Her chronic symptoms satisfied the Rome IV criteria for cannabinoid hyperemesis syndrome, but her acute vomiting symptoms were more extreme. Evaluation was significant for mild abdominal tenderness and fullness of the epigastrium. Contrast abdominal CT demonstrated moderate gastric and proximal duodenal distention with tapering of the lumen between the SMA and the aorta, consistent with SMAS.
Conclusions: To our knowledge, this is the first reported case of SMAS occurring as the result of CHS. Clinicians should be aware of this possible juxtaposition, when a patient with a history of chronic excessive cannabis use, stereotypical vomiting resembling cyclic vomiting syndrome, and considerable rapid weight loss presents with a sudden exacerbation of symptoms, even when a normal BMI is maintained.
Keywords: superior mesenteric artery syndrome, cannabinoid hyperemesis syndrome, Rome IV criteria, THC, case report BACKGROUND Superior mesenteric artery syndrome (SMAS) is a rare condition that occurs when the third portion of the duodenum is compressed between the superior mesenteric artery (SMA) and the aorta due to a narrowing of the angle between these vascular structures. The most common predisposing factor for the development of SMAS is significant weight loss leading to a diminution in retroperitoneal adipose tissue, including the fat pad between the SMA and the aorta, resulting in worsening acuity of angulation between these vessels and subsequent complete or partial obstruction of the duodenum. SMAS has been seen in association with conditions such as malignancy, malabsorption, trauma and burns, spinal cord injury, prolonged bedrest, and anorexia nervosa. Symptoms include postprandial epigastric pain, bilious emesis, and nausea (1, 2).
CASE PRESENTATION
A 17-year-old female presented to our emergency department (ED) with acute voluminous bilious emesis. Prior to this acute presentation, she had endorsed over 6 months of chronic nausea, especially upon awakening, progressing to protracted non-bilious emesis every 1-2 weeks. Symptoms were frequently relieved by prolonged hot baths. Her recurrent vomiting exacerbations were generally followed by 1 to 2 weeks of symptom reduction and self-imposed limitations in oral intake. Four months prior to her acute presentation, she had norovirus infection with mesenteric adenitis and mid-ileal small bowel-to-small bowel intussusception requiring laparoscopic reduction without resection. Evaluation of her chronic nausea and vomiting 3 months prior to admission revealed normal inflammatory, liver, and kidney markers, negative ß-hCG, and no evidence of sexually transmitted disease. An upper endoscopy performed at that time showed no anatomic abnormalities and was normal apart from a small hyperplastic antral polyp and microscopic noneosinophilic esophagitis (Figure 1).
Her past medical history otherwise included hepatomegaly [seen on computed tomography (CT) scan several years prior to presentation, normal liver enzymes] and intermittent hyperglycemia, glucosuria, and ketonuria with a normal HgbA1c and no other clear clinical or laboratory evidence of diabetes. Of note, her prior CT scan noted no abnormalities in the proximal small bowel. Her social history was significant for over 5 years of heavy and accelerating cannabis use. At the time of presentation, she endorsed smoking 5-6 marijuana cigarettes daily for over 1 year, a habit she attributed to life stressors both at home and at school.
At presentation, the patient reported a rapid weight loss of more than 9 Kg. She denied constipation, diarrhea, or intentional Abbreviations: SMA, superior mesenteric artery; SMAS, superior mesenteric artery syndrome; CHS, cannabinoid hyperemesis syndrome; CT, computerized tomography; BMI, body mass index; THC, tetrahydrocannabinol; AOM, aortomesenteric; NJ, nasojejunal. vomiting or weight loss. She was noted to have a normal body mass index (BMI) of 23.46 Kg/m 2 , despite her weight loss. Physical examination was remarkable for diffuse abdominal tenderness and fullness to palpation, worst in the epigastrium. ß-hCG was again negative and urine toxicology screen was positive for tetrahydrocannabinol (THC). Abdominal x-ray was unremarkable. CT scan with contrast of the abdomen and pelvis revealed moderate gastric and proximal duodenal distention, with tapering of the duodenal lumen at the level of the SMA. The aortomesenteric (AOM) angle was 10 degrees and AOM distance was 6 mm. Taken together, these findings were suggestive of SMAS (Figures 2, 3) (3). There was no evidence of biliary or pancreatic pathology, adenopathy, or malignancy. A nasogastric tube was inserted for gastric and duodenal decompression and a nasojejunal (NJ) tube was placed to bypass the obstruction and provide nutrition and hydration.
While the patient's acute presentation and CT findings were attributable to SMAS, this did not explain her chronic antecedent symptoms of nausea, episodic vomiting, and weight loss. The patient's heavy chronic cannabis use, the longstanding nature of her complaints (>6 months), her otherwise reassuring evaluation, and her stereotypical pattern of episodic vomiting, satisfied the Rome IV criteria for cannabinoid hyperemesis syndrome (CHS), a well-defined disorder, characterized by prolonged cannabis use, cyclical nausea and vomiting with reduction or even complete cessation of symptoms between events, abdominal pain, and often compulsive hot water bathing for relief of symptoms (4). It was postulated that the weight loss she sustained as the result of frequent protracted CHS resulted in a diminution of the fat pad between the SMA and the aorta and eventual obstruction of the third portion of the duodenum leading to her acute presentation with voluminous bilious emesis. Acute symptoms improved with gastroduodenal decompression for several days. She was monitored for refeeding syndrome as NJ feedings were advanced to goal. The patient and her guardian demonstrated good understanding of her acute and chronic conditions and the goals for both her acute and long-term management. She did not follow-up with the Gastroenterology service but sought regular care with her primary care provider and was able to tolerate oral meals and have the NJ removed within 8 weeks of hospitalization. During this period, she was reportedly cannabis-free and had no further episodes of vomiting. Three months after admission, the patient presented again to the ED with intractable nausea and vomiting. She endorsed progressively escalating use of cannabis that approached the quantity and frequency at her prior admission. The resolution of symptoms during prolonged cessation of cannabis and the prompt resumption of symptoms with recurrent use further supported the diagnosis of CHS.
DISCUSSION AND CONCLUSIONS
The patient's chronic illness satisfied the Rome IV criteria for CHS with stereotypical episodic vomiting resembling cyclic vomiting syndrome for >6 months, excessive cannabis use, and abatement of symptoms after prolonged cessation of cannabis use (4,5). Her reliance on hot baths for relief of nausea and vomiting was supportive of CHS. While not all individuals with CHS experience significant weight loss, this patient's rapid loss following the progression of CHS precipitated the development of SMAS. While SMAS is most frequently seen in individuals with a low BMI, this case illustrates how an abnormally low BMI may not be necessary. In fact, a recent report describes the case of an adolescent male who, despite stable weight and BMI, acquired SMAS during a growth spurt whereby a gain of lean body mass occurred in tandem with a demonstrable loss of adipose tissue, including the fat pad between his SMA and aorta (6, 7).
The differential diagnosis for our patient's acute illness, in addition to SMA syndrome, included: small bowel obstruction, gastric or duodenal dysmotility (e.g., gastroparesis, ileus), collagen vascular disease (e.g., scleroderma), chronic idiopathic pseudo-obstruction, and peptic ulcer disease. The decreased AOM angle of less than 22 to 28 degrees and AOM distance between 2 and 8 mm in association with the patient's symptoms, however, were diagnostic of SMA syndrome (8-10). The initial treatment for SMAS is conservative, with relief of the patient's obstructive symptoms through the passage of a nasogastric tube to decompress the stomach and proximal duodenum, followed by nutritional support with nasojejunal feeding distal to the site of obstruction, as in the present case (11). For those patients who can tolerate oral nutrition, frequent small feedings in the left lateral decubitus position are encouraged. Total parenteral nutrition may be required when enteral feeding of any sort is not tolerated (12). The goal of conservative therapy is to promote sufficient weight gain to increase the volume of the mesenteric fat pad and thus the AOM angle, alleviating bowel compression. When conservative measures to treat SMAS have failed, surgery may be indicated. Strong's procedure is the least invasive, as it does not involve bowel anastomosis. This surgical approach involves mobilizing the duodenum by dividing the ligament of Treitz and positioning the duodenum to the right of the SMA, allowing it to drop away from the aorta (13). Unfortunately, the procedure has a failure rate of up to 25 percent (13). Strong's procedure has been largely replaced by performing a duodenojejunostomy by dividing the fourth portion of the duodenum and creating a side to side anastomosis between the third portion of the duodenum and the end portion of the jejunum (14). A duodenojejunostomy restores bowel continuity and obviates the risk of blind loop syndrome. Both surgical procedures can now be performed laparoscopically (13, 14).
To our knowledge, this is the first reported case of SMAS occurring as the result of CHS. The patient's presentation with voluminous bilious vomiting in the setting of heavy marijuana use could have simply been attributed to exacerbation of her chronic CHS symptom pattern, leading to less comprehensive evaluation which might have missed or delayed the diagnosis of concurrent SMAS. Clinicians should be aware of the potential juxtaposition of cannabis hyperemesis and superior mesenteric artery syndromes in patients with a history of persistent cannabis use, stereotypical episodic vomiting resembling cyclic vomiting syndrome, and consequent weight loss, who present with a sudden increase in symptom severity or consistently bilious emesis.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. | 2022-02-21T14:22:02.236Z | 2022-02-21T00:00:00.000 | {
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118030585 | pes2o/s2orc | v3-fos-license | Forward Neutral Pion Cross Section and Spin Asymmetry Measurements at STAR
The STAR endcap electromagnetic calorimeter (EEMC) was designed to allow measurement of cross sections and spin observables in the forward direction, $1<\eta<2$ and with the full azimuth. Using the EEMC to measure double longitudinal spin asymmetries in photonic channels---such as inclusive neutral pions, prompt photon, and prompt photon + jet---allows access to $\Delta G$ within a low Bjorken-$x$ region ($0.01<x<0.33$ at $\sqrt{s}=200$) where $\Delta G$ is poorly constrained. Additionally, transverse spin asymmetries, shown to be zero at $\eta$ near zero and as large as 10\% at $\eta$ near 4, occupy a previously unmeasured region in the 3D pseudorapidity, transverse momentum, and $x$-Feynman phase space when measured with the EEMC. The neutral pion cross section measurement verifies that the signal of interest can be properly reconstructed and isolated from the background. Pion reconstruction techniques in the STAR EEMC will be discussed and preliminary cross-section and transverse single spin asymmetry measurements presented.
Introduction
The production of π 0 -mesons in p + p collisions at √ s = 200 GeV accesses quark and gluon distributions within the proton coupled with π 0 fragmentation functions. At intermediate pseudorapidity, the quark-gluon sub-process dominates over gluon-gluon and quark-quark sub-processes, and the lower Bjorken-x is associated with the gluon. The measurements described in these proceedings, taken at an intermediate pseudorapidity (0.8 < η < 2.0), cover previously unmeasured regions of the η and transverse momentum p T phase space.
The longitudinal double-spin asymmetry is sensitive to the gluon polarization distribution ∆g(x). The longitudinal single-spin asymmetries are parity violating and are thus expected to be very small. While ∆g(x) in the range 0.05 < x < 0.2 is becoming more constrained [3], little is known for x < 0.05. The analyzed data cover 0.01 < x < 0.33 at √ s = 200. Preliminary results for the longitudinal single-spin and double-spin asymmetries have been shown previously [4]. This document will focus on the new cross-section and transverse spin asymmetry results.
The measured cross section can be compared with perturbative QCD (pQCD) calculations and add information regarding the gluon to π 0 fragmentation function. Previous cross section measurements at nearby kinematics [1,2] are typically within the pQCD prediction scale uncertainty, lying at about 0.6 to 0.8 of the central scale prediction in the region of 5 < p T < 12 GeV/c. Several processes may contribute to the transverse single-spin asymmetry, A N , for x F > 0, including the Sivers and Collins effects at twist-2 and higher twist effects. Measurements in different kinematic regions may help elucidate the underlying mechanisms. The A N measurements herein cover the previously unmeasured region 0.06 < x F < 0.27 and 5 < p T < 12 GeV/c.
Analysis
The data were taken during the 2006 RHIC run using the STAR detector [5]. A luminosity of 8.0 pb −1 was used for the cross section results and 2.8 pb −1 for the transverse asymmetry results. The average transverse beam polarization was 0.55. The endcap electromagnetic calorimeter (EEMC) is used to measure the energy and position of photons from π 0 decays. The EEMC is a lead-scintillator sampling calorimeter [6], with 60 azimuthal and 12 radial segments (denoted towers). Position information is determined through a shower maximum detector (SMD), consisting of two layers of tightly packed 1 cm wide scintillating strips. The response of each SMD plane is smoothed [7], and energy clusters are identified by a strip above 2 MeV with three strips of monotonically decreasing energy on either side. Clusters from each of the two SMD planes are paired to determine the 2D position on the EEMC, and energy deposited in the towers are used to determine the energy of the incident particles, with a tower energy resolution of δ E/E = 0.16/ √ E. Pion candidates are formed by making all possible pairs of incident particles passing selection requirements.
The π 0 signal fraction among the π 0 candidates was determined by fitting a linear combination of template functions to the two-photon invariant mass (M γγ ) distribution over the range 0.0 < M γγ < 0.3 GeV/c 2 for each p T (or x F ) bin. A typical bin is shown in Figure 1. Three template functions were determined by fitting Pythia Monte Carlo data and represent (a) the π 0 signal, (b) the conversion background where the two reconstructed "photons" of the π 0 candidate were actually the two leptons from a photon that converted in upstream material, and (c) all other backgrounds, including combinatorial backgrounds. When fitting the weights of the three template functions an additional factor was also included to account for the energy scale difference between the data and the Monte Carlo.
To compute the cross section, the number of background-subtracted π 0 s was corrected for p Tbin smearing by applying the inverse of a smearing matrix, obtained from a Pythia/GEANT Monte Carlo simulation. The final cross section was then computed using Eq. 2.1, where N is the corrected number of π 0 s, L is the sampled luminosity (including dead-time corrections), ε is the product of reconstruction and trigger efficiencies, BR is the branching ratio π 0 → γγ [8], p T is the average p T for the particular p T bin, ∆p T is the width of the p T bin, and ∆φ = 2π and ∆η = 1.2 are the φ and η phase space factors. The trigger efficiency is below 10% for π 0 s with 5 < p T < 6 GeV, and plateaus above 40% at p T ≈ 9 GeV/c. The reconstruction efficiency is around 30% for 5 < p T < 9 GeV/c, and decreases to around 20% for 12 < p T < 16 GeV/c. The transverse spin asymmetry was computed by binning with respect to φ , the angle between the azimuthal angles of the π 0 and the spin polarization vector. The raw cross ratio E (φ ) was computed per φ bin, using Eq. 2.2, where N is the number of counts and ↑, ↓ denote the spin direction. In a right handed system wherê z is defined by the beam momentum, spin up (down) points in the positive (negative)ŷ direction." The quantity E N (φ ) was fit to the equation C + ε sin φ . The background was subtracted from ε using the signal fraction per bin and an estimate of the background asymmetry, computed as the average of the asymmetry in the two sideband M γγ -mass regions. The background asymmetry was [1,9]. Error bars are total uncertainty. Right panel, upper section: π 0 cross section (black markers) is shown compared with a pQCD calculation [10] with three options for the scale parameter. Statistical uncertainties are shown by the error bars, which are indistinguishable from the marker in most bins. Systematic uncertainties are shown by the error boxes. The right panel, lower section presents the ratio of the data to the theory curves for the various scales.
found to be less than 1σ from zero, with σ ≈ 0.01. The final result for A N was obtained by dividing by the luminosity weighted polarization. Figure 2 shows the cross section results of this analysis in comparison with previously published STAR results in other pseudorapidity and transverse-momentum regions, highlighting both the broad spectrum of coverage of the STAR detector and that this result lies in a previously unmeasured region. In Fig. 2, the cross section is also compared with a theory curved based on pQCD and global fits of distribution and fragmentation functions [10]. The EEMC π 0 cross section data points are observed to lie between the p T and 2p T scale. This is qualitatively consistent with published mid-rapidity STAR [1] and PHENIX results at √ s = 200 GeV and √ s = 500 GeV [2] where the cross section is lower than the p T -scale theory curve in the region of 6 < p T < 16 GeV/c.
Results
The A N results versus x F for 0.06 < |x F | < 0.27 and 5 < p T < 12 GeV/c are shown in Fig. 3. Systematic uncertainties include the uncertainty on the signal fraction and background asymmetry estimate, and single beam backgrounds. A N is consistent with zero for x F < 0, as expected. As anticipated from the previous results at lower p T and similar x F [11,12], A N is also consistent with zero for x F > 0. The A N results versus p T , over the same range of x F and p T , are also shown in Fig. 3. Within the x F region of this measurement, A N is consistent with zero and no strong conclusions about the p T dependence can be made. | 2013-09-12T16:54:20.000Z | 2013-09-12T00:00:00.000 | {
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111390715 | pes2o/s2orc | v3-fos-license | Deficiency in the secreted protein Semaphorin3d causes abnormal parathyroid development in mice
Primary hyperparathyroidism (PHPT) is a common endocrinopathy characterized by hypercalcemia and elevated levels of parathyroid hormone. The primary cause of PHPT is a benign overgrowth of parathyroid tissue causing excessive secretion of parathyroid hormone. However, the molecular etiology of PHPT is incompletely defined. Here, we demonstrate that semaphorin3d (Sema3d), a secreted glycoprotein, is expressed in the developing parathyroid gland in mice. We also observed that genetic deletion of Sema3d leads to parathyroid hyperplasia, causing PHPT. In vivo and in vitro experiments using histology, immunohistochemistry, biochemical, RT-qPCR, and immunoblotting assays revealed that Sema3d inhibits parathyroid cell proliferation by decreasing the epidermal growth factor receptor (EGFR)/Erb-B2 receptor tyrosine kinase (ERBB) signaling pathway. We further demonstrate that EGFR signaling is elevated in Sema3d−/− parathyroid glands and that pharmacological inhibition of EGFR signaling can partially rescue the parathyroid hyperplasia phenotype. We propose that because Sema3d is a secreted protein, it may be possible to use recombinant Sema3d or derived peptides to inhibit parathyroid cell proliferation causing hyperplasia and hyperparathyroidism. Collectively, these findings identify Sema3d as a negative regulator of parathyroid growth.
The parathyroid glands synthesize and secrete parathyroid hormone (PTH) 3 to regulate serum calcium concentration in the body (1)(2)(3)(4). In mice, there are two parathyroid glands located bilaterally in the neck and near the superior border of the thyroid gland. The parathyroid glands develop from the common parathyroid/thymus primordia, derived from the third pharyngeal pouch (5,6). As the development proceeds, parathyroid is separated from the thymus and becomes situated near or embedded within the thyroid gland (7,8). The early development and patterning of the parathyroid and thymus domains are controlled by a set of common regulatory genes (9 -13). However, once the domains are specified within the same primordium, parathyroid cells express specific transcription factors such as glial cells missing 2 (Gcm2) (8). Gcm2 is essential for the differentiation and survival of parathyroid cells. Mice with Gcm2 deficiency develop aparathyroid phenotype because their parathyroid precursor cells fail to proliferate and differentiate and die via programmed cell death (8,14,15). Parathyroid hormone (PTH) gene expression is detected in the parathyroid domain before its separation from the thymus and is maintained throughout development (14).
Primary hyperparathyroidism (PHPT) is a common endocrine disorder caused by excessive secretion of PTH from the parathyroid glands (16,17). PHPT is the third most common endocrine disorder, with a prevalence of 0.1-1.0%. It is more common in elderly females, with a prevalence of up to 2.1% in postmenopausal women (16,18). In most cases of nonfamilial origin PHPT, hyperparathyroidism results from either benign single adenoma (80 -85% cases) or multiglandular parathyroid hyperplasia (15-20% cases) (16,17). In rare cases (Ͻ1%), parathyroid carcinoma has been associated with PHPT (19). The parathyroid gland maintains calcium homeostasis by sensing fluctuations in extracellular calcium levels through the calciumsensing receptor (CaSR) and responding to changes in PTH secretion (1)(2)(3)(4). PHPT is characterized by increased parathyroid cell proliferation and calcium-insensitive hypersecretion of parathyroid hormone (20). Another form of hyperparathyroidism is secondary hyperparathyroidism (SHPT), in which parathyroid glands become enlarged and hyperactive in response to a condition outside of the parathyroid causing hypocalcemia. SHPT is most frequently seen in patients with chronic kidney disease (21). In most primary or secondary hyperparathyroidism cases, increased serum PTH levels are associated with increased serum calcium levels, causing complications such as renal stones, neuropsychiatric disorders, and bone abnormalities (16). Parathyroidectomy is the most common treatment for hyperparathyroidism. However, reoperative treatment for persistent or recurrent hyperparathyroidism remains technically challenging due to fibrotic scar and distorted anatomy that make it more difficult to identify the abnormal parathyroid glands (17,22).
Semaphorins are highly conserved secreted membranebound glycoproteins originally identified as axon guidance molecules in the developing nervous system (23). However, work in recent years has implicated them in a wide range of developmental, physiological, and pathological processes outside of the central nervous system, including tumor progression (24 -26). The primary receptors for semaphorins are plexins and neuropilins. Most membrane-bound semaphorins bind directly to plexins, whereas secreted class 3 semaphorins may require neuropilin as obligate plexin co-receptors (26,27). Increasing evidence has shown that semaphorins also can signal through a nonplexin receptor complex (28,29).
We have previously shown that Sema3d is expressed in the proepicardial organs and Sema3d-expressing proepicardial cells give rise to coronary vascular endothelium. While studying epicardial function of Sema3d, we noticed that Sema3d was expressed in the third pharyngeal pouch, giving rise to the parathyroid gland. Here, we report that Sema3d is expressed in the developing parathyroid gland and required for its proper development. Genetic deletion of Sema3d leads to PHPT due to the enlarged parathyroid gland. Molecular and biochemical analyses suggest that Sema3d inhibits parathyroid cell proliferation by decreasing the EGFR/ErbB signaling pathway. We demonstrate that EGFR signaling is elevated in Sema3d Ϫ/Ϫ parathyroid glands, and pharmacological inhibition of EGFR signaling can partially rescue the parathyroid hyperplasia phenotype. Because EGFR/ErbB signaling pathway is activated in the parathyroid glands in both PHPT and SHPT, the Sema3d-EGFR/ErbB signaling axis may be targeted to treat parathyroid hyperplasia, especially in persistent or recurrent hyperparathyroidism after parathyroidectomy.
Sema3d-deficient mice develop primary hyperparathyroidism
To determine the expression of Sema3d during parathyroid development, we performed expression and lineage tracing analyses in Sema3d GFPCre/ϩ mice (30). Bright field and direct GFP fluorescence in E9.5 Sema3d GFPCre/ϩ embryos demonstrated that Sema3d is expressed in the third pharyngeal pouch. Stronger GFP signals were observed in the dorsoanterior domain of the third pouch that represents the parathyroid domain (Fig. 1A). Similarly, when we crossed the Sema3d GFPCre/ϩ line with the R26R lacZ reporter line, strong -gal activity was observed in the third pharyngeal pouch (Fig. 1B). Our results are consistent with a previous report suggesting that Sema3d is expressed in the parathyroid-specific domain of the common primordium that gives rise to the thymus and parathyroid (31). Thus far, Sema3d Ϫ/Ϫ mice survive and have abnormal pulmonary vein patterning causing left-toright shunt, as characterized by severely enlarged right atria and ventricle (30). To demonstrate the functional requirement of Sema3d in parathyroid development, we performed histological and molecular analyses on the parathyroid gland in both control and Sema3d Ϫ/Ϫ mice. Morphological and histological examinations of the parathyroid glands from postnatal day 0 (P0), P6, and 12-month-old control and Sema3d Ϫ/Ϫ mice revealed a marked increase in size and hypercellularity, suggesting hyperplasia (Fig. 2, A and B and Fig. S1). Only one of the two parathyroid glands was hyperplastic, suggesting adenomatous transformation. Gross morphological and histological abnormalities resembling parathyroid hyperplasia were observed in 40% (19 of 46) of Sema3d Ϫ/Ϫ mice. Gcm2 immunohistochemistry was performed to mark the parathyroid glands in control and Sema3d Ϫ/Ϫ mice (Fig. 2C). Significant increase in the parathyroid area was observed in Sema3d Ϫ/Ϫ mice (Fig. 2D).
To determine whether parathyroid hyperplasia in Sema3d Ϫ/Ϫ mice leads to hyperparathyroidism, we measured serum PTH in 6-month-old mice. We observed elevated PTH levels in Sema3d Ϫ/Ϫ mice compared with their littermate controls (Fig. 2E). In most cases of PHPT, increased serum PTH levels are associated with increased serum calcium levels. However, in some cases of normocalcemic PHPT, PTH levels are elevated but serum calcium levels remain normal (18,32). To determine whether elevated PTH levels in Sema3d Ϫ/Ϫ mice are associated with changes in calcium levels, we measured serum calcium levels in the same set of animals. We observed elevated calcium levels in Sema3d Ϫ/Ϫ mice compared with their littermate controls, suggesting PHPT (Fig. 2F). To determine the molecular changes associated with parathyroid hyperplasia, we isolated RNA from dissected parathyroid gland from control and Sema3d Ϫ/Ϫ mice and performed qPCR analysis for parathyroid-specific genes such as parathyroid hormone (PTH), glial cells missing homolog 2 (Gcm2), and calcium-sensing receptor (CaSR). Expression levels of PTH and Gcm2 were significantly elevated in Sema3d Ϫ/Ϫ parathyroid compared with controls. However, the expression of CaSR was not altered in Sema3d Ϫ/Ϫ parathyroid glands (Fig. 2G).
Sema3d and parathyroid hyperplasia Sema3d inhibits parathyroid cell proliferation
To determine whether observed hyperplasia in Sema3d Ϫ/Ϫ mice is due to abnormal parathyroid cell proliferation, we performed Ki-67 immunostaining on P0 and 6-month-old parathyroid sections from control and Sema3d Ϫ/Ϫ mice. A dramatic increase in the number of Ki-667-positive cells was noted in the Sema3d Ϫ/Ϫ parathyroid sections compared with controls at both time points. This suggests that abnormal cell proliferation is the primary cause of parathyroid hyperplasia (Fig. 3A). To determine the molecular changes associated with increased parathyroid cell proliferation and hyperplasia in Sema3d Ϫ/Ϫ mice, we analyzed the expression of cell cycle regulators, including cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitor (CDKIs) by qPCR on RNA isolated from microdissected parathyroid glands. We observed a 4-fold increase in the expression of Ccdn1 (cyclin D1), an oncogene known to be overexpressed in 20 -40% of parathyroid adenomas (33)(34)(35). We also observed an increase in Cdkn2a (p21) and a modest decrease in Cdk4 expression in Sema3d Ϫ/Ϫ parathyroid (Fig. 3B). Increased expression of Cdkn2a has been associated with tumor progression (36). Growing evidence suggests that, depending on its environment, Cdkn2a can act as either tumor suppressor or an oncogene (37,38). No significant changes were observed in Ccdn2 (cyclin D2), Cdkn2c (p18), Cdkn1b (p27), and Cdkn1c (p57). To determine total RNA quality isolated from the microdissected parathyroid tissues, we performed qPCR analysis for thyroid-stimulating hormone receptor (TSHR), troponin T1 (Tnnt1), and Sox9. This allowed us to detect possible contamination from thyroid, skeletal muscle, and cartilage tissues, respectively. No significant changes were observed (Fig. S2). Together, these results suggest that Sema3d inhibits parathyroid cell proliferation in vivo by regulating the expression of cell cycle genes.
Because parathyroid tissues from mice are limited, we used a human parathyroid cell line, sHPT-1, to dissect the molecular mechanism of Sema3d-mediated inhibition of parathyroid cell proliferation. sHPT-1 is established from a hyperplastic parathyroid gland surgically removed from an SHPT patient (39). To determine whether sHPT-1 cells are sufficiently similar to primary parathyroid cells with regard to their semaphorin sig-
Sema3d and parathyroid hyperplasia
naling pathways, we treated sHPT-1 cells with recombinant Sema3d and analyzed proliferation by Ki-67 staining. Consistent with the in vivo data, we see significant reduction in sHPT-1 cell proliferation in Sema3d-treated samples compared with controls. This suggests that Sema3d inhibits parathyroid cell proliferation under both in vivo and in vitro conditions (Fig. 3C).
Sema3d inhibits EGFR signaling pathway to control parathyroid cell proliferation
Semaphorins signal through their classical Plexin and neuropilin receptors and other receptors, such as receptor tyrosine kinases (28,40,41). Sema3d binds and signals through Neuropilin-1 (Nrp1) to regulate endothelial cell patterning during pulmonary vein development (30,42). However, Nrp1 is not expressed during parathyroid development, suggesting that Sema3d may signal through other receptors expressed by the parathyroid gland (31). In a candidate-based screening, we found that Sema3d inhibits the epidermal growth factor receptor (EGFR) signaling pathway. EGFR is a member of the ErbB family of receptor tyrosine kinases that includes ErbB2, ErbB3, and ErbB4. Ligand binding induces homo-or heterodimerization of EGFR with other family members, including ErbB2, and activation of intracellular tyrosine kinase through formation of an asymmetric kinase dimer. Activation of ErbB receptors results in activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling, leading to enhanced proliferation, primarily due to aberrant changes in cell cycle gene expression, including cyclin D1 (43,44). Enhanced EGFR signaling has been associated with parathyroid adenomas and hyperplasia in both PHPT and SHPT (45)(46)(47)(48)(49).
To determine whether Sema3d inhibits parathyroid cell proliferation through the EGFR signaling pathway, we treated sHPT-1 cells with control and alkaline phosphatase (AP)tagged Sema3d (Sema3d-AP) conditioned medium and analyzed the expression of EGFR signaling components, such as pEGFR-Y1045, pEGFR-Y992, EGFR, pErbB2-Y1248, ErbB2, pAkt, Akt, pErk1/2, Erk1/2, and cyclin D1, by Western blotting. Sema3d-AP conditioned medium treatment reduced the amount of total EGFR, as well as pEGFR-Y1045 and pEGFR-Y992, suggesting that Sema3d may inhibit EGFR signaling by inducing receptor endocytosis/internalization and degradation. In parallel with EGFR down-regulation, ErbB2 and pErbB2-Y1248 levels were also decreased after nanomolar quantities of Sema3d treatment. To determine whether the downstream components of EGFR pathway were also affected after Sema3d treatment, we analyzed expression and activation of Akt and Erk1/2. The phosphorylation level of Akt and Erk1/2 was reduced. However, the total amount of Akt and Erk1/2 was not changed. Cyclin D1, one of the known targets of ErbB signaling in cancer cells, was significantly reduced in Sema3d-APtreated cells (Fig. 4A). Next, we treated sHPT-1 cells with recombinant Sema3d at different time points and analyzed EGFR signaling components. Consistent with earlier observation, all signaling components were significantly down-regulated after Sema3d treatment (Fig. 4B). Treatment of sHPT-1 cells with increasing recombinant Sema3d concentrations affected the EGFR signaling pathway in a manner similar to that described above (Fig. 4, C and D).
Inhibitory effects of Sema3d on ErbB2 are EGFR-dependent
Although Sema3d does not bind to ErbB2 alone, it can bind as part of a receptor complex. This suggests that changes in pErbB2 levels in sHPT cells after Sema3d treatment were likely from the impact of EGFR signaling pathway ( Fig. 4 and Fig. S3) (29). To determine whether the inhibitory effect of Sema3d on ErbB2 activation was EGFR-dependent, we co-transfected HEK293T cells with WT ErbB2 and a plasmid expressing either WT EGFR or kinase-inactive EGFR. The transfected cells were then treated with recombinant epidermal growth factor (EGF) in the presence/absence of Sema3d. Consistent with previous observations, when WT EGFR and WT ErbB2 were co-expressed, both receptors were phosphorylated in response to EGF (Fig. 4E). Sema3d exposures inhibited EGF-induced phosphorylation of both EGFR and ErbB2. However, when kinaseinactive EGFR and WT ErbB2 were co-expressed, no EGFR phosphorylation and a low level of ErbB2 phosphorylation were observed after EGF stimulation (Fig. 4E). The addition of Sema3d did not change ErbB2 phosphorylation, suggesting that the inhibitory effect of Sema3d on ErbB2 activation is EGFRdependent (Fig. 4E).
Next, we tested whether Sema3d can inhibit the EGFR-activated pathway, a condition associated with parathyroid hyperplasia in both PHPT and SHPT (45)(46)(47)(48)(49). We treated sHPT-1 cells with different doses of NSC 228155, an EGFR activator (50), and performed Western blotting analyses on pEGFR-Y1045 and pEGFR-Y992 to determine the optimal dose required to activate the pathway. We observed that 100 M NSC 228155 significantly increased the level of pEGFRs (Fig. 5A). Recombinant Sema3d inhibited NSC 228155-induced EGFR activation (Fig. 5B). Cyclin D1 expression was also increased with NSC 228155 treatment, but it was decreased following Sema3d treatment (Fig. 5B). To determine whether increased cyclin D1 expression correlated with increased cell proliferation, we treated sHPT-1 cells with NSC 228155 in the presence/absence of recombinant Sema3d and analyzed their proliferation (Fig. 5C). Activation of the EGFR pathway by NSC 228155 resulted in a significantly increased proportion (Ͼ80%, p Ͻ 0.001) of Ki-67-positive cells. However, the addition of Sema3d significantly decreased (ϳ30%, p Ͻ 0.001) the number of Ki-67-positive cells induced by NSC 228155 treatment. This suggests that Sema3d regulates cell cycle progression by signaling through the EGFR (Fig. 5C). Together, both in vivo and in vitro results suggest that Sema3d inhibits the EGFR signaling pathway to restrict parathyroid cell proliferation. A recent study by Aghajanian et al. (29) demonstrated that Sema3d activates the ErbB2 signaling pathway in endothelial cells during coronary vasculature patterning. This indicates that, depending on its biological context, Sema3d can act as either agonists or antagonists for the ErbB signaling pathway (29).
Blocking EGFR signaling can partially rescue the parathyroid hyperplasia phenotype in Sema3d ؊/؊ mice
To determine whether Sema3d inhibits the EGFR pathway in vivo, pEGFR immunohistochemistry was performed on para- (C and D). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; NS, not significant (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test). E, EGFR signaling in cells expressing mixed WT and kinase-inactive EGFR/ErbB2 heterodimers. HEK293T cells expressing the indicated pairs of EGFR and ErbB2 receptors were treated with recombinant EGF with and without recombinant Sema3d for 30 min. Cell lysates were prepared for Western blots to detect pEGFR, EGFR, pErbB2, ErbB2, and -actin levels. Western blots were quantified for pEGFR and pErbB2 relative to -actin levels. Six bars in the graph represent six conditions labeled above the blots. Experiments were repeated three times and quantified. Error bars, S.D.
Sema3d and parathyroid hyperplasia
thyroid sections from control and Sema3d Ϫ/Ϫ mice. We observed elevated levels of pEGFR in Sema3d Ϫ/Ϫ parathyroid sections compared with controls (Fig. 6A). Next, we tried to determine whether hyperplasia seen in Sema3d Ϫ/Ϫ mice was due to an abnormal increase in the EGFR signaling and whether blocking EGFR signaling can rescue the hyperplasia phenotype. To examine whether elevated EGFR signaling resulted in parathyroid hyperplasia, we treated Sema3d Ϫ/Ϫ mice with either vehicle or erlotinib, an EGFR signaling pathway inhibitor, and analyzed their parathyroid glands. We observed a significant size reduction of the parathyroid glands in Sema3d Ϫ/Ϫ mice treated with erlotinib compared with vehicle-treated controls (Fig. 6B). Consistent with reduced parathyroid size, we also observed that Ki-67-positive cells were reduced in erlotinibtreated samples when compared with controls (Fig. 6C). To determine whether the reduced parathyroid glands impact PTH levels, we measured serum PTH levels in Sema3d Ϫ/Ϫ mice treated either with vehicle or erlotinib. PTH levels were significantly reduced after erlotinib treatment (Fig. 6D). Together, these results indicate that blocking EGFR signaling could partially rescue the hyperplasia and hyperparathyroidism phenotype seen in Sema3d Ϫ/Ϫ mice.
Discussion
The pathogenesis of parathyroid gland hyperplasia in hyperparathyroidism is poorly understood. Thus, a better molecular understanding is essential for prevention and therapeutic inter-vention. In this study, we demonstrate that Sema3d Ϫ/Ϫ mice develop PHPT from parathyroid hyperplasia. However, only ϳ40% of Sema3d Ϫ/Ϫ mice develop hyperplasia. We suspect that this incomplete phenotype is contributed by the genetic background, as phenotypes in other class 3 semaphorin knockouts are also strain-dependent (51). Expression of parathyroidspecific developmental genes such as PTH and Gcm2 was increased in Sema3d Ϫ/Ϫ mice. However, CaSR expression was not altered. Expression of CaSR enables the parathyroid cell to respond to changes in extracellular calcium concentration. CaSR activation by high extracellular calcium results in reduced PTH secretion. On the other hand, deactivation of CaSR by low extracellular calcium induces PTH secretion. Normal expression of CaSR in Sema3d Ϫ/Ϫ mice suggests that elevated PTH levels are primarily caused by increased gland size and not from individual parathyroid cells secreting more PTH.
Genetic inactivation of Sema3d leads to an activated EGFR signaling pathway and cyclin D1, a cell cycle regulator and an oncogene. This results in increased parathyroid cell proliferation. Cyclin D1 binds and activates CDKs that phosphorylate several downstream proteins to ensure cell cycle progression through the G 1 /S checkpoint. The role of cyclin D1 in parathyroid tumorigenesis is well-established. Cyclin D1 is overexpressed in 20 -40% of parathyroid adenomas (33)(34)(35). However, these numbers are much higher (ϳ80%) in sporadic parathyroid adenomas from Asian Indians. Cyclin D1 is also
Sema3d and parathyroid hyperplasia
activated in a subset of parathyroid adenomas from chromosomal rearrangement, bringing the cyclin D1 gene under the influence of the PTH promoter region (34). Studies in transgenic mice have demonstrated that targeted overexpression of cyclin D1 in parathyroid tissues can lead to parathyroid adenomas (33).
Semaphorins signal through their classical plexin and neuropilin receptors. However, semaphorins can also signal through other receptors, such as receptor tyrosine kinases (28,40,41). Our data suggest that Sema3d can modulate cell behavior by regulating receptor tyrosine kinases activities. Growth factor receptor tyrosine kinases, including EGFR and ErbB2, play an important role in mediating cancer growth and survival. EGFR is a member of the ErbB family of receptor tyrosine kinases that also includes ErbB2, ErbB3, and ErbB4. Upon ligand binding, ErbB family members can form homo-or heterodimer and activate downstream mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/Akt signaling pathway to regulate cell proliferation. Our data demonstrate that Sema3d can inhibit both EGFR and ErbB2 signaling pathways. Down-regulation of both total and phosphorylated form of EGFR suggests that Sema3d may inhibit EGFR signaling by inducing receptor endocytosis/internalization and degradation. Different posttranslational modifications, such as tyrosine and serine/threonine phosphorylation, ubiquitylation, and acetylation, regulate EGF receptor endocytosis (52). Among the class 3 semaphorins, both Sema3a and Sema3e induce endocytosis of plexin recep-tor/receptor complex during axon guidance (53,54). Recent work by Aghajanian et al. (29) demonstrated that Sema3d does not bind to ErbB2 alone. However, it can bind as part of a receptor complex, suggesting that inhibitory effects of Sema3d on ErbB2 activation are likely from the EGFR signaling pathway (29). Using plasmids expressing either the WT or kinase-inactive form of EGFR, we demonstrate that the inhibitory effects of Sema3d on ErbB2 are EGFR-dependent. In contrast to the inhibitory effect of Sema3d on RTKs, another class 3 semaphorin, Sema3C, can activate RTKs during prostate cancer progression. This suggests that, depending upon its biological context, class 3 semaphorins can act as either agonists or antagonists on the RTK pathway (40).
Our results demonstrate that pharmacological blocking of EGFR signaling can partially rescue the parathyroid hyperplasia phenotype seen in Sema3d knockout mice. It would be interesting to explore whether Sema3d-EGFR signaling can be therapeutically targeted to inhibit parathyroid hyperplasia in both PHPT and SHPT conditions. Parathyroidectomy is the most common treatment for hyperparathyroidism. Because the neck's anatomy was distorted with fibrosis following the initial surgery, there were technical challenges in identifying and safely removing the abnormal parathyroid glands. Therefore, pharmacological treatment for persistent or recurrent hyperparathyroidism may be a good option to address this issue. Administration of EGFR inhibitors has shown beneficial effects. However, systemic inhibition could also suppress renal EGFR signaling and compromise kidney function (49). In contrast to its broad expression during embryonic development, Sema3d expression in adults is enriched in the spleen and thyroid gland (55). In this context, Sema3d may be a potential therapeutic target to treat parathyroid hyperplasia, given its more limited tissue expression in adult mice. Nonetheless, the possible off-target effects of systemic or targeted delivery of Sema3d need to be determined.
Because class 3 semaphorins are secreted proteins, it is possible to use recombinant semaphorins or derived peptides to inhibit parathyroid cell proliferation causing hyperplasia. A recent study by Casazza et al. (56) demonstrated that systemic and targeted delivery of Sema3A inhibits tumor progression in multiple mouse models. However, the inhibitory effect of systemic or targeted Sema3d delivery on parathyroid tumor progression remains to be investigated. Our finding that Sema3d inhibits EGFR signaling to restrict aberrant parathyroid tumor cell proliferation may also be relevant to other tumor types. Aberrant activation or overexpression of EGFR is reported in many tumors (57). For example, overexpression of EGFR is observed in 60% of the high-grade gliomas (glioblastoma) (58). SEMA3D has been shown to inhibit glioblastoma growth. The SEMA3D expression is reduced in glioblastoma as compared with low-grade gliomas, suggesting that its loss is involved in tumor progression (59). However, the signaling pathways mediating these effects of SEMA3D in gliomas have not been identified. Thus, future work in this direction is required for the development of anti-tumor therapeutic strategies targeting the semaphorin signaling pathway.
Histology and immunohistochemistry
After genotyping of the pups from Sema3d heterozygous intercross, we randomly selected 12 control and 12 Sema3d Ϫ/Ϫ mice from the cohort for histological analysis. Histology and immunohistochemistry were performed as described previously (61)(62)(63)(64)(65). Briefly, the thyroid, parathyroids, and trachea tissues were dissected en bloc in PBS and fixed in 4% paraformaldehyde overnight at 4°C. The tissues were washed with PBS, dehydrated in an ethanol series, and stored in 100% ethanol at 20°C. To examine the parathyroid glands, serial paraffin sections were cut to a thickness of 5-10 m and processed for either morphological hematoxylin/eosin staining or immunohistochemistry. For immunohistochemistry, the sections were deparaffinized in xylenes, and slides were deparaffinized in xylene, dehydrated in ethanol, and rehydrated in water. Antigen retrieval using Bull's eye decloaker (Biocare Medicals, catalogue no. BULL1000 MX) or Tris-based solution (Vector Laboratories, catalog no. H-3301) was performed according to the manufacturer's instructions. Endogenous peroxidase activity was blocked with hydrogen peroxide (3%) treatment. Slides were washed in PBS and blocked in blocking buffer (5% serum) for 1-2 h at room temperature. Slides were incubated overnight with primary antibody diluted in blocking buffer. The next day, slides were washed, and a secondary antibody was applied for 2 h at room temperature. Primary antibodies used were anti-GCM2 goat polyclonal (Santa Cruz Biotechnology, Inc., catalogue no. sc-79495), anti-Ki-67 rabbit monoclonal (Abcam, catalogue no. ab16667), and anti-pEGFR-Y1045 rabbit polyclonal (Cell signaling, catalogue no. 2237). Secondary antibodies used were ImmPRESS horseradish peroxidase anti-goat (Vector Laboratories, catalogue no. MP-7405-50) or anti-rabbit (Vector Laboratories, catalogue no. MP-7401-50). The sections were washed, and staining was visualized using the DAB substrate kit (Vector Laboratories, catalogue no. SK-4100).
Biochemical analyses
At the indicated age, control and Sema3d Ϫ/Ϫ mice were anesthetized, and blood was collected by cardiac puncture for plasma separation. Plasma PTH levels were measured using the mouse intact PTH ELISA kits (Immutopics, Inc., catalogue no. 60-2300). Plasma calcium levels were measured using a calcium detection kit (Abcam, catalogue no. ab102505).
sHPT cell culture
Human parathyroid tumor cell line sHPT-1 was established by Björklund et al. (39), as previously described. sHPT-1 cells were cultured and expanded in Dulbecco's modified Eagle's medium (DMEM) (Gibco, catalogue no. 11965-092) containing 1% penicillin/streptomycin and 10% FBS. After reaching 70 -80% confluence, cells were cultured and starved overnight in DMEM supplemented with 1% penicillin/streptomycin and 1% FBS. They were subsequently treated with Sema3d conditioned medium or recombinant Sema3d (Abnova, catalogue no. H00223117-P01) in the desired concentrations at the indicated time points with appropriate controls. For the proliferation assay, sHPT-1 cells were treated with or without recombinant Sema3d at 10 nM for 48 h. In another experiment, sHPT-1 cells were stimulated with NSC 228155 (100 M) or in combination with 10 nM recombinant Sema3d for 24 h. After treatment, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and evaluated for cell proliferation by Ki-67 (eBioscience, catalogue no. 14-5698-82) immunofluorescence staining. Briefly, cells were fixed and washed with PBS and incubated for 2 h at room temperature with Ki-67 antibody diluted at a concentration of 5 g/ml in PBS containing 2% BSA. After washing with PBS, cells were incubated for 30 min at room temperature with goat anti-rat IgG, Alexa Fluor 586 (Invitrogen, catalogue no. A-11077). The cells were then washed again and incubated with DAPI for nuclear staining visualized with fluorescence microscopy (Olympus IX71S1F3).
EGFR activation assay
sHPT-1 cells were incubated with either DMSO as vehicle or NSC 228155 compound (Calbiochem, catalogue no. 5.30536.0001CN) for 30 min at 25, 50, 100, 200, and 300 M and analyzed for EGFR members by Western blotting. In another experiment, sHPT-1 cells were treated with either NSC 228155 (100 M) or in combination with recombinant Sema3d (5, 10, and 15 nM) for 30 min and analyzed for EGFR with Western blot analysis.
Mice and erlotinib
12-week-old Sema3d Ϫ/Ϫ mice were randomly assigned to two treatment groups: vehicle (DMSO) and erlotinib (10 mg/kg body weight) (Selleckchem, catalogue no. S1023). Treatments were administered every 2 days by intraperitoneal injection. After 4 weeks of treatment, the mice were sacrificed, and serum and parathyroid tissue were collected for histologic and molecular analyses.
Preparation of Sema3d conditioned medium
HEK293T cells were cultured and expanded in DMEM (Gibco, catalogue no. 11965-092) supplemented with 1% penicillin/streptomycin and 10% FBS. HEK293T cells were transfected with either Sema3d pAP-Tag4 or empty vector plasmid using the FuGENE 6 transfection reagent (Promega, catalogue no. E2691) according to the manufacturer's protocol. After 24 h, the conditioned medium was changed to DMEM (Gibco, catalogue no. 11965-092) supplemented with 1% penicillin/ streptomycin and 0.5% FBS. The cells were then incubated for 72 h, and the conditioned medium was collected. The conditioned medium was filtered using a 0.2-m syringe filter and centrifuged at 5000 rpm for 5 min. AP activity in the conditioned medium was measured with a colorimetric assay as Sema3d and parathyroid hyperplasia described previously (30). The conditioned medium was then used for the experiments.
Kinase activation assay
HEK293T cells were transfected with either plasmid expressing WT ErbB2, a WT EGFR, or a kinase-inactive EGFR (K721A) using Lipofectamine transfection reagent (Thermo Fisher Scientific, catalogue no. 11668019) according to the manufacturer's protocol. Following transient transfection, cells were incubated for 48 h and starved for 4 h with serum-free DMEM supplemented with 1% penicillin/streptomycin. Cells were then treated with/without 200 ng/ml EGF (PeproTech, catalogue no. AF-100-15) and with/without 10 nM recombinant Sema3d for 30 min. Cell lysates were collected for Western blot analysis. Plasmids expressing WT EGFR and kinase-inactive EGFR (K721A) were kindly provided by Sara Sigismund from Pier Paolo Di Fiore's laboratory (European Institute of Oncology, Milan, Italy). The plasmid expressing ErbB2 was a gift from Martin Offterdinger (Addgene plasmid 40268) (66).
RNA extraction and quantitative RT-PCR
Parathyroid glands were dissected from control and Sema3d Ϫ/Ϫ mice. Because only ϳ40% of the Sema3d Ϫ/Ϫ mice had parathyroid hyperplasia, we decided to use hyperplastic (enlarged) parathyroid tissue from Sema3d Ϫ/Ϫ mice. Total RNA was isolated from glands using TRIzol (Life Technologies, Inc., catalogue no. 15596-018). RNA was reverse-transcribed using random hexamers and the SuperScript III First-Strand Synthesis system (Life Technologies, catalogue no. 18080-051). Gene expression was measured by quantitative RT-PCR (ABI PRISM 7900 or ViiA7 Real-Time PCR System) using the Power SYBR Green master mix (Life Technologies, catalogue no. 4368702). Both signals and relative gene expression were normalized to corresponding glyceraldehyde-3-phosphate dehydrogenase controls. PCR conditions and primer set sequences are available upon request.
Statistical analyses
Statistical analyses were performed using two-tailed Student's t test. Data were expressed as mean Ϯ S.D. Differences were considered significant with p Ͻ 0.05. One-way analysis of variance (ANOVA) was used to assess statistical differences between groups. Significant ANOVA results were further analyzed by Tukey's multiple-comparison test (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; NS, not significant). | 2019-04-14T13:02:47.025Z | 2019-04-12T00:00:00.000 | {
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608932 | pes2o/s2orc | v3-fos-license | Migraine: treatments, comorbidities, and quality of life, in the USA
This study sought to characterize the experience of stress, treatment patterns, and medical and disability profile in the migraineur population to better understand how the experience of migraines impacts the social and psychological functioning of this group. A 30-minute self-report survey was presented via a migraine-specific website with data collection occurring between May 15 and June 15, 2012. Recruitment for the study was done through online advertisements. In total, 2,907 individuals began the survey and 2,735 met the inclusion criteria for the study. The sample was predominantly female (92.8%). Migraine-associated stress was correlated with length of time since first onset of symptoms (P<0.01) and number of symptoms per month (P<0.01). Disorders related to stress, such as depression (P<0.01) and anxiety (P<0.01), were also positively correlated with the measured stress resulting from migraines. Migraine-associated stress must be understood as a multidimensional experience with broader impacts of stress on an individual correlating much more highly with negative mental and physical health profiles. Stress resulting from frequent migraine headaches may contribute to the development of medical and psychological comorbidities and may be a part of a cyclical relationship wherein stress is both a cause and effect of the social and medical impairments brought about by migraine.
Introduction
Migraine headache is a chronic pain condition that afflicts millions of Americans, with prevalence estimates ranging between 16.2% and 22.7% for USA adults aged 18 years or older. 1 Migraines are disabling, with a majority of migraineurs reporting impairments in their ability to engage in work activities, perform household chores, attend social activities, and maintain relationships due to their migraines. 2 The social costs of migraine are felt in the form of lost productivity as well as increased health care costs. 3 The disorder is further complicated by the multiple and varied symptoms that have been reported, including sweating, allodynia, photophobia, phonophobia, vomiting, and nausea. 4 In light of the high prevalence rate and significant impairment of daily living, it is unsurprising that patients use many treatment approaches to alleviate the impact of migraine. Pharmacological treatments are common, with both abortive and prophylactic medications used; however, these medications are burdened by side effects and cost. 5 Non-pharmacological interventions, such as behavioral therapy, 6 relaxation therapy, and cognitive behavioral therapy, have also been employed, with varying degrees of success. 5 Despite the variety of treatment approaches available, migraineurs report that they feel their treatment is insufficient or unsatisfactory. 7 Frequent migraine headaches also impose a heavy psychological burden on migraineurs. Both stress and anxiety levels are higher among migraineurs when compared with healthy controls. 8 Other research has found a reinforcing cyclical relationship between stress and migraine headaches. 9 The relationship between stress and migraine may also have a deeply personal component, as suggested in recent findings, ie, an individual's response to stressors, rather than the stressors themselves, may be the predisposing factor to developing migraine. 10 Previous research has treated the experience of stress in the context of migraine headache as principally a prodromal indicator of onset of migraine and a strictly internal experience on the part of the migraineur. 8 The experience of migraines is further complicated by comorbid medical and psychological disorders. Psychological disorders, especially depression and anxiety, have been found to be prevalent among migraineurs. 2 Chronic pain, stroke, gastrointestinal disorders, cardiovascular disease, and other medical conditions have also been found to be more prevalent in migraineurs compared with the general population. 11 Given the high prevalence of migraine headaches, it is unsurprising that a large variety of comorbid medical and psychological disorders is found in the migraine population.
The high social and personal costs of migraine as well as complications in delivering effective migraine treatment emphasize the need to understand treatment patterns in this highly prevalent clinical population. The current study extends the available literature by describing the relationship between stress, treatment patterns and satisfaction, and comorbid disorders in a large online sample of migraineurs. This study elaborates on previous research into migraineassociated stress by viewing the stress experience as a social as well as internal impact of migraine headaches.
Patients and methods Recruitment
Participants were recruited from a well-known online migraine headache resource. Adults aged 18 years or older and currently living in the USA were invited to participate. The presence of migraines was validated through agreement with the statement "Have you ever been diagnosed with migraine headaches by a physician?"
Procedure
This study was approved by the University of Massachusetts institutional review board. Between May 15 and June 15 2012, an online survey was presented via a migraine-specific community website. 12 Participants were informed that their participation was voluntary, information would be collected anonymously, the anticipated completion time of the survey, and that they would not receive compensation for participating. No personal identifiers were collected nor solicited from participants. The survey contained questions related to demographic information, migraine history and symptoms, social information, and treatment history and satisfaction. The survey host used embedded cookies to prevent any participant from taking the survey multiple times. The survey employed an adaptive methodology. Data were collected by the survey host and stored on a secure server.
A composite variable termed "negative live events" was created using items from the survey that examined specific areas of life impacted by participant migraineurs. The negative life events score was further divided into variables examining internal areas of life, such as mental state and well-being, and external areas of life, such as social relationships.
Data analysis
All data analysis was performed using Statistical Package for the Social Sciences version 22.0 (IBM Corporation, Armonk, NY, USA). Participant demographics were analyzed using descriptive statistics and analysis of variance, and Pearson's R correlation analyses were used to examine relationships among the survey data. Path analyses were conducted to examine differential relationships among the length of time since onset of migraine symptoms, the number of reported comorbidities, the frequency of migraine symptoms, the number of migraine symptoms, and composite scores reflecting impacts resulting from migraines (negative life impact internal score and negative life impact external score).
Participants
The survey was started by 2,907 individuals. A total of 172 individuals were excluded due to: being younger than 18 years (n=32), not diagnosed with migraines (n=19), or not currently living in the USA (n=121). Of the remaining 2,735 individuals who started the survey, 2,632 completed it (giving a completion rate of 96.2%). The mean time taken to complete the survey was 23 minutes (± standard deviation
539
Migraine, treatment, comorbidities, and quality of life
Demographics
The sample was 92.8% female and nearly half of the sample (47.8%) was aged 40-54 years, which is demographically similar to national rates for migraineurs. The majority of the sample (75.9%) had experienced their migraine symptoms for more than 10 years. The majority of the sample (68.3%) was diagnosed with chronic migraine. A quarter of the sample (25.1%) reported experiencing migraine symptoms on more than 20 days per month; the remainder of the sample experienced migraine symptoms less frequently. Additional demographic information can be found in Table 1. Table 2 shows the data relating to reported comorbid disorders. Depression and anxiety were found to be the most commonly reported comorbid conditions, at 63.8% and 60.4%, respectively. Chronic (non-headache) pain was reported by 39.5% of the sample. All remaining disorders were reported by less than onethird of the sample. A minority of participants (16.4%) reported suffering from a disorder not listed, and of those participants, the vast majority reported multiple comorbid disorders (89.9%).
negative life events
Nearly three quarters (74.1%) of the sample reported that people in their social network do not believe that the migraines are as severe as the participants report. The migraineurs reported that their employer knew about their migraines (54.8%) and the migraines negatively impacted their professional advancement (50.2%). A composite variable was created by summing the number of negative life events that participants endorsed in order to examine the extent to which migraine headaches impact their life (Cronbach's α=0.848). The negative life event score was found to have a mean of 4.023 (standard deviation 27; median 4; range 0-13). The results of questions identifying the impact of migraine on personal aspects of life can be found in Table 3.
The negative events were also examined according to whether the migraineurs perceived that the negative impact was caused by internal (psychoemotional) stress factors or external (social-interactive) stress factors. The negative life impact internal score was composed of questions asking about the migraineurs' feelings of worry or shame about their migraine headaches. The negative life impact external score was composed of questions examining the social, professional, and relational impact of migraines. Pearson's R correlational coefficient path analyses examining the relationship between the characteristics of migraine and the internal of participants who were found to have a negative life event score of 10 or higher also reported 15 or more migraine symptoms per month.
Migraine triggers
Most of the participants (78.2%) were able to identify at least one trigger for their migraine headaches. Of those participants who did identify a trigger, 78.3% identified environmental causes (such as weather) and 76.7% identified stress as a trigger. Of those participants who identified triggers for their migraines, 88.3% reported that they take steps to avoid those triggers. Additional results are reported in Table 5.
Migraine treatments
In total, 96.7% of the sample reported consulting a physician specifically for treatment of migraine headaches at some point and 81.3% of the sample were currently seeing a physician about their migraine (Figures 3, 4 and Table 6). Of those who do not see a physician about migraine, 47.7% reported financial cost or health insurance to be the preventing factor. Approximately half of the sample (45.5%) reported that they avoided taking a prescribed migraine medication due to the cost. Ninety percent of the sample reported trying a prescription oral migraine medication at some point and 67.2% reported currently being treated with a prescription oral migraine medication. Approximately three quarters of the sample (73.3%) reported withholding medication to save for later. Participants also reported behavioral therapies
Migraine frequency and symptoms
Migraine frequency data can be found in Table 4. The most commonly reported symptoms of migraine experienced by the sample were head pain (98.0%) and sensitivity to light (91.1%). Participants who reported a higher frequency of migraine symptoms were also more likely to report more negative life impact due to migraine. Seventy-five percent that they had tried, with "dark room" (92.2%) and "hot/cold therapy" (75.2%) being the most commonly tried; however, many participants report initiating a behavioral or complementary therapy (eg, yoga), but not continuing with the practice in the long term.
Treatment satisfaction
When asked about their current migraine treatment, most individuals appeared to be less than satisfied with their treatment. Approximately one-third of participants (31.2%) reported feeling "neutral" on satisfaction and a further
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Malone et al 27.0% reported being "satisfied" with their current migraine treatment (see Figure 5). One-third of respondents (33.7%) reported being satisfied with their treating physician and a further 29.6% were neutral on their satisfaction with their treating physician. Participant satisfaction with behavioral or complementary therapies was found to be predominantly neutral (38.7%).
Relationship between migraine measures and comorbid disorders
The results of the Pearson's R correlational analyses identifying relationships between the negative life events score, length of time since the onset of migraine symptoms, and the monthly frequency of migraine symptoms by presence of comorbid disorders can be found in Table 7. The negative life events score was found to be significantly correlated (P,0.01 or P,0.05) with all comorbid diseases except for diabetes and chronic obstructive pulmonary disease. The reported length of time since first migraine symptoms was found to be significantly correlated (P,0.01) for all disorders except stroke and epilepsy. Further, the reported frequency of migraine symptoms per month was found to be significantly correlated (P,0.01 or P,0.05) for all disorders except for stroke. The negative life event score, the length of time since onset of migraine, and the monthly frequency of migraine symptoms were found to be significantly correlated with each other (P,0.01). Additional analyses were conducted according to age and sex; however, these analyses were not found to be statistically significant. The negative live events score was separated into tertiles, ie, high (score 10-13), medium (score 4-9), and low (score 0-3) scores. Analyses of variance comparing these groups found that those in the higher tertile group of negative life impact were more likely to have a higher frequency of migraine symptoms, a longer history of migraine symptoms, and were more likely to experience comorbid disorders. The results of these analyses of variance can be found in Tables 8-10.
Discussion
The present study characterized migraineurs and examined their treatment patterns, stress attributes, migraine disorder characteristics, and disability profile. As in previous research and clinical data, 1 the vast majority of migraineurs in this study were female. Respondents in this survey tended to be younger than in other large migraine studies, with a higher proportion of participants aged 18-39 years but this is most likely due to the online mode of delivery rather than a change in the migraineur population. 13 Similar to previous findings, migraineurs at every degree of symptom frequency reported that their migraine symptoms negatively affected their lives. Migraineurs in this study were found to experience elevated levels of bodily pain, anxiety, and depression, and were able to identify physiological aspects of their migraines in proportions similar to those in other studies, 14 but a substantial minority appeared to be ignorant of the prodromal features
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Migraine, treatment, comorbidities, and quality of life of their condition. This is significant in that while migraine headaches commonly confer negative impacts, the precipitating factors are poorly understood by a large percentage of migraineurs, casting doubt on the ability of some patients to be knowledgeable about which treatment may be most efficacious for them. 2,7,15 Similar to previous findings, migraineurs at every degree of symptom frequency reported that their migraine symptoms negatively affected their lives. The length of time since the participant's first migraine and the number of migraine symptoms were found to have a stronger relationship with negative interpersonal and situational impacts than with negative affective and psychological impacts, such as shame or embarrassment related to migraines. While this relationship may be a result of misperception of treatment by others or poor self-knowledge of emotional state, this observation is significant in that it suggests that migraineurs may attribute a wide variety of negative social interactions to their disorder rather than other factors, such as the dispositions of others or situational factors. Previous research has treated the stress resulting from migraines as a purely affective or psychological experience on the part of migraineurs; 8 however, our research indicates that migraine-associated stress negatively impacts social and professional areas even more strongly than psychological states. While the findings of this study suggest that the negative impacts of migraine related to the number and frequency of migraine symptoms may be more closely related to the interpersonal impact of migraine, this may not be a simple static relationship. It is possible that the interpersonal impact of migraines may be more easily attributed to specific events whereas the self-perceived internal stress may result from a general sense derived from recent or distant personal experiences. Future research should further explore the relationship between the impact of migraines on an individual's social network and the negative affective and psychological impacts on migraineurs.
Despite the fact that the vast majority of the participants in this study sought the care of a physician for their migraine headaches, only a subset of these participants continued their treatment and an even smaller proportion were satisfied or extremely satisfied with their treatment. Our study found that migraineurs tended to be dissatisfied with their migraine treatment regardless of the intervention used. These results continue the trend of low patient satisfaction with migraine treatment found in previous studies; however, the current sample tended to be even more dissatisfied than noted in previous research. 7,16 Similar to previous studies, the current study found that migraineurs often attempt many treatments, but are discouraged by factors such as cost, side effects, and efficacy. 17 Even for highly efficacious, cost-effective treatments, limited availability of treatment or lack of treatment providers may prevent an individual from learning about and using low-risk and potentially high-benefit practices. 18 The finding that a proportion of migraineurs found some benefit for each treatment suggests that the differential effectiveness of intervention may be due to idiosyncratic patient characteristics rather than specific treatment ineffectiveness. This emphasizes the need for a migraine practitioner to understand the interaction between psychological, social, and biological characteristics in the individual patient in order to accurately match a patient with the treatment approach most suited to their specific disorder pattern and personal circumstances. The most salient difference in how migraineurs perceived their disorder was in the symptomatic manifestation of the disease. Although migraine headaches are frequently associated with aura, our sample reported migraine with aura at a lower level than would have been anticipated. 19 Respondents in this study reported symptoms of nausea/vomiting and sensitivity to light to a higher degree than has been found in previous studies. 20 In addition, participants in our study reported migraine symptoms that have not been widely studied to date, such as puffy eyelids and food cravings. The variety of symptoms reported by migraineurs attests to the individualized nature of the disorder. Further, the diverse manifestations of migraine symptoms lends further support to the view that migraines must be considered in the context of the individual, because different symptoms may have varying degrees of impact on an individual's daily life. It is not unreasonable to consider symptoms such as vomiting and sensitivity to light as potentially eliciting a greater change in a sufferer's behavior than symptoms such as food cravings. Different symptoms may exert differential degrees of impact on a sufferer's day-to-day life, even when experienced at similar frequencies, and as mentioned previously, the individual characteristics of the migraineur may determine the level of stress experienced as a result of these symptoms. The present study probed the areas of daily life impacted by migraine headaches and found that these headaches impose a substantial biological, psychological, and social burden on migraineurs. The pervasive and destructive impact of migraine headaches on a migraineurs' life has been well documented in the literature, but previous research has not closely examined how the stress of these negative impacts is moderated by the time since onset and the frequency of the disorder. 7 The stress of chronic migraine may be a cyclical process which contributes to the development and fermentation of comorbid disorders, additional stress, and more migraines. This insidious cycle may be exacerbated by a higher number of symptoms, a higher migraine frequency, or pre-existing psychological or medical conditions. The psychological impact of migraines may inhibit the ability to engage in healthy practices that would reduce the prevalence of other disorders, decrease the use of adaptive coping with biological symptoms, exacerbate the pain experience, and may discourage adherence to treatment. Psychological distress, as well as pain, have been found to be strongly associated with development of depression or anxiety disorders, 21 and these were found to be the most common comorbid disorders in our study. The high medical and personal burden reported by participants in the present study is significant in terms of emphasizing the pervasive onus that migraine headaches confer. This raises the question of how much medical misery can someone endure? Competing health priorities created by comorbid diseases can exacerbate the everyday cost of migraine symptoms by inhibiting a migraineur's ability to cope biologically and psychologically. High levels of stress have been shown to lead to a higher prevalence of an array of medical and psychological disorders. 22 This is true of both depression and anxiety, which have both been found to be more prevalent in the migraineur population. 23 The findings of this study underscore the clinical importance of understanding the social and psychological impact of migraine headaches on a migraineur. Participants found to have a higher degree of stress attributable to migraine symptoms were also found to have more comorbid disorders, and importantly, the most common comorbid disorders found in this sample are also known to be highly susceptible to stress.
Our results highlight the clinical relevance of understanding the impacts of migraine headaches on patients. Previous research has identified chronic psychological stress, such as in sufferers of a chronic disorder, to be a major suppressor of the human immune system. 24 Healthy social relationships may serve as a protective factor with regard to the development of additional disorders in an already medically and psychologically vulnerable population; however, our study sample reported that they experienced a variety of negative impacts to their social relationships due to migraine, which suggests that migraineurs may lack social capital when compared with other chronically ill groups. Use of adaptive coping strategies and healthy lifestyle choices can also act as protective factors for individuals with migraine headaches. The active engagement with biological, psychological, and social protective factors can help to reduce the "suffering" experienced by migraineurs and give patients more control over their headache pain and symptoms. Migraine practitioners should make efforts to understand and alleviate the impact of stress on the diverse areas of life in migraineurs. Where this is beyond the scope of the prescriber's medical practice, they should consider referrals to pain psychology specialists, other mental health providers with specific training in the biopsychosocial models of pain treatment, or pain support groups.
limitations
There are some limitations to the present study. The principle limitation is the bias, such as self-selection and selfidentification, inherent in studying an online sample. The lack of information collected relating to participant race, education, and socioeconomic status also limit the generalizability of this study.
Conclusion
The present study extends the body of literature characterizing treatment patterns, disorder characteristics, and disability profile in migraineurs. Migraine-associated stress was found to be a much more diverse experience than previous researchers have described. Negative social and personal impacts of migraines were found to be common in this sample. Migraineurs were found to have tried many different treatments but were, overall, unsatisfied with their efficacy. The highly personal experience of migraine is seen in the variety of negative life impacts secondary to migraine headache, diverse comorbid disorder profiles, and variable responses to migraine treatments. Each of these factors contributes to the idiosyncratic experience of migraine. Practitioners must understand the personal experience of each migraineur in order to match their patient to a treatment which will alleviate the social, biological, and psychological experiences of migraine headache. Migraine headaches were found to impart a wide variety of possible negative impacts on an individual's life. Migraineurs who experienced more frequent symptoms tended to experience more negative life events related to migraine, as well as more medical or psychiatric comorbidities. The vast majority of participants reported having seen a physician about their migraine headaches, with financial concerns being the most commonly reported reason as to why participants had not contacted a physician about their migraines. Similarly, many participants had attempted various medications in the past; however, these medications were frequently discontinued due to side effects, and the cost of medication was reported to be the most common reason for discontinuation. While our study sample reported common migraine symptoms in similar proportions to previous research, some symptoms reported by our study participants, such as puffy eyelids and food cravings, have not been examined in previous migraine research. Future research into migraine headaches should explore the social network impacts of migraine headache and examine this impact on the effectiveness of treatment and delivery of care for migraineurs.
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Submit your manuscript here: http://www.dovepress.com/journal-of-pain-research-journal The Journal of Pain Research is an international, peer-reviewed, open access, online journal that welcomes laboratory and clinical findings in the fields of pain research and the prevention and management of pain. Original research, reviews, symposium reports, hypothesis formation and commentaries are all considered for publication. 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. Disclosure CDM, ABW, and AB declare that they have no competing interests to report. AB is an employee of Health Union, LLC, which owns Migraine.com. No other relationship, bias, or ethical conflict exists among the authors of this work. | 2016-10-26T03:31:20.546Z | 2015-08-12T00:00:00.000 | {
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258257719 | pes2o/s2orc | v3-fos-license | Biofertilizer effect of some zinc dissolving bacteria free and encapsulated on Zea mays growth
Crop nutrition depends on zinc for enzymatic, oxidative, and metabolic processes. In the current study 20 different bacteria were isolated from five soil samples collected from different fields in Egypt. Bacterial isolates were screened for their ability to solubilize insoluble zinc oxide and zinc carbonate. The ability of selected isolates to tolerate soluble zinc was determined using different concentrations of (ZnSO4). Three bacterial isolates were selected with efficiency in solubilizing zinc oxide and zinc carbonate while tolerating high levels of soluble zinc. Molecular identification by 16S rRNA sequencing of the chosen isolates identified them as B3 (Acinetobacter calcoaceticus), B5 (Bacillus proteolyticus) and C6 (Stenotrophomonas pavanii). Sodium alginate beads formulated with the isolated bacteria were tested for stability under different storage conditions for 3 months. A pot experiment was conducted to study and compare the effect of using chosen isolates as an in vivo Zn solubilizer with amended ZnCO3 either alone or embedded in beads as carrier in the soil and its effect on growth parameters of Zea mays after 2 months. There was an increase in Zn uptake in all treatments compared to the control. However, plants grown in a pot treated with ZnCO3 and Acinetobacter calcoaceticus showed the highest zinc content and plant dry weight as compared to the control. Finally, selected isolates in both free and encapsulated forms showed improved plant growth parameters and higher zinc content and can be applied as biofertilizers to enhance soil fertility. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-023-03537-5.
Introduction
Soil zinc deficiency affects millions of cropland worldwide, and is particularly prevalent in developing countries. In plants, Zinc is involved in carbohydrate metabolism (Alloway 2008). Zinc is imperative for both human development and crop production. Egypt's soils are among those that tend to be deficient in zinc (Khafagy et al. 2017), soils in areas where wheat is grown often have extremely low levels of plant-available P and Zn, causing widespread P and zinc deficiency in this crop (Kotb 2007). Zn deficiency can affect the development of plants and animals since Zn is a regulatory co-factor and structural constituent in proteins and enzymes involved in many biochemical pathways (Cakmak et al. 2017). In addition to carbohydrate metabolism, photosynthesis, and glucose metabolism, these enzymes are implicated in starch and sugar metabolism. In addition, Zn is also vital for protein metabolism, auxin metabolism, pollen formation, maintenance of the integrity of biological membranes and those related to resistant to pathogens (Rashid 1996). Zn acts as a significant antioxidant. The lack of zinc in plants can lead to retarded shoot growth, chlorosis, reduced leaf size, susceptibility to heat, light and fungal infections, as well as affecting grain yield, pollen formation, root development, and water uptake (Tavallali et al. 2010). Almost half of the world's cereal crops are grown on zinc-deficient soils; as a result, zinc deficiency in humans is a widespread problem. Soil Zn content is determined by the geochemical composition and weathering of the parent rock. Insoluble Zn cannot be assimilated by crops, resulting Communicated by Yusuf Akhter. in Zn deficiency; more Zn fertilizers are applied to crops to combat Zn deficiency. However, this technique is costly and can be harmful to both human health and the natural environment. Thus, eco-friendly and cost-effective agrotechnologies are needed to increase crop yield and reduce Zn deficiency (Khanghahi et al. 2021). When the concentration of Zn available in soil becomes less than 0.5 mg Zn/ kg dry soil, deficiency symptoms become visible (Alloway 2001). Despite most soils having a total Zn content of around 50-100 mg l −1 , soil solutions are usually extremely low in Zn concentration (0.002-0.196 mg l −1 ). (Srivastava and Gupta 1996). Fertilization appears a quick solution to rectify the nutrient deficiency but the cost of micronutrient fertilizers is high. The continuous use of inorganic fertilizers may cause damage to the physical, chemical and biological properties of soil, which could lead to the decline of soil fertility. Various methods have been used including zinc sulfate (White and Broadly 2005) or Zn-EDTA (Karak et al. 2005) as zinc fertilizers. However, their usage is not economical feasible or environment friendly and they are transformed into insoluble complex forms within 7 days of fertilizer application (Rattan and Shukla 1991). The use of plant growth-promoting microorganisms is a novel approach in this respect (Alavi et al. 2008). As bacteria release chelating metabolites in the rhizosphere of plants, siderophores are considered important reserves of micronutrients, like Zn and Fe that are easily available to plants. (Ahemad and Kibert 2014). Microbial siderophores make complexes with Zn and increases plant uptake (Madsen et al. 2012). There have been previous reports on transforming insoluble Zn into plant-accessible, soluble forms by soil microbiome. Bacterial genera, such as Pseudomonas, Bacillus, Acinetobacter, Azotobacter, Azospirillum, Gluconacetobacter, Burkholderia, and Thiobacillus have shown their ability to solubilize Zn (Bhakat et al. 2021). ZSB have the ability to improve crop quality via producing various phytohormones and soluble nutrients (e.g., P and K), synthesizing exopolysaccharides and siderophores, and reducing environmental stresses (Gupta et al. 2022). Using a pot experiment as a model, this study investigated the selection of powerful Zn solubilizing bacteria that could infiltrate edible parts of the crop and improve its Zn accumulation. The chosen bacteria will also be formulated in sodium alginate beads and their viability will be tested over 3 months during storage.
Determination of zinc solubilization activity by isolated bacteria
The zinc-solubilizing potential of twenty selected bacteria was evaluated using two insoluble zinc sources: zinc oxide (ZnO), zinc carbonate (ZnCO 3 ), and a combination of the two. The bacterial isolates were spotted on media in triplicate and incubated at 28 ℃ for 5 days to check for clear halo zones. By measuring the colony and halo zone diameter, hydrolysis capacity (HC) was calculated as follows: diameter of clear zone/diameter of colony (Omara et al. 2016).
Determination of zinc tolerance by zinc-solubilizing bacterial isolates
At low concentrations, zinc is a nutrient, but at high concentrations, it is toxic. The ability of selected bacterial isolates to tolerate solubilized zinc was determined under in vitro conditions in nutrient broth containing different concentrations of soluble zinc (ZnSO 4 ). The nutrient broth was prepared and ZnSO 4 was integrated into the broth in such a manner that the final concentration of zinc was (20,40,50,60,80,100,150,200,300,400 and 500) mg kg −1 . These solutions were divided in 10 ml quantities in test tubes, sterilized and inoculated with 0.1 ml of each of the tested isolates. An un-inoculated control was also maintained. The total zinc-solubilizing bacterial population was assessed by plating on nutrient agar medium. The highest concentration at which poor growth was observed was taken as a tolerance level (Nandal and Solanki 2017).
Molecular characterization of bacteria
The most efficient and tolerant isolates were chosen for molecular identification at Sigma Scientific Services Co., Giza, Egypt. DNA of the test bacterial isolates grown in nutrient broth was extracted with GeneJet Bacterial Genomic DNA Extraction Kit (Fermentas). The 16S rRNA gene of the isolate was amplified using universal primers forward and reverse (F, 5-AGA GTT TGA TCC TGG CTC AG-3 and R, 5-GGT TAC CTT GTT ACG ACT T-3) used to obtain a PCR product of ∼1.5 kb. The sample was placed in a hybrid thermal reactor thermocycler (Maxima Hot Start PCR Master Mix (Fermentas), initially denatured (enzyme activation) for 10 min at 95 ℃ for one cycle and denaturated for 30 s at 95 ℃, annealing for 1 min at 65 ℃ then extension for 1 min at 72 ℃. This was followed by a final elongation step for 10 min at 72 ℃. The PCR products were analyzed on 1% (w/v) agarose gels and sent to GATC (Germany) for sequencing by ABI 3730xl DNA sequencer. Sequence data were imported into the BioEdit version 5.0.9 sequence editor; base-calling was examined, and a contiguous sequence was obtained. Sequences used in the phylogenetic analysis were obtained from the RDP and GenBank databases. A phylogenetic tree was constructed using the neighbour-joining method (Omara et al. 2016).
Preparation of inoculums
A loop full of each tested zinc-solubilizing isolate on nutrient agar slant was individually inoculated in 250 ml nutrient broth medium in 500 Erlenmeyer flask at 28 ℃ in a shaking incubator for 24 h at 150 rpm.
Formulation of selected zinc-solubilizing bacteria with Alginate beads preparation
Alginate was prepared according to Bashan (1998) and Draget et al. (1997). Sodium alginate solution (6% w/v) were autoclaved at 121 ℃ for 20 min, then cooled, and mixed slowly with growth of each strain at the rate of (1:1 v/v). The alginate-cells mixture was stirred gently for 30 min at 100 rpm to be homogenous. For preparing the beads, the mixture was added drop wise, with the aid of a micropipette, to 0.1 M CaCl 2 solution. The CaCl 2 was then removed from the solution and the beads were washed twice with distilled sterile water. The beads were stored on sterile plates at room temperature and in the fridge. Viability of bacteria was tested every month for 3 months where 1 g alginate beads samples were rehydrated with NaCl solution (0.8% W/V, pH 7) while shaking based on Ivanova et al. (2005) then serial dilution of rehydrated bead sample and plate counting method was used for 10 7 dilution of each (Mohamed et al. 2016).
Pot experiment to test the effect of biofertlizer on plant growth
Using sterile soil, the experiment was conducted in the greenhouse of the Microbiology department at Ain Shams University. The temperature during the experimental period (September and October) ranged from 30 ℃ to 37 ℃ with 10-12 h daylight and 60-65% relative humidity. According to the experiment plan, eight treatments referred to in (Table 1). Experiment was conducted in 18 cm pots filled with 2 kg of soil inoculated with 3 ml of each bacterial inoculant (B3, B5 and C6) and 120 beads. Each bead was 25 mm (B3 beads, B5 beads and C6 beads). The experiment was done in duplicate and arranged in a random pattern. Grain of maize (Zea mays) surface sterilization was performed in a 3.5% (w/v) solution of calcium hypochlorite for 10 min then submerged in distilled water twice, and afterwards each pot was seeded with one grain (Javed et al. 2018). Soil sample was sent to the research laboratories complex in Cairo University, Egypt for physical and chemical analysis of soil (Vaid et al. 2014). After 2 months, plants were harvested and dried in an electric oven at 70 ℃ for 72 h and dry weight was measured.
Zinc content assay of plant materials
Samples were sent to Research laboratories complex in Cairo University, Egypt, for Zn content analysis using atomic absorption technique (Thermo Scientific iCE 3300, German) according to Christian and Feldman (1970).
Isolation and screening of zinc-solubilizing bacteria on solid medium
20 isolates obtained and purified from soil samples were screened for hydrolysis capacity (HC) according to the diameter of the clear zone and of the colony on modified Bunt and Rovira solid medium containing insoluble ZnO, ZnCO 3 and a combination of both. The results showed that most isolates were able to solubilize zinc to some degree but the most potent Zn-solubilizing isolates were A1, A5, B1, B3, B5, C1, C6 and D4, so they were selected for further studies (Fig. 1).
Determination of Zn tolerance for the selected isolates
The solubilization of zinc might limit the growth of bacteria at higher levels. In vitro testing was performed on selected isolates using nutrient broth containing different concentrations of soluble zinc (ZnSO 4 ) in order to determine their ability to tolerate solubilized zinc. The results in Table 5 showed that most of the isolates were tolerant isolates as they were able to grow and tolerate up to 400 mg kg −1 of ZnSO 4 where only B3, B5 and C6 were able to grow at 500 mg kg −1 of ZnSO 4 .
Characterization of isolates
Bacterial isolates designated as B3, B5 and C6 were isolated from mature compost. Colony morphology and Gram stain proved that: B3 is white, flat, entire colonies with dry surface and Gram negative cocco-bacilli, B5 is white, raised, entire colonies with smooth surface and Gram positive spore forming and C6 is transparent, raised, entire colonies with glossy surface and Gram negative rod-shaped. The selected isolates
Storage experiment
Since it is imperative the biofertilizer agent be viable during storage, the viability of the selected efficient isolates was determined with formulation in sodium alginate beads (Fig. 6) and at intervals during storage at room temperature and in cold storage for 3 months (Table 7). The results showed that the viable count of isolate B3 was 8.8 log 10 CFU/ml at the start then was 8.8 log 10 CFU/ ml in the 1st month and remained 8.8 log 10 CFU/ml after 3rd month of storage in room temperature also in fridge. It showed stable viability then decrease slightly (0.1 log 10 CFU/ml) after the 3rd month, For the number of B5 bacteria it started from 9.2 log 10 CFU/ml and maintain the same count till the last month of storage in room temperature while, it increased by 0.1 log 10 CFU/ml after the 3rd month in the fridge. Similarly, the number of C6 bacteria started from 8.9 log 10 CFU/ml reached 9.15 log 10 CFU/ml the 1st month. Then, it decreased to 8.9 log 10 CFU/ml in the last month at room temperature while, it increased to 9.1 log 10 CFU/ml after the 3rd month in the fridge.
Pot experiment
Plant experiment was carried out using soil, which was analyzed to determine its physical and chemical characteristics (Table 8). Figure 7 showed the pots at different growth stages. The results showed considerable increase in shoot and root lengths, fresh and dry weights in all treatments over the negative control. This is because no zinc or bacteria was added to the sterile soil. As showed in (Table 9)
Zinc content assay of plant materials
The results showed that negative control plants where no zinc or bacteria was added to the soil had far lower zinc levels than positive control plants (Fig. 8). On the other hand, all treatments resulted in an increased zinc content of the analyzed plants. In addition, plants grown in the pot with free B3 isolate as a biofertilizer had the highest zinc content (370.20 mg kg −1 ) while plants with B3 in beads had only 212.61 mg kg −1 ; meanwhile, plants with free B5 inoculum showed higher zinc concentrations than those grown in beads formulated with the same isolate. The zinc content of beads loaded with isolate C6 was 358.28 (mg kg −1 ), while the zinc content of free C6 isolate inoculum was 299.13 (mg kg −1 ). . 7 Pot experment soil inoculated with C6 beads compared to negative control during different growth stages a 1 week, b 2 weeks, c 3 weeks, d 4 weeks, e 5 weeks, f 6 weeks, g 7 weeks and h 8 weeks Finally, it is clear that both selected isolates and isolates embedded in sodium alginate beads improved zinc content and growth of plants.
Discussion
Globally, zinc deficiency in soil is a major problem because plants' Zn requirements are rarely met (Sillanpaa 1990). There is an estimated 50% shortage of plant-available zinc in soils used for cereal production around the world (Graham and Welch 1996). Almost all crops and pastures worldwide suffer from zinc deficiency, leading to severe yield losses and nutritional deficiencies. This is particularly the case in areas of cereal crop (Alloway 2008). Fertilizers are not always effective in correcting Zn deficiency due to economic and agronomic factors. There are several factors that contribute to reduced Zn availability in developing countries, including topsoil drying, soil constraints, disease interactions, and fertilizer costs. (Graham and Rengel 1993). Bacteria play a vital role in the processes of environmental cycling such as metal solubilization into soluble forms that are more suitable for uptake by plants. This study is focused on the isolation and identification of zinc dissolving bacterial strains and their ability to be used as biofertilizers based on their ability to solubilize zinc. Twenty zinc-solubilizing bacteria were isolated from soil collected from different farms in Egypt. Bhatt and Maheshwari, (2020) have previously isolated zinc solubilizing bacteria from cow dung using serial dilution protocols on Bunt and Rovira media containing 0.1% zinc sources, ZnO and ZnCO 3 , incubated for 1 week at 30° C. In contrast, Javed et al. (2018) stated that the plate method is limited and used both qualitative and quantitative methods to isolate zinc solubilizing bacteria. This study showed the ability of most isolates to tolerate soluble zinc in the form of (ZnSO 4 ) up to 400 mg kg −1 only B3, B5 and C6 were able to survive 500 mg kg −1 . Metal resistance mechanisms are highly dependent on metal interactions with cells, and the most likely cause of such high resistance is either bioaccumulation or biosorption. Atomic absorption spectroscopy studies revealed zinc bioaccumulation by zinc tolerant bacteria (ZTB). The ZTB strains were found to produce a significant amount of exopolysaccharide (EPS) under Zn stress. EPS mediated Zn biosorption mainly occurs due to the interaction between positively charged Zn ions and negatively charged EPS on the cell surfaces (Gupta and Diwan 2016). Which was supported by Pramanik et al. (2018) they isolated three bacterial strains and selection was based on their ability to grow in media containing 500 mg kg −1 of soluble zinc (ZnSO 4 ). Similarly, Saravanan et al. (2003) results showed that both test isolates were able to survive 500 mg kg −1 but one was inhibited after 8 days incubation. On the contrary, Nandal and Solanki. (2017) showed that most of their isolates were only able to grow up to 100 ppm of ZnSO 4 and only one isolate was able to tolerate 200 mg kg −1 . Solubilization of Zn compounds by bacteria depends on organic acid production, especially 2-ketogluconic acid and H + , as well as other metabolic products, siderophores, and CO 2 (Nautiyal et al. 2000). Sodium alginate was chosen as a carrier and tested for the ability to keep the biofertilizer agent viable during storage. Viability was compared during storage at both room temperate and fridge. Viable counts results showed that biofertlizer agents remained viable in both storage conditions during 3 months, where only B3 fridge sample showed slight decrease from 8.8 log 10 CFU/ml to 8.7 log 10 CFU/ml by the end of the storage period while all other samples showed stable counts. These results agreed with the results by Mohamed et al. (2016) who, recommended sodium alginate beads as best carrier as it is a cheap and easily used and stored. Encapsulation using alginate exerted high stability densities up to the end of 6 months storage period at 5, 20 and 30 ℃, likewise this study showed that sodium alginate beads are able to maintain viability of bacteria for 3 months in room temperature and fridge. The results agreed with those of Bashan (1998) who mentioned that bacteria could survive in alginate beads for long periods. Moreover, Ivanova et al. (2005) found that total number of bacteria decreased quickly in the first 7 days of storage, but total number of viable bacteria remained stable for 6 months of storage. Also, plant growth-promoting bacteria, Bacillus subtilis and Pseudomonas corrugata, immobilized in sodium alginate-based formulation were evaluated for t heir sur vival, viability and plant growth-promoting ability after 3 years of storage at 4 ℃ (Trivedi and Pandey 2008). Populations of both bacterial isolates recovered from the immobilized sodium alginate beads were in the order of 10 8 cfu/g. The plant-based bioassay indicated that the plant growth promotion ability of both of the bacterial isolates was equal to those of fresh broth-based formulations. The bacterial isolates retained root colonization, and antifungal and enzyme activities in the alginate-based formulation during storage (Trivedi and Pandey 2008). In this study, three bacteria were selected based on their ability to solubilize zinc in different forms and their ability to tolerate high amounts of zinc and were identified as B3 (Acinetobacter calcoaceticus), B5 (Bacillus proteolyticus), C6 (Stenotrophomonas pavanii). Plant experiment was very helpful for analyzing the effects of microbial species on various plant growth parameters as well as on zinc content and evaluating their efficacy under simulatory field conditions when maize seeds were planted in soil inoculated with these strains, they showed enhanced growth as well as increase in zinc content over both negative control where no zinc or bacteria was added as well as positive control where no bacteria was added only ZnCo 3 was added. The results varied with inoculation of bacterial isolate alone or as beads with ZnCo 3 . There were instances when inoculation of bacterial isolate showed better results than the addition of beads as with B3 as well as B5 with B3 isolate having highest zinc continent of 370.20 Zn (mg kg −1 ), while C6 isolate the beads had better results where encapsulated C6 had second highest zinc content 358.28 Zn (mg kg −1 ). Similarly, Goteti et al. (2013) demonstrated that inoculation with plant growth-promoting rhizobacteria significantly enhanced the growth of maize in all dimensions. While, Omara et al. (2016) indicated that inoculation with E. cloacae alone or with different zinc applications variably increased zinc percentage of Zea mays with the highest content of Zn in plants reported for inoculation with E. cloacae combined with ZnO. Similar approach by Zaheer et al. (2019) showed that Pseudomona ssp. strain AZ5 and Bacillus sp. strain AZ17 inoculation improved considerably the number and dry weight of nodules, grain, and straw weight, and uptake of P and Zn of the chickpea cultivar compared to non-inoculated control. A similar finding was made by Kamran et al. (2017) using Zinc solubilizing Pseudomonas fragi, E. cloacae, and Rhizobium sp. When they inoculated wheat plants with these strains, showed enhanced shoot and root length and weight as well as zinc content. While, Yasmin et al. (2021) findings showed that In the treatment with Zn-solubilizing bacteria P. protegens RY2 and ZnO as zinc source, zinc content in both root and shoot was significantly (P 0.05) higher than in the control treatment.. Plant growth and development are enhanced by zinc-solubilizing bacteria colonizing the rhizosphere and intensifying zinc bioavailability by solubilizing complex zinc compounds. Enhancement of the soil microbiome has the potential to increase crop production (Yuan et al. 2019), and reduce chemical inputs (Thijs et al. 2016), resulting in more sustainable agricultural practices. In conclusion, the current study findings represent contribution to achieve sustainable agriculture by introducing a cheap biofertilizers of three selected different bacteria formulated in stable commercial form. Sodium alginate encapsulated biofertilizers displayed viability at both room and fridge temperatures during storage and lasting for 3 months. And also, they showed enhanced zinc content and increased growth parameters ofthe plants during a pot experiment conducted in greenhouse. Hence, can be used for biofortification of Zea mays, which in turn improves human and animal health.
Authors contribution
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by AAYAS and HAIY. The first draft of the manuscript | 2023-04-22T06:16:07.336Z | 2023-04-21T00:00:00.000 | {
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37595627 | pes2o/s2orc | v3-fos-license | Can Insects Develop Resistance to Insect Pathogenic Fungi?
Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25 th generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses. Copyright: ß 2013 Dubovskiy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. VK) and the DFG Priority Program 1399 ''Host-Parasite-Coevolution – rapid reciprocal adaptation and its genetic basis'' to AV (VI 219/3-1).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
Insects are predominantly dependent upon cuticular, humoral and cellular defenses to resist fungal pathogens. The cuticle is the primary and possibly the most important barrier to fungal infection. Fungistatic fatty acids, phenoloxidases and melanins can help resist penetration of the cuticle [1]. If the pathogen is able to breach the cuticle, it then has to contend with the host's humoral and cellular defenses [2]. The latter consist of hemocytes, which will participate in wound healing and encapsulate fungal elements too large to be phagocytosed. Key humoral elements include phenoloxidase, reactive oxygen species and antimicrobial peptides [3]. Phenoloxidase synthesizes melanin, a highly fungitoxic compound which is deposited on the fungal surface and may block fungal development by encapsulating the pathogen in a melanic sheath [4,5]. A wide range of antimicrobial peptides (AMPs) have been reported in insects with most of them showing antibacterial activity while relatively few have been identified with antifungal activity [6,7,8,9]. Some AMPs appear to be peculiar to specific insect species while others have been reported in several insect species suggesting that some share a common ancestry while others have evolved independently [10].
Insect pathogenic fungi, of which there are over 700 species, have evolved to counter the host's defenses using a combination of enzymes to penetrate the cuticle and access the nutrient rich contents of the hemocoel [11]. During the colonization phase, the pathogen will produce a wide range of secondary metabolites that may suppress the host's immune system [12,13] and concomitantly prevent secondary infections. The type and quantity of metabolite produced in vivo is dependent upon the host species and fungal strain [14,15]. Fungi have evolved additional strategies to evade the host immune defenses. For example, hyphal bodies of Metarhizium anisopliae, during colonization of the hemocoel, will mask the cell wall by coating it with collagen-like material [16]. The integument, therefore, may represent the best opportunity for the insect host to detect and incapacitate the fungal pathogen.
There is much interest in insect pathogenic fungi because they are considered to offer an environmentally friendly alternative to chemical pesticides, which have been withdrawn or to which pests have developed resistance. Strains of fungi have been identified that kill both crop pests as well as pests of veterinary and medical importance such as ticks, midges and mosquitoes [17,18,19]. Of these fungi, the hypocrealean ascomycetes M. anisopliae and Beauveria bassiana are the best characterized and the most widely used in biological control programs. About thirteen species or subspecies of both fungi have been formulated and registered as mycoinsecticides or mycoacaricides [20]. Of 171 products reviewed by [20] ca. 68% were products based on Beauveria and Metarhizium. The use of these fungi is expected to increase following new EU legislation particularly EC regulation 1107/ 2009 and Directive 2009/128/EC which make it obligatory for EU member states to implement the principles of Integrated Pest Management with priority to be given to non-chemical methods of pest control.
The increased use of insect pathogenic fungi raises the question: will insects develop resistance to these agents in the same way that they developed resistance to chemical pesticides? Resistance is a major concern of producers of these fungi as biological control agents (BCAs) as they need to recoup development costs, but also of end users who are trapped between a diminishing number of chemical pesticides and the lack of safe alternatives.
Insect species, populations and intrapopulation groups differ in their susceptibility to fungal infections, which possibly reflects adaptation of the immune defenses to local conditions. Differences may be linked to a wide range of factors including the presence or absence of symbionts, density-linked melanism or genetic variation [21,22,23,24,25,26]. The darkened cuticle of melanic insects intrinsically confers some degree of resistance to insect pathogenic fungi [27,28,29].
Studying induced resistance poses many challenges because of the large number of parameters that need to be considered, often involving complex physiological responses. One approach is to select for increased resistance to a natural pathogen under specific laboratory conditions, then to identify correlated responses to selection and associated cost-benefits. Kraaijeveld and Godfray [30] used such an approach to explore the evolution of resistance to B. bassiana in Drosophila melanogaster and found no evidence of resistance after fifteen generations of selection. The authors did note increased late-life fecundity in selected lines suggesting evolved tolerance of fungal infection. In the absence of fungal infection, these selected flies had lower fitness than control flies indicating a trade off with increased tolerance [30]. In another study, exposure of D. melanogaster over twenty six generations to an antagonistic (but not true insect-pathogenic) fungus Aspergillus nidulans, resulted in selected lines with no increase in resistance but a reduced sensitivity to sterigmatocystin, a toxin produced by this fungus [31]. The underlying mechanism(s) for the increased toxin specific tolerance is unclear.
In this paper, we report an artificial selection experiment designed to explore the evolution of resistance in a melanic morph of the Greater wax moth, Galleria mellonella, to natural topical infection by B. bassiana. We ask whether this morph possesses additional traits that allow increased resistance to evolve, and, if so, at what cost. We recognise that resistance is not entirely mediated by an immune response and may involve multiple factors including stress management, energy re-allocation and tissue specific resource distribution. We therefore compared several of these parameters in G. mellonella larvae selected for resistance versus a non-selected line. In addition, we tested specificity by measuring the insects' responses to another pathogenic fungus, M. anisopliae.
Selection and Insect Survival following B. bassiana and M. anisopliae Topical Treatment
Larvae of a melanic phenotype of the Greater wax moth, Galleria mellonella, were selected for resistance to B. bassiana over 25 generations (Table S1). Survival assays conducted on cohorts of the selected (S) and non-selected (NS) lines revealed significantly increased resistance (survival times) of the 25 th generation of the S line to B. bassiana compared with the NS line ( Figure 1A); (P = 0.01). Importantly, this S line did not show statistically significant cross-resistance to M. anisopliae than the NS insects ( Figure 1B). The defense responses of the S and NS lines to B. bassiana and M. anisopliae were investigated further to elucidate the underlying mechanisms for the increased resistance at species level.
Cuticular, Cellular and Humoral Immune Defenses following Topical Inoculation
The NS and S larvae had identical levels of basal (uninfected) PO activity in the hemolymph and integument (Figure 2A, 2B). However, during the early stages of topically-applied fungal infection, the cuticular PO activity in infected S, but not infected NS larvae, became elevated above uninfected larval levels at 24 h post inoculation (pi) for both B. bassiana and M. anisopliae infections (P,0.05, P,0.001 Figure 2A), with this being more pronounced in M. anisopliae treated insects. This coincides with peak fungal germination and penetration activities.
In contrast, hemolymph PO activity was only elevated significantly in NS larvae exposed to M. anisopliae (P,0.01 Figure 2B) while B. bassiana failed to trigger significant changes in PO activity in either line relative to the uninfected controls ( Figure 2B). Lysozyme-like activity was unchanged in all samples, irrespective of the insect line or treatment ( Figure 2C).
An elevated capacity for encapsulation was observed in uninfected S larvae compared with the NS line (P,0.05; Figure 2D), evidenced by a strong melanotic encapsulation of the nylon implant. However, this activity was significantly lower 24 h pi with B. bassiana for both lines ( Figure 2D; 1.16 times for S line P,0.01; and 1.30 times for the NS line larvae P,0.05 relative to the uninfected controls). Unlike B. bassiana, infection by M. anisopliae did not lead to changes in the encapsulation response in either line ( Figure 2D).
Immunity-and Stress-related Gene Expression after Topical Infection
Expression of seventeen immunity and putative stress management genes was investigated in the integument and fat body of uninfected control and fungal infected insects from both the S and NS lines. The expression data were complex, with some genes behaving differently under each experimental parameter. However, several important trends can be reported. The majority of the studied genes were expressed at a lower basal level in uninfected control S larvae compared with the NS larvae, in both the integument and fat body. However, a group of genes coding for four putative stress-management factors (Contigs 704, 17373, 15256, 03093) and one AMP (Galiomicin) exhibited different expression patterns, being slightly higher expressed in the integument (but not the fat body) of control S larvae compared with control NS insects (1.5-to 2.5-fold increase; Figure 3A, Figure S1 and S2).
Following topical fungal infection, the pattern of gene expression in the S-line insects infected with B. bassiana was strikingly different from the other three groups of infected insects. In the other groups (i.e. NS insects infected with either fungal species, and in S insects infected with M. ansipoliae), fat body expression was characterized by strong upregulation of the genes coding for AMPs, transferrin and Hsp90 (with M. anisopliae triggering the strongest responses; up to 910-fold in the case of Galiomicin and 50fold for Gallerimycin; P,0.0001 compared with uninfected larvae), while genes with putative roles in stress-management exhibited minimal changes or were mildly downregulated in the fat body after infection. The expression of most stress-management and immunity-related genes in the integument of these infected insects was also either unchanged or downregulated, although some AMP genes were upregulated in the integument of M. anisopliae infected NS insects. In contrast, in S-line insects infected with B. bassiana, seven of the nine putative stress management genes and six of the eight immunity-related genes were preferentially upregulated in the integument (1.5 to 4.3 fold), concomitant with very low or even downregulated expression of the same genes in the fat body ( Figure 3B, 3C, Figure S1, S2, S3, S4). The significance of this overall trend was confirmed by mega-analysis (P,0.01; Table S4). Furthermore, differences between gene expression in the two tissue locations was significant, i.e. B. bassiana induced significantly higher gene expression in the S-line integument compared with M. anisopliae (P,0.05; Table S5).
It should be noted that overall, and irrespective of the infection state of the insect, expression levels of the genes in the S-line fat body was significantly lower than in NS insects (P,0.01, megaanalysis; Table S4). Upregulation of AMP genes (Gallerimycin, Galiomicin, Gloverin, Cecropin-D, 6-tox) and Transferrin (a siderophore) was 5-10-times higher in fat body of NS and S fungi infected
Cost of Resistance on Life History Traits
Selected insects with enhanced resistance to B. bassiana did not display differences in pupal biomass, but larval development time was slightly increased (P,0.05) in S line insects compared with the NS line ( Figure 4A, 4B). Both NS and S lines exhibited reduced fecundity (as a consequence of melanism) but this was at equivalent levels in both lines (summarized in Table S2).
Discussion
The present study shows that insects can develop resistance to insect pathogenic fungi but the resistance is not absolute and is at a cost. A melanic morph of G. mellonella was found to have increased resistance to the insect pathogen B. bassiana following exposure of successive generations to this fungus. A combination of mechanisms was identified that can account for this resistance, some of which were specific for B. bassiana and others which were non-specific antifungal defenses. Both the S (resistant) and NS (susceptible) lines of this morph expressed a wide repertoire of inducible immune and putative stress management genes. In the S line these resources were re-focussed to the integument. These ''front line'' defenses were only activated by B. bassiana.
By concentrating its energies to the first and most important barrier to infection, the host delayed pathogenesis by B. bassiana but not M. anisopliae, demonstrating a lack of cross-resistance. Similarly, infected S line insects exhibited higher cuticular PO activity but not plasma PO or lysozyme. It is possible the S line had evolved an enhanced Beauveria recognition apparatus allowing for a more coordinated and targeted response which would account for the increased activity in the integument but subdued activity in the fat body, the main hemopoietic organ. The fat body occupies a large portion of the insect hemocoel and is the principle site for the synthesis of AMPs in insects exposed to immunogens, including pathogens, irrespective of point of contact or entry [32,33,34]. As far as we are aware, there are to date no reports of pathogen specific defense mechanisms involving re-allocation of systemic resources to localised tissues under or at risk of attack.
An insect's repair and stress pathways limit the damage inflicted by either pathogenesis and/or the host's own immune response, but little is understood about the relative contribution of damage to the outcome of pathogenesis and the mechanistic links between the immune system and remedial pathways [35,36]. This study shows elevated integumental expression of all but two of the examined putative stress management genes (i.e. 80%) in the S line in response to Beauveria but not Metarhizium. The fact that so many putative stress related genes were upregulated signifies their importance in defense responses yet their role is poorly understood and often overlooked. By working in concert with the AMPs they probably mitigate damage and initiate repair resulting in the increased resistance observed in the S line to B. bassiana. The importance of the putative stress-response genes is further emphasized in the M. anisopliae infected NS insects where upregulation of the AMPs without concomitant upregulation of the stress management genes failed to confer any resistance to the pathogen.
The current study suggests that the resistance in the S line is heritable and multi-factorial, comprising several different physiological traits prioritised not only in terms of location but also timing as they were activated concomitant with the period of fungal penetration of the integument. For example, the elevated cuticular PO activity would inhibit fungal growth through synthesis of melanin and its precursors and through melanin partially shielding cuticular proteins from degradation by Pr1, a major cuticle degrading protease and virulence determinant produced by B. bassiana and M. anisopliae during the infection process. Elevated expression of IMPI specifically induced by B. bassiana in the S line could inhibit metalloproteases produced by insect pathogenic fungi during the infection process [37,38,39,40]. Genes coding for antioxidants are also highly represented under the same conditions and may defend the host against reactive oxygen species generated by PO activity during cuticle penetration [41]. Interestingly, cecropins do have antifungal activity but have been reported as ineffective against B. bassiana in other experimental insect systems (e.g. [8]). Current knowledge is very limited regarding the role in G. mellonella of 6-tox, although related X-tox AMPs are considered to primarily perform opsonisation roles [42]. Gloverin is not currently known to have any potent antifungal activity, however, putative antifungal and antimicrobial peptides may fulfil different and currently unrecognized roles in different hosts and under specific infection conditions. In isolation, each of these responses are unlikely to sufficiently impart the observed resistance, however, their impact may be amplified by a synergy between these and other as yet unidentified traits.
There is growing evidence that insects can acquire long-term protection against pathogens through immune priming or transfer from the parent to the offspring, a phenomenon referred to as transgenerational immune priming [43,44]. A wide range of immunogens including pathogens enhance the host's immune system conferring greater resistance to subsequent exposure to pathogens but the underlying mechanisms of the response remains unclear [45]. The current study shows the 25 th generation S line larvae exhibit specific, enhanced resistance to B. bassiana without prior exposure to the fungus, suggesting the resistance may be due to specific transgenerational immune priming but with an unexpected degree of specificity and complexity. An earlier but weaker resistance was also observed in the S line which suggests a heritable and amplified immunocompetence. These observations warrant further investigations of possible underlying genetic and epigenetic mechanisms of resistance.
Melanism is strongly correlated with general pathogen resistance with this trait often being accompanied by a trade off in fecundity, development time and even expression of selected immune components such as lysozyme [22,23,29,46]. In the current study, the pupal weight of the melanic S and NS lines (which is directly correlated with fecundity) was similar and already pushed close to minimum. Therefore, the additional drain on resources required to boost antifungal defenses still further, comes not from compromising this life history trait but mainly via a re-allocation of the insect's immune defenses. It could be argued that resistance in S line insects would increase further with time but to meet the increased demand on resources may result in untenable sacrifices. This is in marked contrast with insects developing resistance to synthetic chemical insecticides where a slight change can have a profound effect [47,48,49] whereas resistance to B. bassiana clearly involved multiple array of interdependent traits. Increased insect resistance to a strain of B. bassiana is not a major threat to the use of insect pathogenic fungi as biocontrol agents for several reasons. Firstly, we show that there is no cross resistance to other fungi so the extra investment in defense offers no benefit against other pathogens (introduced or natural). Secondly, the investment in defense is at the expense of fecundity. Thirdly, the downregulation of the AMPs will probably predispose the insect to opportunistic microbial pathogens.
In conclusion, this work reports a previously overlooked adaptation strategy of an insect to a widespread, natural pathogenic fungus. Suppression of systemic responses allows for re-allocation and concentration of resources to the integumental ''front line'' defenses with an array of immune and stress management factors. This directional selection with B. bassiana was specific to this pathogen and not M. anisopliae. However, the less fecund insects are probably at no evolutionary advantage in the wild, and we postulate that the risk is small of fungal biological control agents failing in the field.
Insects
For artificial selection we used insects from a laboratory population of a melanic morph of the Greater wax moth, Galleria mellonella, from the Institute of Systematics and Ecology of Animals SB RAS. The starting population was separated into two lines: one line was exposed to the insect pathogenic fungus Beauveria bassiana and selected for increased resistance to the pathogen (S line), and the other line was the non-selected untreated control (NS line). The pertinent phenotypic attributes of these lines are summarized in Table S2. The defense responses of the 25 th generation of the S and NS insects to B. bassiana were compared to elucidate the resistance mechanism(s). Full details of insect rearing and selection are provided in the Text S1.
Fungal Infections
The susceptibility of S and NS lines to B. bassiana isolate Sar-31 was determined by natural topical application of conidia. Unlike the previous generations, insects from the 25 th generation were not exposed to fungi until they were used for these experiments. Each insect was dipped in an aqueous suspension of the pathogen for 10 s using a concentration of 7.5610 7 conidia/ml. Dipped insects were kept in Petri dishes (10 larvae/dish) until sacrificed. The uninfected control insects were dipped into distilled water (n = 60). To determine if there was cross-resistance to other species of fungal pathogen, repeat assays were performed as above, using the insect pathogenic fungus Metarhizium anisopliae isolate P-72. Larvae were observed daily for 10 days (up to pupation) for both B. bassiana and M. anisopliae infections. The emergent adults were monitored for several days to see what percentage were infected with the pathogen. All dead insects were removed and examined to confirm the cause of death. Both fungal isolates were from the Institute of Systematics and Ecology of Animals SB RAS culture collection. All insects used in these experiments were 6 th instar larvae raised in the same cohort. The experiment was repeated independently three times. The total number of individuals used from each line was 390 for the B. bassiana experiment and 285 for M. anisopliae.
Phenoloxidase Activity in Plasma and Cuticle
Larvae were topically infected with the fungal pathogens as described above, and at 24 post-inoculation (pi), cell-free hemolymph plasma samples and homogenized integuments were prepared for spectrophotometric analysis of phenoloxidase enzymatic activity using L-DOPA as a substrate, and expressed as a change in absorbance/min/mg protein. Uninfected insects were used as a control. The experiment was repeated independently three times. Full details are provided in the Text S1.
Encapsulation Response
To determine melanotic encapsulation responses to fungal infection, encapsulation assays were performed in NS and S larvae. Nylon monofilament implants were retrieved from the hemocoel and examined using image analysis software to quantify the extent of melanization. Full details are provided in the Text S1 and experiments were repeated independently three times.
Plasma Lysozyme-like Activity
Antibacterial activity in hemolymph plasma was determined by a zone-of-clearance assay using freeze-dried Mirococcus lysodeikticus as a substrate suspended in agarose. The radius of the digested zone was compared with a standard curve made with Egg White Lysozyme [50] and expressed as an EWL equivalent (mg/ml). The experiment was repeated independently three times. Full details are provided in the Text S1.
QRT-PCR Analysis of Insect Immunity-related Gene Expression
The expression of a range of G. mellonella immunity-related genes was quantified in fat body and integument samples dissected from S and NS larvae at 24 h after topical infection with B. bassiana or M. anisopliae. Gene expression was measured by realtime quantitative RT-PCR using normalised cDNA samples with the Rotor-Gene 6000 (Corbett Research), with Rotor-Gene SYBR Green PCR mix (Qiagen), relative to two reference genes, 18S rRNA (AF286298) and Elongation Factor 1-alpha (EF1; AF423811). Seventeen target genes were investigated, coding for the antimicrobial peptides gallerimycin, galiomicin, gloverin, cecropin D and 6-tox, the siderophore transferrin, the insect metalloproteinase inhibitor (IMPI), one linked to immune signaling (Contig 20004), three coding for heat-shock proteins (HSP-90, contig 21310 and 1489) whose activities ameliorate stress [51], two coding for enzymes dealing with oxidative stress (Contigs 17373 and 03093), one linked to G-protein coupled receptor activity and stress response (Contigs 15265), one involved in anti-apoptosis activity (Contig 5976) and two involved with cell proliferation (Contigs 704 and 233). Full details are provided in the Text S1 and Table S3.
Life History Traits
The following life history traits were monitored in NS and S insects: larval development time (from egg hatching to onset of pupation) and pupal weight.
Data Analyses
Data analyses were performed using GraphPad Prism v4.0 (GraphPad Software, USA) and Statistic v6.0 (StatSoft Inc., USA). Data were checked for normal (Gaussian) distribution using the Agostino-Pearson omnibus test, and if abnormally distributed a more conservative non-parametric analysis was applied. In Q-RT-PCR data with a Gaussian distribution, Grubbs' extreme studentized deviate (ESD) test was used to exclude extreme outliers. In order to assess overall trends associated with selection on basal and induced gene expression, the data from three independently repeated experiments were pooled for megaanalysis after confirming (by 2-way ANOVA) that ''experiment'', as treated as a variable, had no significant effect on the outcome. Individual gene comparisons were made with non-parametric oneway ANOVA (Kruskall-Wallis with Dunn's post test). Cox's proportional hazards survival regression was used to quantify differences in mortality rates after fungal infections between NS and S larvae. Larvae from bioassay experiment uninfected with the fungi all survived the duration of the experiment, for this reason, the uninfected treatment was ignored in the statistical analysis. One-way ANOVA (Kruskall-Wallis with Dunn's post test) was used to assess differences between lysozyme, PO and encapsulation responses in S and NS insects. Differences in life history traits were compared by non-parametric t-test (Mann-Whitney). Differences between NS and S larvae, or between treated and control samples, were considered significant when P,0.05. Expression of genes was assayed in fat body tissue by Q-PCR in uninfected animals, and in animals at 24 h after topical infections. Basal expression in uninfected S larvae (bar 1) is calculated as a fold change relative to NS uninfected larvae. Fold induction in NS larvae infected with Bb (bar 2) and Ma (bar 5) is also calculated relative to NS uninfected larvae. Fold induction in S larvae infected with Bb and Ma is calculated both relative to the S uninfected expression (bars 3 & 6) and relative to the NS uninfected baseline to indicate overall expression (bars 4 & 7). The mean DDCt values of 3 independent experiments are reported +/ 295% CI. a-P,0.05, b-P,0.01, compared with fold induction in NS infected same fungus (i.e. comparing S vs NS); c-P,0.05 compared with fold induction in the same line infected by Bb (i.e. comparing Bb vs Ma).
(TIF)
Table S1 Susceptibility of G. mellonella selected and non-selected lines to B. bassiana. Susceptibility of Galleria mellonella larvae of selected and non-selected lines to topical fungal infection with Beauveria bassiana (7.5610 7 conidia/ml). (DOC) | 2018-05-08T17:50:28.205Z | 2014-01-13T00:00:00.000 | {
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119072065 | pes2o/s2orc | v3-fos-license | Bound states of L-shaped or T-shaped quantum wires in inhomogeneous magnetic fields
The bound state energies of L-shaped or T-shaped quantum wires in inhomogeous magnetic fields are found to depend strongly on the asymmetric parameter $\alpha =W_{2}/W_{1}$, i.e. the ratio of the arm widths. Two effects of magnetic field on bound state energies of the electron are obtained. One is the depletion effect which purges the electron out of the OQD system. The other is to create an effective potential due to quantized Landau levels of the magnetic field. The bound state energies of the electron in L-shaped or T-shaped quantum wires are found to depend quadratically (linearly) on the magnetic field in the weak (strong) field region and are independent of the direction of the magnetic field. A simple model is proposed to explain the behavior of the magnetic dependence of the bound state energy both in weak and strong magnetic field regions.
I. INTRODUCTION
Recently, quasi-1D structures, such as quantum wires attract much attention due to the enhanced confinement of the reduced dimension and the possibility of tailoring the electronic and optical properties in applications [1]- [9]. Among the structures considered, the opened quantum dot (OQD) is one of the simpler mesoscopic systems in which the essential physics can be studied in great details. An OQD can be formed by additional lateral confinements [10,11] or by applying certain magnetic fields [12,13]. Electrons and holes are trapped at the L-shaped or T-shaped intersections because the singleparticle confinement energy can be found to be lower in the intersection of the arms. These OQDs are quite different from the traditional quantum dots, since there remain openings in such OQDs. Electrons in OQD systems are classically unbounded. However, recent experimental photoluminescence spectroscopy analyses [1,2,3] have manifested that there are bound states in such OQDs. The existence of bound states in OQDs essentially shows the confinement effect of the mesoscopic geometry in quantum mechanical region.
The exploration of the properties of bound states is a key to understand some recent optical and electrical experiments on T-shaped quantum wires and quantum dots [2,3,8,9,10,11]. The magneto-photoluminescence of T-shaped wires were measured recently [4]. The energy shift ∆E of PL peaks with magnetic field B applied perpendicular to the wire axis and parallel to the stem wire was measured. In these experiments, the information of exciton binding energy can be provided from the photoluminescence spectroscopy. However, it is unable to identify exactly the exciton binding energies unless we have the knowledge of the confinement energy of either an electron or a hole in quantum wires or quantum dots. Because they can not be extracted directly from magneto-optical data due to the nonlinearity of the systems. In a theoretical calculation of magneto-excitons in T-shaped wires [14], the observed field dependence of the exciton states for weak confinement was reproduced, however, the diamagnetic shifts calculated from perturbation theory is fail to describe the experimental results.
In this work, we consider two-dimensional OQDs which are formed at the intersection of the arms of L-shaped or T-shaped quantum wires when additional magnetic fields are applied perpendicular to the plane of arms. A T-shaped quantum wire can be obtained by first growing a GaAs/Al x Ga 1−x As superlattice on a (001) substrate, after cleavage, a GaAs quantum wire is grown over the exposed (110) surface, resulting in a T-shaped region where the electron or hole can be confined on a scale of 5-10 nm. The bound state energy of a charged particle (e.g. electron) in such an opened quantum dot will be affected by the asymmetric geometry of the system and the applied inhomogeneous magnetic fields. Intuitively, when the confinment along one arm of the quantum wire is increased, confinment along the orthogonal arm will decrease, because squeezing the electron or hole in one arm will result in pushing the electron or hole out of the quantum wire through the other arm. These pheonomena are not only interesting in physics but also have no classical correspondence. To our knowledge this squeezing effect have not been studied thoroughly. Furthermore, T-shaped semiconductor quantum wires could be exploted as three-terminal quantum interference devices, thus the study on the L-shaped or T-shaped quantum wire is also important in practical applications.
II. FORMULATION
In the present work, a two-dimensional T-shaped (TOQW) or L-shaped opened quantum wire (LOQW) is considered. A quantum dot with an area of W 1 × W 2 is formed in the intersection region while magnetic fields B 1 , B 2 and B 3 are applied perpendicularly to the other subregions of the TOQW as shown in Fig. 1(a). The LOQW as shown in Fig. 1(b) can be regarded as a transformation of TOQW in which the arm 2 is cut off. For simplicity, the boundaries are assumed to be a hardwall confinement potential, leading to the formation of a magnetically confined cavity in which the confinement of electron is enhanced. The transverse potential inside the TOQW or LOQW is assumed to be zero. The magnetic fields are assumed to be uniform in each individual subregion. Landau gauge is chosen for the vector potential in different subregions: The form of gauge guarantees the continuity of the vector potential at each interface. The origin is chosen at the center of the intersection region.The wavefunctions of the bound state n of an electron for different subregions I, II, III.IV are in region I; in region III; is the first subband energy in arm 1. One can note from the figure that the bound state energy becomes smaller as the arm ratio α becomes larger. For α = 1 (i.e. W 1 = W 2 ), r m = t m at zero magnetic field, the bound state energy is 0.92964E 1 . The bound state energy ε goes down and behaves like the curve 1/α 2 as the α is increased larger than 1.14. A deviation from the curve 1/α 2 is observed in the region of α ≤ 1.14 as shown in the inset of Fig. 2(a). The result can be ascribed to the fact that the bound state energy of the electron matches the subband energy of arm 2 due to the lateral confinement of region II. Since in this circumstance, 1/α 2 (π/W 1 ) 2 is equal to (π/W 2 ) 2 , which is the first subband level of the vertical wire. As the width W 2 becomes larger and larger, the energy level becomes lower and lower, and gradually coincides with the bound state level of the electron. Thus electron is unable to be bounded in the corner region any more. As the asymmetry becomes more prominently, the electronic energy becomes larger than the bottom of the subband of the wider arm. However, if the energy of the electron state is less than or just equal to the subband bottom, the electron is still bounded inside the corner and does not move to the right or to the left. Fig. 2(b) shows the bound state energy of the electron in a TOQW as a function of α. The bound state energy approaches unity as the width of the vertical arm becomes very small, and behaves like the curve 1/α 2 while α becomes larger. This is similar to the case of a LOQW. The reason of this result can be understood intuitively that the wavefunction of the electron is purged out of the vertical arm when it becomes very narrow, therefore, the energy of this state is close to the first threshold energy E 1 of the horizontal arm with a width of W 1 . This bound state of the electron exists as long as the vertical arm is infinite long, and is expected to disappear owing to the effect of leakage if the arms is finite in length.
The calculated bound state energy of a symmetric LOQW in magnetic fields as a function of the field strength f =hω c /E 1 is shown in Fig. 3(a) and (b), where ω c is cyclotron frequency of the electron. One can observe that the bound state always exists when the magnetic field is applied to both arms. The bound state depends linearly on the magnetic field in weak field region while quadratically in strong field region. increases monotonically as the magnetic field increases. However, the energy of the bound state is pushed up by the applied magnetic field, and then it goes up to E 1 when the magnetic field is applied to only one arm. Thus, the electron can escape via the field free arm. Fig. 4 presents the confinement energy in a symmetric TOQW versus the field strength when (a) all arms are acted by the same magnetic field B, (b) the two horizontal arms are acted by the same magnetic field, and (c) only the vertical arm is acted by the magnetic field. The same quadratic dependence of magnetic field of the bound state energy is revealed again for weak field, and the linear dependence appears in the strong field region as the case of LOQW. Obviously, the bound state of the electron in a TOQW system locates deeper than that in a LOQW, thus, the TOQW system has a weaker confinement potential than the LOQW system.
The magnetic fields introduce a depleting effect on electrons and add an extra potential surrounding the intersection region. The effective potentials introduced by the magnetic fields are k-dependent. For the bound state, these effective potentials are complex due to the pure imaginary {k}. One expects intuitively that the magnetic field adds the lowest Landau levelh ωc 2 =h eB 2m * directly to the quantum dot system an extra potential. Such levels are added into the wire arm regions. However, the field plays another role due to the essential physics of the magnetism. Qualitatively, one can understand the effect induced by the magnetic field on the bound state by considering an one-dimensional shallow quantum well with finite height U 0 . In the limit of shallow well, there is only one bound state exists in the well. Its level energy is given by E 0 = U 0 −(m * W 2 /2h 2 )U 2 0 , which is near the top of the well. As the magnetic field applies to the system, the bound state energy changes because the potential height is changed to U 0 + 1 2h ω c . The variation of the state level depends linearly on the potential height, i.e.
The variation of the state level by taking account the depletion effect of the magnetic field is assumed as Obviously, once we take the well shrunk into account, the quadratic form of the dependence of magnetic field has to be considered also. This simple model manifests the important geometric effect and the essential properties of magnetism at the same time. Since the shrinking of the geometric scale is no longer prominent in strong magnetic field region, the influence of the magnetic field on the electron becomes smaller. Thus, the bound state energy depends simply on the added effective potential, such that it seems likely to depend linearly on the magnetic field in the strong magnetic field region.
IV. SUMMARY
The effects of the asymmetric geometry and surrounding inhomogeneous magnetic fields on the bound state of L-shaped or T-shaped quantum wires are studied. When α increases, the bound state energy of the electron is lower as expected. On the other hand, when the applied magnetic field increases, the bound state level of the electron is pushed higher and higher and the electron begins to be unbounded if there is an arm with finite length which offers a passway for electron to leak out. Generally, the bound state level of an electron in a TOQW system is lower than that in LOQW system. This fact reflects the weaker confinement of the geometry. Parabolic dependence of the bound state energy of the electron in weak field region on the field strength is understood as a result of the depletion effect. In the contrast, linear dependence in high field region is found to be resulted from the additional effective potential due to the magnetic field.
This work is supported partially by National Science Council, Taiwan under the grant number NSC90-2112-M-009-018. | 2019-04-14T02:01:53.165Z | 2003-01-08T00:00:00.000 | {
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101090209 | pes2o/s2orc | v3-fos-license | Treatment of Olive Oil Mill Wastewater by UV-Light and UV/H2O2 System
Olive oil mills generates large volumes of wastewaters (OMW). The absence of legislation, which manages and obligates the treatment of OMW has led to the creation of large build-evaporation ponds as a solution for this environmental problem. The function of these ponds is the evaporation of wastewater during summer months. However, evaporation isn't enough to eliminate all wastewater in the rafts and this fact allows the generation of a great volume of concentrate wastewater with high organic load (COD = 0.538 g O2/L). The environmental impact produced by the accumulation of polluted wastewater demands the design of processes for OMW treatment. In this sense, the influence of ultraviolet light (UV) and the combined system of UV/H2O2, in the degradation of organic matter of OMW, were studied. UV-light application at a short time (<30 min) implies a removal values in COD = 15-22%, total carbon (TC), total organic carbon (TOC) and total nitrogen (TN) in the range 34% to 43%. The turbidity elimination was registered in the range 68% to 70%. In the case of combined UV/H2O2 system the removal percentages were 40-48% for COD, 39.451.9% for TC, 33.0-48.0% for TOC, 37.0-53.1% for TN, and 66.8-93.4% for turbidity.
INTRODUCTION
Currently, worldwide production of olive oil reaches three millions of tons per year, where Spanish production represents 45% and Andalusia region occupies 60% of total Spanish area dedicated to olive cultivation [1,2].
The main product of olive tree cultivation is olive oil, which is rich in phenolic compounds and thus very beneficial for human health [3][4][5]. Pressing (traditional system) and continuous (three-or two-phases) are the most important extraction processes, applied for olive oil production. Three-and two-phases extraction technologies differ in the water supplies. Large amounts of water are required to an extraction process with a three-phase decanter and large amount of a liquid byproduct is generated. This wastewater is characterized by its high organic load, which is not easily biodegradable and contains toxic constituents (e.g., phenols). Phenolic compounds inhibit OMW's biodegradation and are toxic at high concentrations, especially for microorganisms [4]. OMW are usually disposed to aqueous receptors or to soil directly (untreated or inadequately treated) [6]. * Olive-oil production processes were upgraded at the beginning of the 1990s through the introduction of twophases continue centrifugation system. In this system, a 'decanter' with two outputs is used. The first output is for olive oil (with some impurities) and the second output for a wet residue (named as 'Alperujo' in Spain) [7]. This system is more ecological friendly than previous other systems because of the reduction of water consumption leading on less volumes of wastewater generation. For this reason, it has been implemented in almost all Spanish olive oil mills [8]. However, they are generated 5.4 10 6 m 3 olive oil mill wastewater (OMW) on the world per year. Management of these large volumes is difficult because of the presence of high organic load [9]. Nowadays, Andalusia Government Good Practice Guidelines allow the accumulation and control of this kind of wastewaters into evaporation rafts [10].
However, it would be recommended recycle these large amount of wastewater in the same process or in irrigation. Many researchers are investigating about efficient and low-cost wastewater treatment methods. There are different kinds of treatment technologies: (i) biological methods; for example, aerobic or anaerobic digestion, composting or co-digestion [2,[10][11][12] and (ii) physico-chemical methods such as filtration, flocculation, membrane technologies and chemical oxidation [13,14]. OMW are successfully treated by advanced oxidation processes (AOPs). AOPs are usually simple methods, which generate no residues [15][16][17].
In this work advanced oxidation processes (AOPs), based on UV-light and UV/H 2 O 2 system were used for the treatment of OMW in order to determine the potential of UV-light alone and in combination with hydrogen peroxide in the degradation of organic matter. In this sense, the effect of UV-light versus time (up to 3 h) has been studied, as well as the influence of UV/H 2 O 2 system by applying different concentrations of H 2 O 2 (2.5%, 5%, 7.5%, 10%, 15%, 20% and 30%, w/v). Also, the composition of the wastewater during the campaigns of olive harvest 2013/2014 and 2014/2015 has been determined.
OMW Samples
The wastewaters used in this work were directly collected, in summer months (April-June), from accumulation rafts of three different olive oil mills in Seville (Spain), operating with two-phase continuous centrifugation process. These wastewaters are formed by olives and olive oil washing wastewaters plus all other wastewaters generated in the mill.
Installation
All experiments were performed in a photoreactor composed of a 1 L reactor tank with a UV-lamp covered by a quartz immersion tube and a quartz cooling jacket, a magnetic stirrer inside the reactor for blending the OMW and chemical reagent, PolyScience Chiller was used to eliminate the heat created by the UV lamp and controlling the temperature of wastewater in the reactor. A thermometer was located in the interior of the reactor to indicate the operating temperature.
The UV-lamp used has the following characteristics: UV Immersion lamp (model TQ 150; No 5600 1725; brand HNG Germany G4) with a length of total immersion 384 mm, length of the luminous part of the immersion 303 mm, position of the emission centre of the lamp 44 mm, power of the lamp 150 W and nominal level of emission intensity 200-280 nm.
Methodology
Firstly, the composition of the crude OMW during the campaigns of olive harvest 2013/2014 and 2014/ 2015 it has been studied.
Secondly, two experimental series were performed. In the first set the influence of UV-light alone in the degradation of OMW was examined. In this sense, two OMWs with different loads of organic matter (COD = 1944.2 and 6598.9 mg O 2 /L, TOC = 3929 and 1058.9 mg/L) were studied. In the second set, the effect of UV/H 2 O 2 system on the degradation of organic matter of OMW was studied. In this case, experiments with different concentration of hydrogen peroxide (2.5%, 5%, 7.5%, 10%, 15%, 20% and 30%, w/v) were carried out. In all experiments, samples were taken at different times (3, 10, 20, 30, 60, 120 and 180 min) and the following parameters were determined: pH of OMW, total carbon (TC), total organic carbon (TOC), inorganic carbon (IC), total nitrogen (TN), and NO 3 +NO 2 . In addition, at the end of experiments chemical oxygen demand (COD) and turbidity were determined.
To develop the experiment OMW was placed in the photo-reactor. The circulation of water refrigerated by the quartz-cooling jacket around the lamp was activated, the UV lamp was switched on, and the wastewater temperature was controlled. Once the desired temperature was reached (within a few minutes) the hydrogen peroxide (in the case of using UV/H 2 O 2 system) was added and time was started from zero.
In all experiments, the operation conditions were: pH = 3, temperature 293.15 K and agitation rate 150 rpm. The pH was adjusted to an initial value of 3 with 5 mol HCl L -1 .
Analytical Methods
The OMW used in this investigation was characterized by measuring pH, total solids (TAPPI T 11 m-59), ash (TAPPI T 15 os-58), electric conductivity, total iron concentration, total phenols, chemical oxygen demand, total carbon, total organic carbon, inorganic carbon, total nitrogen and NO 3 +NO 2 .
The pH-values were measured using a CRISON pH-meter, mod. LPG 22.
Electric conductivity was assessed with a CRISON conductivity meter, mod. Basic 30.
Turbidity was determined directly by using a HANNA turbidity meter, mod. HI93703.
The total solids were determined according to the weight loss of the sample after being placed in an oven at 378 ± 1K until reaching constant weight. The difference in weight with respect to the original sample, expressed in %, determined the moisture content. The total solids were calculated by subtracting between 100 and the moisture [15].
Ashes correspond to the mineral salts remaining after a waste sample was incinerated at 873 ± 25K for 3 h [15].
Organic matter was determined by subtracting between total solid and ash.
All iron ions were reduced to iron ions (II) and, in a thioglycolate medium with a derivative of triazine, formed a reddish-purple complex that was determined photometrically at 565 nm [18,19].
Total phenols and phenol derivatives reacted with a derivative thiazol, giving a purple azo dye, which was determined photometrically to 475 nm [18,19].
Chemical oxygen demand (COD) was determined by the photometric determination of the concentration of chromium (III) after 2 h of oxidation with potassium dichromate/sulfuric acid/silver sulfate at 421K [20]. At the time of the determination of COD was used manganese oxide to remove residual hydrogen peroxide, which remained unreacted in samples taken from the reactor [21].
Finally, total carbon (TC), total organic carbon (TOC), inorganic carbon (IC), total nitrogen (TN), and NO 3 +NO 2 were determined by using a Skalar total carbon and nitrogen analyser, mod. Formacs HT and Formacs TN .
CHARACTERIZATION OF OLIVE OIL MILL WASTEWATER (OMW)
The characterization of OMW can be seen in Table 1. First, in all cases it can be observed pH values above 7. These values are higher than normal values (pH < 7) for these wastewaters [22] probably due to mixed different waters in the raft. As water from washing olive-oil tanks (drinking water plus NaOH), or the wastewater from table olives industry with OMWs, which increases pH values. Moreover, high values of the parameters total solids, COD, TC, TOC, TN and electric conductivity were detected which is due to the concentration of them by evaporation effect [16,17]. Solely in the case of OMW from Seville 2 the COD value (510.9 mg O 2 /L) is low because the OMW, which accumulates in this raft, was only waters from olives washing. On the other hand, the high values of electric conductivity registered in the rafts Seville 3 and 4 is due to mixture of wastewater from olive oil industry with wastewater from table olives industry, which is rich in NaCl salt. Based on the characterization of raw wastewaters, obtained from evaporation rafts, and considering that the initial mean COD values of olives and olive oil washing wastewaters is 3000-4000 mg O 2 /L [16], it has chosen to use a wastewater with an initial COD value equal to 6454.8±307.4 mg O 2 /L for the experiments performance. Therefore, there have been mixtures of raw wastewaters to achieve this representative initial COD value (M1 to M5). Also, another mixture (M6) was prepared with a COD value equal to 1944.2 mg O 2 /L ( Table 2).
Direct UV-Light Effect in Wastewaters
It is well-known UV-light is able to improve natural oxidation [23]. Ultraviolet oxidation is a photochemical process where organic components are partially mineralized because of the absorption of this high-energy irradiation. As happens in natural system, organic matters are transformed into suite of unstable free organic radicals. Excited short-lived oxygen species are also formed in the water. Decarboxylation by oxygen-dependent pathway occurs, obtaining harmless inorganic photoproducts, such as dissolved inorganic species. In this sense, UV effect in the degradation of organic matter has been studied in photoreactors. Figure 1 shows UV-light effect on TC, TOC and TN parameters during the experiments using M5 wastewater. It can be observed that degradation occurs in one step by an instantaneous reaction in the first minutes (< 4 min). After that, no significant degradation was recorded (Figure 1). Similar behaviours were obtained in all experiments realized in this work. These results are similar to that reported by other authors which indicate UV oxidation allows sudden decomposition of toxic substances as nitrosodimetylamine (NDMA), hydrazine, 1,4-dioxane and methylthrethylbutaneethyl (MTBE) [24,25]. (1) Figure 2 shows the behaviour of normalized values of TC, TOC and TN versus time in both wastewaters (M5 and M6). Similar behaviour were obtained for all parameter (TC, TOC and TN). This similar behaviour obtained, for all parameters, confirm the independence of removal grade from the initial organic matter when direct UV-light was used to treat OMW. Table 3 shows the percentage removal of all parameters determined at the end of experiments. It can be seen that there are no significant differences between the removal percentages for the different parameters registered. In this sense, Hajjouji et al. [26] have obtained a COD removal percentage value slightly higher (%COD removal = 22%) than determined in this work when OMW was treated by photocatalysis using TiO 2 under UV-light during 24 h.
Effect of UV/H 2 O 2 System in Olive Oil Mil Wastewaters
Direct UV-oxidation is not enough for a complete oxidation of organic matter in OMW. In this sense, combined system UV/H 2 O 2 has been studied in order to improve degradation percentages of organic matter present in OMW.
Combined system UV/H 2 O 2 is based on two main reactions: direct conversion by UV-light (photolysis) and conversion by hydroxyl radicals. In direct photolysis, organic compounds absorb light energy in the UV spectra range (in this work UV-light has a nominal level of emission intensity in the range 200-280 nm and lamp power equal to 150W). This fact lead organic molecule into an excited state and results in photochemical transformation and degradation. On the other hand, the direct photolysis of hydrogen peroxide leads to the formation of OH-radicals [27,28]: These radicals react quickly with organic compounds converting them to other products, water and carbon dioxide, Ec. 3: In spite of using the same initial amount of H 2 O 2 , the initial hydrogen peroxide concentration in the solution used can have different effects [16]. For this reason different initial H 2 O 2 concentrations have been prepared and used in the photo-oxidation experiments. Figure 3 shows the oxidation of organic matter present in OMW by using direct UV-light or UV/H 2 O 2 system at different H 2 O 2 concentration. Specifically, the behaviour of the parameters TC, TOC and TN versus time. In all experiments it were observed higher organic load degradation using the combined system of UVlight/H 2 O 2 . The best results, when the initial oxidant agent was varied from 2.5% (w/v) to 7.5% (w/v) (Figure 3), were obtained. This fact can be explained by "scavenging" effect. According to this effect, high initial H 2 O 2 concentration lead on the production of other less reactive species such as hydroperoxides and organic radicals. The presence of these groups decreases the efficiency of the reaction because the lower formation of hydroxyl radicals [29]. These results indicate a significant reduction in the pollution load of water which means that the application of direct UV-light or the combined systems UV/H 2 O 2 can be of great interest if it's integrated into a complete treatment processes for OMW or other wastewaters. It should be noted, by way of example, their integration in biological processes, a first attack on the organic load allows a significantly reduction on the inhibition growth effect of this wastewater. Also, it can be adding a catalyst to this system if necessary.
CONCLUSION
Wastewater from olive oil mill show great variation in its physico-chemical composition depending on many factors. Mainly, i) The amount of potable water used in washing olives machines, ii) The amount of water added to vertical centrifugal to washing olive oil, iii) The commercial system (press, two phase or three phase) used.
The application of biological processes for OMW treatment has shown little efficacy due to existence of inhibiting growth compounds (mainly, fat matter, polyphenols, wastewater colour (e.g. when microalgae used). Direct application of UV-light or the use of UV/H 2 O 2 system allow a significant reduction in the organic load (up to 50%) and it is of great importance for biological treatment processes considering the elimination of inhibitors growth by these technologies.
ACKNOWLEDGMENT
The Junta of Andalusia and Ministry of Economy and Competitiveness (Spain) are acknowledged for financial support through Project Ref.: AGR-7092 "Application of advanced oxidation technologies for treating of washing wastewaters of olive oil and olives". | 2019-04-07T13:09:24.816Z | 2015-11-24T00:00:00.000 | {
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240558062 | pes2o/s2orc | v3-fos-license | PHARMACEUTICAL WORLD OF PERMEATION ENHANCERS
The drugs with poor solubility results in delayed absorption which consequently affects the bioavailability. There are many drugs which are having good therapeutic value but not used commercially because of this reason. The permeation enhancers are therefore being utilized to counter this problem. There are many such synthetic and natural materials which have the ability to enhance the drug permeation rate. The essential oils, alcohols, terpenes, azoles and many other chemical derivatives have the capability to be used for permeation enhancer. The present review work suggested the role of permeation enhancer in the pharmaceutical world.
INTRODUCTION
Nature has provided the humanities with the wide range of natural substances which can be used in a number of ways. In this context the pharmaceutical market and research too are in one way or the other dependent on nature. The herbal, as well as the biological products, can be utilized in this perspective. The numerous drugs are there which lack its usage and difficult to be formulated because of the poor permeability [1][2][3][4][5][6][7][8]. This hitch has confined the use of many drugs, which is potentially and therapeutically very active. To counter this problem the researchers have tried to incorporate the permeation enhancers. These permeation enhancers can be of natural origin or semi-synthetic or even synthetic too. The large numbers of researchers all over the globe are working hard to bring out the best permeation enhancer with wide compatibility [9][10][11][12][13][14][15][16][17][18][19][20]. The present work tries to focus on the natural permeation enhancer used in the pharmaceutical world.
Superlative properties of permeation enhancer [21]
1. Biocompatible with no toxicity induction properties (least or no chances of toxicity).
2. There should not be biocompatibility issues with incorporated medicaments.
3. Possesses good solvent properties with economical nature. 4. Must have sustainable, reproducible and rapid action.
5.
Minimum adverse reaction with better therapeutic enhancement.
6. Should be physically and chemically inert with no organoleptic involvement with active ingredients and formulations.
7. Should not causes leakage of body fluid or endogenous material with unidirectional flow properties.
8. Must have the capacity to be tested in vitro for better results of final formulations.
Mechanism of permeation enhancers
The mechanism of action of permeation enhancer depends upon the technique through which it works. The permeation enhancers are operated though natural, drug vehicle based, chemical and physical. The natural permeation enhancers like terpenes and essential oils works through partition coefficient, diffusion coefficient, drug solubility, lipid extraction, molecular orientation and macroscopic barrier and perturbation [22,23]. The drug vehicle-based technique is activated through enhancer's interaction with the stratum corneum and initiate permeation.
Fig. 1: Mechanism of action permeation enhancer [22]
International Journal of Current Pharmaceutical Research In chemical permeation enhancers one among the three ways worked out like chemical interaction with intracellular protein, by improving partition of drug in stratum corneum, or by disruption of highly ordered lipids of stratum corneum. In case of the physical permeation enhancement technique the magnetic separation, physical separation and ultrasonic wave are utilized (iontophoresis, sonophoresis, magnatophoresis etc). Gupta et al., 2012 provided evidence through the study that the permeation enhancers provide the better alternate to the conformist forms. The study also revealed the poorly soluble drug sertraline can be easily delivered transdermally using natural permeation enhancers [28].
Categories of permeation enhancers
Tawfeek et al., 2020 formulated the Lornoxicam transfersomes containing sodium deoxycholate. The drug belongs to the family of antiinflammatory drugs and showed poor permeability. The results concluded that the encapsulated formulation showed better permeation along with higher flux and apparent permeability coefficient and superior anti-inflammatory activity compared to non-transfersomal LOR hydrogel and indomethacin gel as a standard NSAID [29].
Aggarwal et al., 2012 carried out the development of transdermal delivery system of olanzapine utilizing natural oils as permeation enhancers. Penetration enhancing the potential of groundnut oil, corn oil and jojoba oil on in vitro permeation of olanzapine across rat skin was studied. The results suggested relative bioavailability of TDDS was 113.6 % as compared to oral administration of olanzapine [30].
Patil and Saragoi 2014 carried out the study of natural products as permeation enhancers. The permeation enhancer proves to be safe, non-toxic, pharmacologically inert, non-irritating, and nonallergenic. Moreover, the SAR studies on the used permeation enhancers provided the results of better bioavailability and significantly enhanced activity [31]. Sharma et al., 2010 designed to develop a suitable matrix type transdermal drug delivery system (TDDS) of olanzapine using blends of two different polymeric combinations, polyvinylpyrrolidone (PVP) and ethylcellulose (EC). Vegetable oils (soyabean oil, olive oil, eucalyptus oil) were employed as permeation enhancers. In vitro skin permeation study was also conducted in a modified Franz's diffusion cell which shows that the maximum permeation was with the formulation C3 and it was 768.64µg/cm2 after 48 h. Optimized formulations were found to be suitable for formulating in terms of physicochemical characteristics and there was no significant interaction noticed between the drug and polymers used [32].
Nan et al., 2018 investigated the enhancement effects of natural transdermal permeation enhancers from Ledum palustre L. var. angustum N. Busch. The outcomes of research recommended that the effectiveness and safety of the natural transdermal permeation enhancers could be improved by understanding their composition and the enhancement mechanisms [33].
CONCLUSION
From the current review work, the critical roles of the permeation enhancers are being accessible. The permeation enhancers are being available in wide varieties and so many options that the researchers can modify the formulations. The recent advancements in the utilization of the enhancers also suggested the commercial role and the marketed value. Further, the future prospective of the penetration enhancers is still not clear and needs more research to strengthen its economical and marketable value.
AUTHORS CONTRIBUTIONS
All the authors have contributed equally.
CONFLICT OF INTERESTS
Declared none | 2021-10-20T15:18:20.976Z | 2021-09-15T00:00:00.000 | {
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11247828 | pes2o/s2orc | v3-fos-license | Alpha-lipoic acid ameliorates the epithelial mesenchymal transition induced by unilateral ureteral obstruction in mice
The epithelial-to-mesenchymal transition (EMT) is one of mechanisms that induce renal interstitial fibrosis. Understanding EMT in renal fibrosis has important therapeutic implications for patients with kidney disease. Alpha-lipoic acid (ALA) is a natural compound with antioxidant properties. Studies for ALA are performed in acute kidney injury with renal tubular apoptosis, renal inflammation, and oxidative stress. We investigated the effects of ALA on EMT-mediated renal interstitial fibrosis in mice with unilateral ureteral obstruction (UUO). UUO mice developed severe tubular atrophy and tubulointerstitial fibrosis, with a robust EMT response and ECM deposition after 7 postoperative days. In contrast, ALA-treated UUO mice showed only moderate injury and minimal fibrosis and also larger reductions in the expression of ECM proteins, inflammatory factors, and EMT markers. ALA was shown to be involved in the suppression of infiltrating macrophages associated with EMT and the progression of interstitial fibrosis. It also lessened the destruction of the tubular basement membrane, by reducing the expression of matrix metalloproteinases. This is the first study to show that ALA modulates EMT in a UUO mouse model. Our results suggest that ALA merits further exploration as a therapeutic agent in the prevention and treatment of chronic kidney disease.
Tubulointerstitial disease is a common histopathologic feature of progressive renal disease of diverse causes 1,2 and it strongly correlates with the deterioration of renal function. Inflammation of the tubulointerstitial compartment leads to fibrosis 3,4 via excessive extracellular matrix (ECM) production, fibroblast activation/proliferation, monocyte and macrophage infiltration, and tubular atrophy 5,6 . Interstitial fibrosis is a hallmark of chronic renal failure and strongly correlates with the deterioration of renal function, regardless of the underlying disease.
Kidneys with ureteral obstruction develop progressive tubulointerstitial damage. Unilateral ureteral obstruction (UUO) is a well-characterized animal model of renal injury leading to tubulointerstitial fibrosis 7,8 . UUO in rats and mice produces the tubulointerstitial inflammation and fibrosis seen in humans with obstructive nephropathy 9 . Damage to the obstructed kidney is accompanied by tubular atrophy and progressive interstitial fibrosis, which reflect the excessive production and deposition of ECM in the interstitium 10 . ECM proteins, such as fibronectin and collagens, are secreted by myofibroblasts. These cells are derived from resident interstitial fibroblasts or from transformed epithelial cells, a process referred to as the epithelial-to-mesenchymal transition (EMT) 11 .
The progression of renal disease in UUO mice is associated with EMT, in which there is reciprocal up-regulation of fibroblast-specific protein-1 (FSP-1) and α -smooth muscle actin (α -SMA) expression and a decrease in E-cadherin expression 12 . With the loss of epithelial cell properties, myofibroblasts proliferate, migrate,
Results
ALA improves the histopathological changes in the kidneys given to UUO. Histological examination by H&E staining showed normal renal cortex in ALA-treated (ALA) and ALA and sham-operated (ALA + Sham) kidneys. There were interstitial inflammatory cell infiltration, partial tubular expansion, a severe tubular atrophy, and swelling epithelial cells in the obstructed kidney (UUO, Fig. 1). The ALA + UUO group significantly exhibited an attenuated inflammatory cell infiltration, reduced tubular expansion and tubular atrophy, and less swelling epithelial cells as compared with UUO group.
ALA ameliorates UUO-induced renal fibrosis. Fibrosis examination of the Masson's trichrome stained sections showed normal renal cortex in both ALA and ALA + sham kidneys. Obvious interstitial fibrosis was seen in the obstructed UUO kidneys, as shown by intense collagen deposition in the interstitium (blue staining in Fig. 2A). Semiquantitative scoring using an image analyzer confirmed that all UUO-only mice developed severe interstitial fibrosis, whereas in UUO + ALA mice only mild renal fibrosis was detected after 7 postoperative days ( Fig. 2A). We then examined the expression of the ECM protein collagen I, a surrogate marker of renal fibrosis, in the kidney. Immunostaining with anti-collagen I antibody showed strong expression in the interstitium (arrow) adjacent to the dilated tubules (asterisk), with more intense staining in UUO mice than in UUO + ALA mice (Fig. 2B).
UUO-induced EMT is ameliorated by ALA. EMT, a key feature in the response to UUO, is characterized by a decrease in the cellular expression of intercellular epithelial adhesion molecules, such as E-cadherin, and the concomitant development of a mesenchymal phenotype, including the expression of α -SMA 20 . UUO led to a marked down-regulation of E-cadherin and the up-regulation of α -SMA in the obstructed kidneys, whereas ALA treatment moderately ameliorated these effects (Fig. 3A). There was widespread expression of α -SMA in in the interstitium (arrow) adjacent to the dilated tubules (asterisk) of the obstructed kidneys, as well as strong staining of intrinsic interstitial fibroblasts. By contrast, in ALA + UUO mice, α -SMA-positive signals were significantly decreased (1.5-fold, UUO vs. ALA + UUO; P < 0.05) (Fig. 3B). ALA decreases UUO-induced TGF-β1 and pSmad expression. EMT is induced by a wide variety of stimuli in the setting of chronic kidney disease (CKD) 21 . TGF-β is a potent pro-fibrotic factor that promotes EMT by directly stimulating the expression of ECM proteins in renal cells 22 . In addition, it enhances the recruitment of inflammatory cells, especially macrophages. Western blot analyses of total kidney lysates showed a robust induction of TGF-β 1 in UUO mice but only a minor response in ALA + UUO mice (Fig. 4A). Specifically, TGF-β 1 expression was significantly higher in the interstitium of UUO kidneys than in ALA + UUO kidneys (2.38-fold; P < 0.05; Fig. 4B). Immunohistochemistry studies showed TGF-β 1 staining predominantly in the renal interstitium of UUO mice (arrow, Fig. 4B) whereas its expression in the renal tubules of the ALA and ALA + sham mice was negligible. EMT induction by TGF-β 1 decreased the E-cadherin expression associated with the nuclear translocation of phosphorylated Smad (pSmad) 23 . pSmad-positive signals were mainly detected in the nuclei of dilated tubular epithelial cells and in a few interstitium (arrow) adjacent to the dilated tubules (asterisk) and were significantly higher in the UUO group than in the other groups (Fig. 4C).
ALA suppresses UUO-induced NF-κB pathway activation, mononuclear cell infiltration, and ICAM-1 protein expression. There is increasing evidence of a role for nuclear factor κ B (NF-κ B) activation in the initial stages of EMT 24 . Because ALA was previously shown to suppress the NF-κB pathway [25][26][27] , we examined whether the NF-κ B pathway was sensitive to ALA. The results showed that the levels of phosphorylated NF-κ B and phosphorylated Iκ B-α , components of the NF-κ B signaling pathway, were induced by UUO and Figure 2. ALA ameliorates renal fibrosis. Masson's trichrome staining (A) and immunohistochemical staining against collagen I (B) were performed. The severity of interstitial fibrosis and densitometric quantification was applied for collagen I positive areas were examined by densitometric quantification. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Scale bar, 50 μ m. UUO1, enlarged image from insert in UUO. Arrow, tubular interstitial area. Asterisk, the dilated tubules. attenuated by ALA in UUO mice (Fig. 5A). Changes in the expression of these phosphorylated proteins were correlated with the ALA-stimulated expression of E-cadherin and α -SMA. Mononuclear cell infiltration is an additional measure of progressive renal injury. ALA is known to suppress the NF-κ B-dependent up-regulation of monocyte chemoattractant protein 1 (MCP-1), a chemokine promoting macrophage infiltration, and of ICAM-1 in vivo and in vitro 26 . It also attenuated the cisplatin-induced increase in the phosphorylation and nuclear translocation of the NF-κ B p65 subunit in kidney tissue 27 . In the current study, a few F4/80-positive cells were observed in the peritubular areas (arrowhead) and interstitial foci (arrow) of ALA and ALA + sham kidneys but the difference was not statistically significant ( Fig. 5B). However, significant amounts of interstitial cellular infiltration were present in the interstitium of UUO kidneys, whereas in the ALA + UUO kidneys the cells were mainly in peritubular areas and there were significantly fewer macrophages (18 vs. 148 in UUO kidneys; P < 0.05; Fig. 5B). In the obstructed kidneys, MCP-1 expression was highly induced whereas ALA treatment caused a dramatic reduction in MCP-1 levels (Fig. 5C).
ALA ameliorates the UUO-induced expression of MMP-2 and 9.
During EMT in the kidney, the integrity of the tubular basement membrane (TBM) is gradually destroyed, in a process that includes the up-regulation of matrix proteinases. Both MMP-2 and MMP-9 participate in EMT-mediated obstructive nephropathy 28,29 . ALA inhibited MMP-9 expression in smooth muscle cells from the thoracic aorta 25 . In the UUO kidneys, both MMP-2 and MMP-9 were expressed in the epithelial cells of the dilated tubules (asterisk) (Fig. 6A and B) and occasionally in interstitial areas adjacent to the dilated tubules (arrow) (Fig. 6B) whereas their expression was reduced by ALA (Fig. 6).
The contralateral kidney has normal morphology and histology. The pathology in the contralateral kidney was similar to that of ALA and ALA + Sham groups (Supplement Figure 1). The contralateral kidney 7 days after UUO operation showed normal architecture and histology.
Discussion
The severe tubular atrophy and tubulointerstitial fibrosis that developed in the UUO mice included a robust EMT and the deposition of large amounts of ECM. However, in UUO mice treated with ALA, only moderate, histologically identifiable renal injury and minimal fibrosis were observed. At the molecular level, the expression of ECM proteins, fibrogenic and inflammatory factors (TGF-β 1 and MCP-1), and EMT markers (E-cadherin, α -SMA, and Immunoblot analysis was performed with a specific antibody against E-cadherin and α -SMA (A). Whole kidney were prepared and processed for immunoblot. β -actin was used as loading control and data were normalized against the density of β -actin by TotalLab TL100 v2006 software. Immunohistochemical staining was performed with a specific antibody against α -SMA (B). Scale bar, 50 μm. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Arrow, tubular interstitial area. Asterisk, the dilated tubules.
Scientific RepoRts | 7:46065 | DOI: 10.1038/srep46065 pSmad) was much lower in ALA-treated than in non-treated UUO mice. This is the first demonstration that ALA modulates EMT in a UUO mouse model of the fibrotic, obstructed kidney.
EMT is a major pathway that leads to fibrosis in the kidney 30 . In humans with fibrotic kidneys, strong mesenchymal marker expression is accompanied by the deposition of collagen I among the renal tubules and massive interstitial fibrosis in the renal cortex 31 . Studies showed that, in UUO mice, fibroblasts and myofibroblasts (identified based on the markers FSP-1 and α -SMA, respectively) are increased after 7 days, indicating EMT activation 32,33 . Decreased E-cad and increased α -SMA expression are typical EMT features. In this study, ALA suppressed UUO-induced tubular interstitial fibrosis through ameliorating down-regulation of E-cad and the up-regulation of α -SMA expression.
Ureteral obstruction causes to up-regulation of MCP-1 and TGF-β 7,34 . TGF-β recruits monocytes (including F4/80-positive interstitial macrophages), induces EMT, and stimulates collagen production 35 . TGF-β is a major contributor to the cellular process that causes renal tubular epithelial cells to become ECM-producing myofibroblasts 20,23,36 . TGF-β 1 binds to its type II receptor and then forms a complex with ALK5, which leads to activation of the intracellular signaling pathway mediated by Smad proteins [37][38][39][40] . The induction of EMT by recombinant human TGF-β 1 decreased E-cadherin expression in association with the nuclear translocation of phosphorylated Smad2 and 3 23 . In this study, we found that ALA significantly suppressed TGF-β 1 expression in the interstitium and pSmad expression in the tubular epithelial cells and in a few interstitial cells adjacent to damaged tubules of the obstructed kidneys.
Macrophages are a key source of the cytokines associated with fibrosis 7,41 . Their interaction with proximal tubular epithelial cells leads to interstitial fibrosis in the injured kidney. Macrophage infiltration is preceded by the local expression of chemokines, such as the proinflammatory cytokine MCP-1, chemokine receptors, and adhesion molecules 8 . In a murine model, macrophage depletion markedly reduced myofibroblast formation and interstitial fibrosis in the kidney 42 . Our data demonstrated that ALA is able to suppress macrophage infiltration and therefore EMT and the progression of interstitial fibrosis.
In the kidney, the obvious initial change of EMT is the destruction of TBM integrity, attributed to proteolysis mediated by MMPs 28,29 . MMP-2 causes structural alterations in the TBM that promote tubular atrophy, fibrosis, Figure 4. ALA ameliorates expression of TGF-β1 and pSmad. Immunoblot analysis was performed with a specific antibody against TGF-β 1 (A). β -actin was used as loading control and data were normalized against the density of β -action by TotalLab TL100 v2006 software. Immunohistochemical staining was performed with a specific antibody against TGF-β 1 (B) and pSmad (C). The TGF-β 1 expression was predominantly in the interstitium of UUO kidneys (arrow). Densitometric quantification for TGF-β 1 and pSmad was applied to each group. Scale bar; 50 μ m. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). UUO1, enlarged image from insert in UUO. Arrow, tubular interstitial area. Asterisk, the dilated tubules. and renal failure. These changes were observed in transgenic mice with renal proximal tubular epithelial expression of active MMP-2 28 . Conversely, in the kidneys of mice genetically engineered with a null mutation of the endogenous MMP-9 gene, interstitial fibrotic lesions did not develop. Similarly, our data also showed that ALA ameliorated upregulated MMP-2 and MMP-9 expression in the tubular epithelial cells and in some interstitial cells adjacent to damaged tubules of the obstructed kidney. Reduced expression of MMP-2 and MMP-9 by ALA well correlated with decreased EMT features as well as less interstitial fibrosis in the obstructed kidney.
Most of studies in kidney injury as well as other tissue damage are focused on antioxidant properties for ALA [14][15][16] . However, we tried to find another ALA's role in this study besides antioxidant properties and our data provide the anti-fibrotic effects of ALA in EMT-mediated renal interstitial fibrosis. There was one limitation to this study. Renal functional study can be an important part of several studies, especially in AKI model. Because we focused on histological and molecular changes of UUO kidney, we initially did not check the biochemical data such as BUN and serum creatinine level. However, many previous studies have reported that BUN and serum creatinine level was not significantly affected by only ipsilateral UUO due to the presence of a contralateral kidney with good renal function [43][44][45][46][47][48] . Practically, we confirmed normal morphology and histology of contralateral kidney. This may suggest that BUN and serum creatinine level are not good markers of renal function in an UUO animal model. Even if the changes biochemical data on renal function exist in our study, we don't think that it would not affect histological changes of UUO kidney.
We found that ALA suppressed UUO-induced tubular interstitial fibrosis through ameliorating the EMT. The plausible mechanisms might be as follows: (1) ALA preferentially inhibits infiltration of mononuclear inflammatory cells expressing F4/80 into the interstitium resulting in mitigating the expression of TGF-β 1 released from these cells in the interstitium of the obstructed kidneys. These anti-inflammatory properties of ALA was also identified by reduction of NF-κ B and MCP-1 expression; (2) MMP-2 and MMP-9 expression, which are induced by TGF-β 1, in the tubular epithelial cells and in the interstitial cells adjacent to damaged tubules of the obstructed kidney was reduced by direct and/or indirect ways through ALA administration. The upregulated MMP-2 and MMP-9 expression in the obstructed kidney might be responsible for degradation of basement membrane finally leading to the tubular epithelial cells dissociation. Areas of basement membrane with dissociated epithelial cells have a mesenchymal phenotype invading the interstitium. Disruption of epithelial cells by proteases like MMP-2 and MMP-9 is sufficient to trigger EMT featured with decreased E-cad and increased α -SMA expression. This expression of MMP-2 and MMP-9 is also positively correlated with the degree of interstitial fibrosis, the Figure 5. ALA reduces renal fibrosis by regulating inflammation. Immunoblot analysis was performed with a specific antibody against phosphorylated NF-κ B and phosphorylated Iκ B-α. Data were normalized against the density of β -action by TotalLab TL100 v2006 software (A). Immunohistochemical staining was performed with a specific antibody against F4/80. A few F4/80-positive cells were observed in the peritubular areas (arrow) and interstitial foci (arrowhead) of ALA and ALA + sham kidneys (B) and MCP-1 (C). Scale bar; 100 μ m. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05).
byproduct of EMT, in the obstructed kidney. But changes including mild or less interstitial fibrosis were detected in ALA-treated obstructed kidneys.
A better understanding of the mechanism by which EMT contributes to the development of CKD will aid in the development of therapeutic interventions in patients with kidney disease. The current study indicates that ALA might be used as a therapeutic agent in the prevention and treatment of CKD.
Methods
Animals, surgery, and tissue preparation. Male C57BL/6 mice (10 weeks of age) were maintained in a 12 h light/dark cycle in a temperature-and humidity-controlled facility. Standard mice chow and water were provided ad libitum. The animal experiments were reviewed and approved by the Gyeongsang National University Guide for the Care and Use of Laboratory Animals (Approved ID: GNU-130813-M0056). The 28 mice were divided into four groups: ALA (50 mg/kg, i.p., n = 7), ALA + Sham (n = λ , UUO (n = 7), and ALA + UUO (n = 7). ALA injection is started 24 hour before the UUO and continued afterward for 7 days. Mice undergoing UUO surgery were anesthetized with inhaled isoflurane (2.5%). In the UUO group, a left flank incision was made in the anesthetized mice, after which the left ureter was ligated completely at the ureteropelvic junction using double silk sutures. The right kidney was also exposed, but the ureter was not ligated. A left flank incision was also made in mice in the sham group, after which the kidney was exposed but the ureter was not ligated. The study Figure 6. ALA decreases the expression of MMP-2 and MMP-9. Immunohistochemical staining was performed with a specific antibody against MMP-2 (A) and MMP-9 (B). Densitometric quantification for MMP-2 and MMP-9 was applied to each group. ALA; only ALA treated group, ALA + Sham; ALA treated and no ureteral ligated group, UUO; no ALA treated, but ureteral ligated group, UUO + ALA; ALA treated and ureteral ligated group. Values are expressed as means ± SE (*P < 0.05). Scale bar; 50 μ m. Asterisk, the dilated tubules. UUO1, enlarged image from insert in UUO. Arrow, tubular interstitial area. Asterisk, the dilated tubules.
Scientific RepoRts | 7:46065 | DOI: 10.1038/srep46065 was performed strictly according to the guidelines developed by the Gyeongsang National University Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize suffering. All of the animals were placed on regular diets, allowed free access to tap water, and euthanized at day 7 post-UUO/sham surgery. This time point was chosen because it is sufficient to observe the development of tubular and interstitial cell apoptosis, as demonstrated in previous studies 43 . At the time of euthanization, both kidneys were removed and tissue samples were either fixed (10% buffered formaldehyde solution) for histopathologic studies or snap-frozen in liquid nitrogen for western analysis.
Renal pathology. Kidneys were routinely fixed in 4% phosphate-buffered paraformaldehyde and paraffin embedded. Tissue sections at 5 μ m were obtained. Paraffin wax was removed with xylene, and sections were rehydrated with ethanol. After washing, the sections were stained with H&E and Masson trichrome. H&E staining was for histopathological analysis. MT staining was to assess the tissue fibrotic changes as well collagen-1 immunohistochenical staining. The semi-quantitative scoring for H&E staining were examined on the degree of interstitial injury that assigned points (0 to 3) for the extent of interstitial fibrosis, tubular atrophy (defined as luminal dilation and flattened tubular epithelial cells) and interstitial inflammatory cell infiltration. Tissue injury (interstitial fibrosis, tubular atrophy, and interstitial inflammatory cell infiltration) has been scored by grading the percentage of affected under a high-powered field ( × 400) with minor modifications from previous study 49 ; 0, 0%; 1, < 30%; 2, 31 to 60%; 3, 61 to 100% and all scorings were summed and represented as average values on the graph. Ten tubulointerstitial fields that were randomly selected were assessed in each section, and the density of trichrome-positive signals was analyzed using NIS-Elements BR 3.2 (Nikon, Japan). Immunohistochemistry. An avidin-biotinylated-HRP (ABC; Vector Laboratories, Burlingame, CA, USA) kit was used for immunohistochemistry studies, together with 5 μ m-thick paraformaldehyde-fixed, paraffin-embedded kidney sections. After their incubation with 1% normal serum, the sections were treated with each primary antibodies to polyclonal anti-Collagen-1 (diluted 1:30; 234167, Calbiochem, Menlo Park, CA, USA), anti-TGF-β 1 (diluted 1:100; sc146, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pSmad (diluted 1:100; SC11769-R, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-F4/80 (diluted 1:100; 14-4801, ebioscience, San Diego, CA, USA), anti-MCP-1 (diluted 1:100; sc1785, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP-2 (diluted 1:50; sc10736, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP-9 (diluted 1:100; sc19016, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and monoclonal anti-α -SMA (diluted 1:500; A5228, Sigma, St. Louis, MO, USA) at 4 °C for 16 h. They were then washed in PBS (pH 7.4), incubated for 90 min with secondary antibody, and then with ABC for 60 min at room temperature. After rinsing the sections with PBS, the reactions were developed using 0.027% 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA) with 0.003% H 2 O 2 . The sections were counterstained with hematoxylin to visualize the cell nuclei. Immunohistochemistry were for the expression levels of EMT-related factors (α -SMA, TGF-β 1, pSmad) and the expression levels of inflammation -related factors (F4/80, MCP-1, MMP-2, MMP-9). Ten fields that were randomly selected were assessed in each section, and the density of target signals was analyzed using NIS-Elements BR 3.2 (Nikon, Japan). The densitometric analysis was performed for quantitative analysis of all data. Statistical analysis. Statistical analysis was conducted using Sigma Plot 7.0. Statistical differences between the experimental groups were determined using analyses of variance and Student's t-tests. P < 0.05 was considered statistically significant. Values are represented as the mean ± the standard error of the mean. | 2018-04-03T04:52:36.170Z | 2017-04-05T00:00:00.000 | {
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235528327 | pes2o/s2orc | v3-fos-license | Micropollutants in Urban Stormwater Runoff of Different Land Uses
: The main aim of this study was a survey of micropollutants in stormwater runoff of Berlin (Germany) and its dependence on land-use types. In a one-year monitoring program, event mean concentrations were measured for a set of 106 parameters, including 85 organic micropollutants (e.g., flame retardants, phthalates, pesticides/biocides, polycyclic aromatic hydrocarbons (PAH)), heavy metals and standard parameters. Monitoring points were selected in five catchments of different urban land-use types, and at one urban river. We detected 77 of the 106 parameters at least once in stormwater runoff of the investigated catchment types. On average, stormwater runoff contained a mix of 24 µ g L − 1 organic micropollutants and 1.3 mg L − 1 heavy metals. For organic micropollutants, concentrations were highest in all catchments for the plasticizer diisodecyl phthalate. Concentrations of all but five parameters showed significant differences among the five land-use types. While major roads were the dominant source of traffic-related substances such as PAH, each of the other land-use types showed the highest concentrations for some substances (e.g., flame retardants in commercial area, pesticides in catchment dominated by one family homes). Comparison with environmental quality standards (EQS) for surface waters shows that 13 micropollutants in stormwater runoff and 8 micropollutants in the receiving river exceeded German quality standards for receiving surface waters during storm events, highlighting the relevance of stormwater inputs for urban surface waters.
Introduction
Urban stormwater runoff from impervious surfaces can be an important source of diffuse pollution contributing to the degradation of urban surface waters [1,2]. This is especially the case in cities with separated sewer systems as dominant drainage networks, as runoff from separated sewers is usually discharged directly to receiving surface waters without further treatment. For example, in Berlin (Germany) each year 70% or 48 million m 3 of stormwater is discharged mostly untreated into Berlin's surface waters via the separated sewer system (own estimate based on urban drainage data). Whereas "classic" pollutants in stormwater runoff such as suspended solids, chemical oxygen demand (COD) or heavy metals have been under investigation for several decades already [2][3][4][5], trace organic substances started to come into focus in recent years only [6][7][8][9][10]. Besides direct discharge into surface waters via separate storm sewers, combined sewer overflows can also be a relevant pathway for stormwater-related micropollutants to enter urban streams [11][12][13]. Sources • presenting a unique data set on stormwater pollution (available open access); • studying potential land-use specificity of micropollutants in stormwater runoff; and • discussing the potential ecotoxicological relevance of these substances based on data in stormwater runoff and in a receiving river.
Materials and Methods
A one-year monitoring program was conducted during which stormwater runoff was sampled in five catchments of different urban land use. Additionally, surface water of an urban stream receiving relevant stormwater inputs from separated storm sewers was sampled during dry and wet weather conditions.
Study Sites
Based on geographic information system (GIS) data of the Berlin Senate, five different urban land-use types were defined that represent the main city structure types of Berlin and other large European cities as summarized in Table 1. The selection was based on the assumption that differences in the selected land-use types (e.g., traffic density, roof area and type, building materials) will result in different concentration patterns of micropollutants in stormwater runoff. Together, the five selected land-use types represent~83% of Berlin's impervious area connected to the sewer network (see Figure 1). In addition to the five monitoring catchments, one monitoring station was installed at the urban river Panke in order to evaluate the impact of stormwater discharges on an urban stream. The Panke river is a small lowland river (mean discharge ~0.5 m³ s −1 ) that is characterised by strong stormwater inputs from impervious surfaces connected via a separate sewer system to the river. Of the 5 investigated catchments, catchment OLD discharges upstream of the Panke river sampling point.
Sampling Strategy for Stormwater Runoff
Four main sampling methods are commonly used to estimate the event mean concentration (EMC) of stormwater runoff [29]: 1. Constant sampling volume at fixed time interval; For each land-use type one representative monitoring catchment was selected to sample stormwater runoff during rain events (Table 1, Figure 1). Selection criteria included location in the area with separate sewer system, high homogeneity in land use of connected catchment area, size (large but homogeneous), monitoring feasibility (accessibility of manhole, sewer condition, suitability for flow measurements) and water level during rain at respective manholes (from sewer modelling of design storms with 1-year return period).
Selected monitoring catchments include an area of typical five-storey perimeter blocks built between 1870 and 1930 (OLD), a newer area of 4-8-storey concrete slab buildings built between 1960 and 1980 (NEW), a residential area characterized by one-family houses and villas with gardens (OFH), a commercial and industrial area of high imperviousness with large flat-roof buildings and yards (COM) and 1.3 km of a busy road including an intersection with traffic lights and bus stops (STR). An overview of the selected monitoring sites is shown in Figure 1. Selected monitoring catchments vary in size from 2.6 to 37 ha (total area) and are very homogeneous regarding the respective land-use type with an average areal ratio of 90% (Table 1).
In addition to the five monitoring catchments, one monitoring station was installed at the urban river Panke in order to evaluate the impact of stormwater discharges on an urban stream. The Panke river is a small lowland river (mean discharge~0.5 m 3 s −1 ) that is characterised by strong stormwater inputs from impervious surfaces connected via a separate sewer system to the river. Of the 5 investigated catchments, catchment OLD discharges upstream of the Panke river sampling point.
Sampling Strategy for Stormwater Runoff
Four main sampling methods are commonly used to estimate the event mean concentration (EMC) of stormwater runoff [29]: 1.
Constant sampling volume at fixed time interval; 2.
Variable sampling volume at fixed time interval with sample volume proportional to runoff flow; 3.
Variable sampling volume at fixed time interval with sample volume proportional to runoff volume; 4.
Constant sampling volume at variable time interval.
The first method gives a composite sample that is likely to be biased since the flow is not considered in the preparation of the composite sample [30]. The error is systematic and cannot be reduced by increasing the sampling frequency [31]. The other methods are conceptually similar regarding their aim at creating a composite sample from a series of subsamples proportional to the runoff flow or volume. Shelley [32] demonstrated that major errors can be expected by estimating EMCs using method 1 and that the other methods provide similar accuracy, with a trend to underestimate the real EMC. The systematic error increases when the sampling frequency decreases but remains below 20% for time intervals of less than 30 min.
Several authors recommend a strategy of variable sampling volume-methods 2 or 3 [30]-with the mixing of the composite sample in the laboratory [29,33,34]. Each subsample is filled in an individual bottle during the event by an automatic sampler device and the composite sample is prepared in the laboratory by mixing a proportion of each subsample, proportional to the measured runoff flow or volume. This approach is preferred to the preparation of the composite sample directly by the automatic sampler since drift or outliers in flow measurement can lead to biased sampling. This is also the main drawback of method 4. The manual mixing enables flow measurement errors in the preparation of the composite sample to be corrected and considered. Additionally, it provides individual grab samples, useful to understand event concentration variability (e.g., maximal event concentration or first flush concentration).
A study by Sandoval et al. [35] estimated the uncertainties of different sampling strategies using online total suspended solids (TSS) and flow measurement from four different stormwater outlets in France, Germany, Austria and Colombia. The results confirm that method 3 delivers the most accurate estimation of EMC, independently of the selected sampling interval or the size of the catchment.
Based on these outcomes, we selected a strategy with fixed time interval and variable sampling volume proportional to the runoff volume (method 3). After the start of the event, each of the eight 2 L bottles would be filled every 20 min in order to cover an event duration of 2 h 40 min. Following the results of Sandoval et al. [35] obtained for stormwater runoff Water 2021, 13, 1312 5 of 18 in Berlin, this strategy would lead to sampling uncertainties below 20%, which seems a reasonable tradeoff between sampling uncertainty and the duration of the events covered by the sampling strategy. Increasing the sampling frequency would reduce the sampling uncertainty but also our capacity to sample the entire duration of most events.
This basic strategy has been further slightly modified in order to • Improve the representativeness of each sub-sample: the first six bottles were each filled with four samples (V = 450 mL) at a fixed interval of 5 min (first two hours of sampling). This solution increases the representativeness of each sub-sample (over a 20 min period, four samples every 5 min are more representative than one single sample). • Cover a longer event duration: if stormwater runoff continued more than 2 h, the sampling interval changed to 15 min for the last two bottles, which leads to a maximum sampling duration of four hours.
A schematic view of preparation of a volume-proportional composite sample is shown in Supplementary Figure S1.
Stormwater Runoff Sampling
At each of the five monitoring catchments one flow meter (PCM4, Nivus, Germany) was installed in the storm sewer and one portable automatic sampler (Sigma SD 900, Hach Lange, Germany) was fixed under the manhole using suspension harnesses. Each sampler was equipped with a set of 8 × 2 L glass bottles and Teflon ® inflow pipes to avoid potential sorption and sample contamination e.g., with phthalates. Automatic samplers were triggered by a preassigned water level threshold, varying between 9 and 15 cm for the different monitoring sites (depending on sewer pipe dimensions and expected water volumes). This setting allowed capturing rain events with a total rain height ≥0.4-2.5 mm, depending on catchment size ( Table 2). For best estimation of EMC, the sampling method of variable sampling volume at fixed time interval with sample volume proportional to runoff volume was chosen after thorough investigation of the literature (see above). An event separation time of two hours was applied; longer gaps of rain were considered as separate events. Samples were retrieved and cooled within 24 h after the sampled rain event. A volume-proportional composite sample was prepared based on runoff measurements (for details see Supplementary Figure S1 On average, about one third of all rain events at each monitoring site was sampled and analyzed in the laboratory (24-41%). Of the 143 samples, 93 samples were analyzed for the complete set of chemical parameters including a large number of organic micropollutants ( Table 3). The remainder was analyzed for a reduced set of standard parameters (TSS, COD, total P) and heavy metals (see Table 3). Criteria for selection of sampled events for full chemical analysis included coverage of all four seasons, coverage of a wide range of rain event characteristics (e.g., regarding rain intensity or rain depth) and coverage of the majority of the rain event (>80% of volume sampled). An overview of the main characteristics of analyzed rain events is shown in Table 2, including rain depth, mean intensity over the rain event, maximum 10 min intensity, and number of antecedent dry days. Sampled events cover a wide range of rain events with local event frequencies between 20 and 1 yr −1 , although no extreme rainfall (>20 mm) occurred during the sampling period. Rain data collected by rain gauge network of Berlin waterworks (>40 gauges)-gauge with best correlation between rain depth and event volume in storm sewer was chosen (distances to monitoring sites: 2-6 km); 1 ADD: antecedent dry days. Table 3. Overview of analyzed substances and parameter groups.
River Sampling
Complementary river monitoring was performed at the Panke river during dry and wet weather conditions to evaluate stormwater-based pollutants in a small urban stream.
This monitoring aimed at peak concentrations rather than EMC. An automatic sampler with 24 glass bottles of 350 mL was installed at the station "Bürgerpark" to sample peak concentrations during rain events, triggered by an online sensor for electrical conductivity κ, as the conductivity in the stormwater runoff (mean in stormwater samples: 190 µS cm −1 ) is much lower compared to the conductivity of the Panke river at dry weather conditions (900-1000 µS cm −1 ). If κ dropped below 550 µS cm −1 because of a high percentage of stormwater runoff during storm events, two samplings 30 min apart were started, each filling 12 bottles subsequently. The set of bottles filled during the interval with lower κ (higher percentage of stormwater runoff in the river) were mixed into a composite sample of~4.2 L, which was analyzed. In total, 20 river samples were taken during rain events, of which 12 were analyzed for the complete set of parameters. In addition, five grab samples were analyzed that were taken during dry weather conditions throughout the year.
Chemical Analysis
Stormwater runoff and river samples were analyzed for a set of 106 parameters, including standard parameters (total suspended solids, biological and chemical oxygen demand, total phosphorous, ammonium, pH and conductivity) and an extensive set of organic micropollutants (including phthalates, biocides/pesticides, organophosphates, PAH, industrial chemicals and heavy metals, see Table 3) in an accredited laboratory in accordance with German standards (DIN). Applied analytical methods and achieved limits of quantification are summarized in Supplementary Table S1.
In general, phthalates, PAH, polychlorinated biphenyls (PCB), polybrominated diphenyl ethers (PBDE), organotin compounds, alkylphenols, bisphenol A + F and heavy metals were extracted before analyses (total concentrations), whereas all other organic micropollutants were analyzed with LC-MS (liquid chromatography-mass spectrometry) directly without extraction. Blank results of tap water cycled five times through one of the applied automatic samplers indicated no particular contamination from sampling devices and/or sample pre-treatment procedure for most pollutants monitored. A low contamination by phthalates could, however, be detected, but values were below or at limit of quantification and far less than levels found in stormwater.
Data Analysis
For data analysis (e.g., calculation of mean values), samples below limit of quantification (LOQ) were calculated with LOQ/2, unless all samples per catchment type were below LOQ, in which case the concentration was set to zero. Mean values in the text are presented with the standard error of mean. For statistical analysis, the Kruskal-Wallis and Dunn tests were applied to determine if concentration differences between the five catchment types are statistically significant. Concentration differences were evaluated for all land-use pairs and chemical parameters. As concentration values for each catchment type (n = 14-41 depending on parameter) were not normally distributed (tested by Shapiro-Wilk-test, see Supplementary Table S2 for results), an analysis of variance (ANOVA) could not be applied.
The international data sets above typically considered (large) stormwater catchments of mixed land use. Thus, similar values of mean/median concentrations across the five land-use types of this data set indicate that monitoring sites in this study were representative for mixed land use.
Land Use-Specific Differences in Stormwater Runoff EMC
While means across the five land-use types were comparable to international data sets, values differed significantly between these land use types. For all heavy metals and standard parameters, except BOD5, significant differences were found among land-use Regarding heavy metals, the highest concentrations were found for zinc with an overall mean concentration of 954 µg L −1 (max: 10,000 µg L −1 ), followed by copper with 253 µg L −1 (max: 5300 µg L −1 ) and lead with 68 µg L −1 (max: 780 µg L −1 ) (see Table 4). Again, means of heavy metal compounds were mostly in line with literature ( Figure 2). Major differences were only found for Ni and Cd, which were clearly higher (outside the 95% interval of the data of this study) in the data set by Brombach and Fuchs [36]. However, the other data sets confirmed the present measurements. On the other hand, Zn and Cu values in this study were clearly higher than in the US data sets by Pitt et al. [28] and Masoner et al. [9]. Again, the other data sets were well within the 95% interval of the Berlin data, even if they were all below the Berlin mean. Sources for zinc and copper were typically traffic-related inputs (tire wear for zinc, brake abrasion for copper) and building materials (metal sheets, gutters).
The international data sets above typically considered (large) stormwater catchments of mixed land use. Thus, similar values of mean/median concentrations across the five landuse types of this data set indicate that monitoring sites in this study were representative for mixed land use.
Land Use-Specific Differences in Stormwater Runoff EMC
While means across the five land-use types were comparable to international data sets, values differed significantly between these land use types. For all heavy metals and standard parameters, except BOD 5 , significant differences were found among land-use types, based on the Kruskal-Wallis test (see Supplementary Table S4). Overall, 38% of all the possible land-use pairs showed significant differences in pair-wise Dunn tests.
Pronounced differences between land-use types could be seen in the concentrations of TSS and COD for land-use type STR, which were significantly higher than concentrations of all other land-use types (significant difference for 88% of land use pairs with STR for TSS and COD, see Supplementary Table S4). As runoff from roads and highways has long been identified as a source of particulates and organics e.g., due to brake and tire wear or street tree inputs, this result is not surprising. Similarly, STR stands out with significantly higher values for a number of heavy metals (Ni, Cr, Ti, V).
In contrast, orthophosphate was significantly higher in OFH, potentially due to fertilizer application in private gardens. Surprisingly, Zn was significantly higher in OLD and COM than STR, which in turn was significantly higher than NEW and OFH. The result can probably be explained by the influence of building materials, such as galvanized rain pipes (OLD) and large tin roofs (COM), in combination with traffic (highest in STR, followed by OLD and COM).
Wet Weather Grab Samples in the River
Most investigated standard parameters and heavy metals also showed clear peaks in the River Panke during stormwater impacts. Differences between wet and dry weather concentrations for Zn and Cu were more than one order of magnitude. On average, wet weather concentrations exceeded those during dry weather by a factor of 7 and 15, for standard parameters and heavy metals, respectively.
EMC in Stormwater Runoff
We detected 63 of 92 analyzed organic micropollutants in at least one sample of stormwater runoff of the investigated catchment types. Only for PCB, PBDE, organotin compounds, MTBE and four biocides/pesticides (imidacloprid, thiacloprid, simazine, dichlorbenzamide [DCBA]) all analyzed samples were below the respective LOQ (see Supplementary Table S1 for LOQs). As shown in Figure 3, concentrations of organic micropollutants were highest for phthalates, dominated by diisodecyl + diisononyl phthalate (DIDP + DINP, technical mixture of phthalates replacing DEHP) with an average concentration for all samples of 12 ± 2.1 µg L −1 . Figure 3 also indicates that the average stormwater sample contained a total of >20 µg L −1 in organic micropollutants (average sum of all analyzed organic micropollutants is 24 ± 2.6 µg L −1 ), with significantly higher concentrations for single events. For 6 parameters of organic micropollutants, the phthalates DIDP + DINP and DEHP, the flame-retardant TBEP, the sum of the 16 PAH and the stimulants caffeine and nicotine, average concentrations >1 µg L −1 were determined over all the samples and land-use types (see Table 4). tration for all samples of 12 ± 2.1 µg L −1 . Figure 3 also indicates that the average stormwater sample contained a total of >20 µg L −1 in organic micropollutants (average sum of all analyzed organic micropollutants is 24 ± 2.6 µg L −1 ), with significantly higher concentrations for single events. For 6 parameters of organic micropollutants, the phthalates DIDP + DINP and DEHP, the flame-retardant TBEP, the sum of the 16 PAH and the stimulants caffeine and nicotine, average concentrations >1 µg L −1 were determined over all the samples and land-use types (see Table 4). Table 3) in stormwater of all 5 catchment types. Boxes show 25% and 75% quantiles with median as thick line, whiskers show 5%/95% quantiles, n is number of samples. Table 3) in stormwater of all 5 catchment types. Boxes show 25% and 75% quantiles with median as thick line, whiskers show 5%/95% quantiles, n is number of samples. Figure 2 compares monitoring results to large European and US datasets for a number of organic micropollutants. In general, reported concentrations are well within 95%prediction intervals of the presented data. However, there are a few noteworthy exceptions.
The most prominent biocide in this study, mecoprop, was found at~100 times lower concentrations in French catchments. In turn, the biocide diuron was reported at 5 to 16-fold higher concentrations by Gasperi et al. [6] and Zgheib et al. [7] for France. Since both biocides are primarily used for algal/plant control on building materials, the difference may be due to regional use patterns of French and German roof/paint suppliers. Concentrations slightly above the 95%-prediction interval of this study were also reported by Gasperi et al. [6] for isoproturon and AMPA (metabolite of glyphosate), two pesticides/biocides, which have a predominant source in classical weed control. Again, different regional use patterns could possibly explain the observation. Generally, biocides of interest (average concentrations >0.05 µg L −1 ) that were found in this study include mecoprop, carbendazim, diuron, glyphosate, AMPA, terbutryn and terbuthylazin.
All PAH compounds reported in the French study by Zgheib et al. [7] are well within the 95%-bounds of this study, with the exception of naphthalene, which was found at 20-fold higher concentrations in Parisian stormwater. In contrast, most PAH compounds reported in the US study by Masoner et al. [9] were clearly above the 97.5% quantile of the concentrations in this study, probably due to higher traffic volumes in the US. PAH concentrations in this study (mean of ∑PAH16: 1.7 µg L −1 ) were dominated by fluoranthene and pyrene with maximum concentrations of 3.2 and 2.9 µg L −1 , respectively.
PAH ratios for source evaluation proposed by Tobiszewski and Namieśnik [38] were determined and clearly show that PAH emissions in all the land-use types of this study are dominated by traffic (e.g., benzo[a]pyrene/benzo[ghi]perylene-ratio to distinguish between traffic (>0.6) and non-traffic (<0.6) emissions resulted in average values of 1.6 for STR and 1.2-1.4 for all other land-use types). Consequently, differences in naphthalene concentrations may be explained by other sources than vehicular combustion in the Paris study.
The phthalate DEHP was detected at significantly lower concentrations than in the US by Pitt et al. (2004), as well as in France by Zgheib et al. [7]. This may be explained by a shift in plasticizer use from DEHP to DIDP + DINP, as reported by Bartolomé et al. [39]. As a consequence, the concentration in DEHP is likely to decrease with every year, following the replacement of plasticizers.
Finally, the concentrations of the insect repellent DEET in stormwater runoff as reported by Masoner et al. [9] were 1-2 orders of magnitude higher compared to concentrations observed in this study, which could indicate a higher usage of DEET in the US compared to Berlin.
Land Use-Specific Differences
Surprisingly, each of the five land-use types showed highest mean concentrations for some organic micropollutants; i.e., the biocide mecoprop was highest in OLD, the pesticide isoproturon in OFH, the flame-retardant TBP in NEW, the phthalate DINP + DIDP in COM and PAH16 in STR. Overall, STR was the dominant land use for 22 out of 48 parameters in Table 4. However, this included 14 PAH. If only the sum parameter of PAH (PAH16) is considered, the five land-use types OLD, NEW, OFH, STR and COM showed the highest mean concentrations for 31%, 8%, 12%, 31% and 19% of the organic micropollutants in Table 4, respectively.
The observed differences among the five land-use types were also statistically significant for all but four tested organic micropollutants, based on the Kruskal-Wallis test (see Supplementary Table S3). Two exceptions were the artificial sweetener acesulfame (possibly from littered consumables such as chewing gums or soft drink bottles) and nicotine (from littered cigarette butts), indicating that littering seems to be equally distributed in Berlin. Of the organic micropollutants with significant differences, 52% of all the possible land-use pairs showed significant differences in pair-wise Dunn tests. This shows that in many cases differences were not just due to one land use with a particularly high or low value, but can be graduated. For instance, the flame retardant and plasticizer TBEP shows significant differences between three groups from highest concentrations in COM and NEW to medium ones in OLD to lowest concentrations in STR and OFH (see Supplementary Table S3).
Plausibility of Differences among Land-Use Types
Since most stormwater pollutants can have multiple sources it was impossible to fully explain the differences among the different monitoring sites. However, we tried to show that observed differences were likely to originate from different land use, based on few examples.
For biocides/pesticides, catchment specificity depends on individual compounds. Mecoprop is contained in high amounts in certain bituminous sealing membranes for roofs [40]. Consequently, it can be found in all catchments with roof runoff (all catchments beside STR, Figure 4 and Table 4). In turn, substances such as the fungicide carbendazim or the herbicide diuron are typical additives to exterior paints [22] and are consequently expected at elevated concentrations from plastered walls/facades (especially on insulated facades). To test this hypothesis, we estimated the area of plastered walls/facades with potential connection to storm sewers (direct connection to impervious surfaces, no front gardens or similar below facade) in all the monitoring catchments and found correlation coefficients R 2 for land-use medians of carbendazim and diuron of 0.85 and 0.68, respectively. Yet another pattern is found for compounds such as isoproturon or glyphosate/AMPA, which are predominantly used as a pesticide for weed control. Consequently, isoproturon is mostly found in OFH, probably from use in private gardens by individual home owners. In Berlin, glyphosate is also used to keep sidewalks free of weeds. As a result, glyphosate and its metabolite AMPA were found at similar concentrations in most land-use types ( Figure 4). 4, such as TBP, 2-Phenylphenol or 4-tert-butylphenol, (dominant) sources are unclear and therefore concentration patterns cannot be explained by known land-use differences
Wet Weather Grab Samples in the River
We detected 54 organic micropollutants also in the Panke river, including all substances which were found in >30% of stormwater runoff samples in Table 3. As shown in Figure 5, concentrations of most compounds in river samples taken during storm events are about one order of magnitude higher compared to concentrations at dry weather conditions, strongly suggesting that stormwater runoff is responsible for concentration peaks of these and many of the other substances during storm events. Exceptions are sewage- Organophosphates (flame retardants) such as TCPP and TBEP are expected mostly from insulation materials on buildings. As a consequence, they were highest in the commercial area, potentially due to more extensive application of insulation materials to the larger industrial buildings, ware houses and large retail stores, followed by the other densely built-up land use types NEW and OLD.
A number of organic micropollutants in stormwater are typically associated with traffic, such as PAH (combustion byproduct), benzothiazoles (vulcanizing accelerators from tire wear) or benzotriazoles (corrosion inhibitor). Also, in this study the compounds in these three groups were found in significantly higher concentrations in STR than for the other land-use types. However, the substances are found in stormwater runoff of all the five monitoring catchments, since the other four land-use types also contain small roads <7500 vehicles per day. Since smaller roads are not covered by the official traffic counts we did traffic estimates by following official counting protocols and found the highest traffic volumes in STR, followed by COM (due to deliveries and store costumers), the two inner city land uses OLD and NEW down to lowest traffic volumes in OFH. By correlating these traffic estimates for the five land-use types with substance medians we found expected correlations for PAH16 and benzothiazoles, with R 2 of 0.83 and 0.85, respectively. Surprisingly, traffic counts showed even higher correlations for phthalates DINP + DIDP (R 2 of 0.89) and DEHP (R 2 of 0.98). After some research one explanation could be the application of plastisols (paste-like polyvinyl chloride, PVC, with high content of plasticizers) for protective undercoating of vehicles (9% of phthalate production in Western Europe, [41]), which might leach phthalates to a higher degree than plastics on buildings or in yards.
The above examples show that observed differences between the five monitoring catchments can be explained plausibly by sources, which are closely associated with the investigated five land-use types. However, for a number of organic micropollutants in Table 4, such as TBP, 2-Phenylphenol or 4-tert-butylphenol, (dominant) sources are unclear and therefore concentration patterns cannot be explained by known land-use differences
Wet Weather Grab Samples in the River
We detected 54 organic micropollutants also in the Panke river, including all substances which were found in >30% of stormwater runoff samples in Table 3. As shown in Figure 5, concentrations of most compounds in river samples taken during storm events are about one order of magnitude higher compared to concentrations at dry weather conditions, strongly suggesting that stormwater runoff is responsible for concentration peaks of these and many of the other substances during storm events. Exceptions are sewage-based tracer substances and benzotriazoles, which are present in the river due to minor upstream inlets of WWTP effluents and consequently are diluted by storm water. All other substance groups showed concentration increases during stormwater impacts by factors between 5 (phthalates and benzothiazoles) and 20 (PAH) with an average of 13. based tracer substances and benzotriazoles, which are present in the river due to minor upstream inlets of WWTP effluents and consequently are diluted by storm water. All other substance groups showed concentration increases during stormwater impacts by factors between 5 (phthalates and benzothiazoles) and 20 (PAH) with an average of 13.
Comparison with Environmental Quality Standards
Concentrations in stormwater runoff were compared with environmental quality standards (EQS) of the European Water Framework Directive (2013/39/EU, regulating
Comparison with Environmental Quality Standards
Concentrations in stormwater runoff were compared with environmental quality standards (EQS) of the European Water Framework Directive (2013/39/EU, regulating DEHP, diuron, terbutryn, the PAH, Pb and Cd) as well as EQS of the National (German) Surface Water Directive ( [42], regulating mecoprop and carbendazim) and EQS-suggestions by the German Federal Environmental Agency ( [43], for Zn and Cu). EQS for surface waters were exceeded by catchment-specific average concentrations for one phthalate (DEHP), four biocides (carbendazim, mecoprop, diuron, terbutryn), four PAH (fluoranthene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[ghi]perylene) and four heavy metals (Zn, Cu, Pb, Cd). For zinc and copper, dissolved fractions were 48% and 33%, respectively, resulting in maximum dissolved concentrations of 3000 µg L −1 and 150 µg L −1 , which is remarkable in regard to environmental quality standards of 33 µg L −1 for zinc and 2.4 µg L −1 for copper recommended by the German EPA as maximum allowable concentrations [43]. However, whether toxicity thresholds are violated in receiving rivers depends on the mixing ratio between storm water runoff and stream flow.
In the case of the Panke river, maximum concentrations measured during storm events also exceeded the higher maximum allowable concentrations values (MAC-EQS) for eight of these compounds (zinc, copper, DEHP, carbendazim, mecoprop, fluoranthene, benzo[b]fluoranthene and benzo[ghi]perylene), highlighting the relevance of stormwater inputs for urban surface waters.
A number of substances in Table 4, such as DIDP + DINP or TBEP, are not regulated through EQS. In some cases, PNEC (predicted no effect concentration) values from literature could be compared, which indicated potentially low relevance for aquatic organisms from TBEP but high relevance from nicotine as PNEC values were exceeded both in stormwater runoff as well as in surface water of the Panke river during rain events.
Comparing the stormwater concentrations directly to EQS neglects dilution with river water as well as other (point) sources of pollution. Moreover, EQS are developed for long exposure times and it remains unclear how short but high concentration peaks during storm events should be assessed. For instance, maximum detected dissolved copper EMC of 150 µg L −1 (dissolved fraction: 33%) is more than 60-fold higher than the EQS of 2.4 µg L −1 proposed by the German EPA, but only occurs for a few hours. An example for organic micropollutants is fluoranthene with maximum concentrations in stormwater runoff of 3.2 µg L −1 , which is more than 25-fold higher than the maximum allowable concentration (MAC) EQS (0.12 µg L −1 ) as set in the European Water Framework Directive and more than 500-fold higher compared to the annual average (AA) EQS (0.0063 µg L −1 ). Another important aspect is the fact that stormwater runoff does not contain single substances but a mix of >50 micropollutants with a total concentration of~24 µg L −1 for organic micropollutants and of~1.3 mg L −1 for heavy metals. Some studies indicated that such a mixture could either amplify or reduce the impact of single compounds [44].
Given these limitations, it is impossible to make a final judgment on the relevance of pollution from stormwater runoff for the ecology of urban surface waters. However, results show that stormwater runoff from very different urban land uses contains a large mix of substances, some of which are known to have detrimental effects on aquatic organisms. Although many compounds were measured in this study, new compounds will be found.
•
We detected 77 of 106 organic and inorganic micropollutants of different chemical groups and sources in 143 event-based samples of stormwater runoff from five different urban land use types.
•
The average stormwater runoff contained a mix of >50 micropollutants with a total concentration in the order of 10 µg L −1 for organic micropollutants and of 1 mg L −1 for heavy metals. • Event mean concentrations showed significant differences between land use types for 90% of tested substances.
• While road runoff was the most important source for some compounds such as polycyclic aromatic hydrocarbons or benzothiazole, all land-use types contained dominant sources for some micropollutants and cannot be neglected in pollution control strategies for stormwater. • For 13 compounds (including 4 heavy metals) average concentrations in stormwater runoff exceeded EQS, indicating a potential relevance of urban stormwater runoff for surface water pollution. For 8 compounds (including 2 heavy metals) maximum concentrations in an urban stream even exceeded maximum allowable concentrations of EQS, demonstrating the relevance of stormwater inputs for urban surface waters.
•
It is suggested that further research is undertaken on: (i) the toxicity of short concentration peaks of mixed substances; (ii) the role of stormwater pollutant loads to surface waters in comparison to other sources, such as waste water treatment plants, combined sewer overflows or agriculture; and (iii) on mitigation strategies, such as substance replacement or treatment at the source or further downstream.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/w13091312/s1, Figure S1: Schematic view of preparation of volume-proportional composite sample incorporating flow data measured in respective storm sewer during sampled rain event. Table S1: List of monitored substances, analytical methods, and limits of quantification (LOQ). Table S2: Results of Shapiro-Wilk test to test for normal distributions of concentrations per catchment type (prerequisite for application of analysis of variance (ANOVA)). | 2021-06-22T17:54:37.911Z | 2021-05-07T00:00:00.000 | {
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160013153 | pes2o/s2orc | v3-fos-license | FTY720/fingolimod decreases hepatic steatosis and expression of fatty acid synthase in diet-induced nonalcoholic fatty liver disease in mice
Nonalcoholic fatty liver disease (NAFLD), a leading cause of liver dysfunction, is a metabolic disease that begins with steatosis. Sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate (S1P), have recently received attention for their potential roles in insulin resistance and hepatic steatosis. FTY720/fingolimod, a prodrug for the treatment of multiple sclerosis, is phosphorylated in vivo to its active phosphorylated form by sphingosine kinase 2 and has been shown to interfere with the actions of S1P and to inhibit ceramide biosynthesis. Therefore, in this study we investigated the effects of FTY720 in a diet-induced animal model of NAFLD (DIAMOND) that recapitulates the hallmarks of the human disease. The oral administration of FTY720 to these mice fed a high-fat diet and sugar water improved glucose tolerance and reduced steatosis. In addition to decreasing liver triglycerides, FTY720 also reduced hepatic sphingolipid levels, including ceramides, monohexosylceramides, and sphingomyelins, particularly the C16:0 and C24:1 species, as well as S1P and dihydro-S1P. FTY720 administration decreased diet-induced fatty acid synthase (FASN) expression in DIAMOND mice without affecting other key enzymes in lipogenesis. FTY720 had no effect on the expression of SREBP-1c, which transcriptionally activates FASN. However, in agreement with the notion that the active phosphorylated form of FTY720 is an inhibitor of histone deacetylases, FTY720-P accumulated in the liver, and histone H3K9 acetylation was markedly increased in these mice. Hence, FTY720 might be useful for attenuating FASN expression and triglyceride accumulation associated with steatosis.
Levels of the ceramide metabolite sphingosine-1-phosphate (S1P) produced by two sphingosine kinase isoenzymes, SphK1 and SphK2, were also elevated in the livers of mice fed a high-fat diet (HFD) (22). Moreover, the SphK1/S1P axis was shown to mediate hepatic inflammation in a mouse model of NASH induced by high-saturated-fat feeding (23), suggesting that enhanced S1P levels in the circulation may mediate some of the systemic effects associated with HFD and obesity (15). Furthermore, increases in plasma S1P in obese humans correlated with fasting plasma insulin level, body fat percentage, and body mass index (24).
To improve understanding of the development of NAFLD and provide insights into future therapeutic avenues, many animal models have been studied (25). However, most do not exhibit the full spectrum of features associated with human NASH (25). A diet-induced animal model of NAFLD (DIAMOND) that recapitulates the key physiological, metabolic, histologic, transcriptomic, and cell-signaling changes seen in humans with progressive NASH has recently been developed (26). DIAMOND is a useful preclinical approach for developing better diagnostic and therapeutic strategies for patients with NASH, targeting those with early-stage disease as well as those with advanced liver fibrosis (27). To better understand the involvement of ceramide and its metabolites in the development of the disease, we used FTY720/fingolimod, an orally available immunosuppressive drug used for treating multiple sclerosis (28) that has been shown to inhibit CerS in vitro (29,30). FTY720 also inhibits de novo ceramide biosynthesis in cultured cells (30,31) and mice (32,33). In addition, FTY720 inhibits and reduces levels of SphK1 and S1P (34,35). Our results suggest that FTY720 could have beneficial effects on the early stage of disease development.
Animals
Isogenic mice derived from C57BL/6J and 129S1/SvImJ backgrounds (B6/129) were kindly provided by Dr. Sandra Erickson (UCSF) and maintained by inbreeding as previously described (26). The mice were housed under a 12-h light/12-h dark cycle in a 21-23°C facility. All procedures were approved by the Animal Care and Use Committee of Virginia Commonwealth University.
Diet and treatments
Genome-wide association mapping of hepatic triglycerides revealed striking differences in the genetic control of hepatic steatosis between female and male mice (36). Female mice were used for this study because the reduced severity of HFD-induced NAFLD in female mice precludes the interrogation of disease pathogenesis in a sex-independent manner. Female mice were fed a standard chow diet (CD; Harlan TD.7012) until reaching 12 weeks of age unless indicated otherwise. Mice were then fed a high-fat, high-carbohydrate diet (Harlan TD.88137) containing 42% kcal from milk fat and 0.1% cholesterol (Western diet; WD) for 16 weeks. Drinking water contained a high amount of fructose (23.1 g/l) and glucose (18.9 g/l) (sugar water; SW) (26). Both food and water were provided ad libitum. FTY720/fingolimod (0.3 mg/kg; Cayman Chemical #10006292) or vehicle (saline) was administered via oral gavage three times a week throughout the 16-week HFD. Where indicated, control mice were fed a standard CD (Harlan TD.7012) with normal drinking water.
Glucose tolerance and insulin tolerance tests
Mice were fasted overnight, and baseline blood glucose levels were measured in tail-vein blood. Glucose (1 mg dextrose/kg body weight) dissolved in sterile PBS was injected intraperitoneally. Blood glucose levels were then determined at 15, 30, 60, 90, and 120 min postglucose injection. All glucose measurements were determined with an Accu-Chek Compact plus glucometer (Roche Diagnostics), and areas under the curve were determined (39). Repeat glucose tolerance tests were performed after several days. For the insulin tolerance test, mice were injected intraperitoneally with insulin (0.75 U/kg body weight), and blood glucose levels were measured prior to and 30 and 60 min after insulin injection.
Nuclear extracts
Livers were homogenized in buffer containing 10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 1 mM Na 3 VO 4 , 1 mM DTT, and phosphatase and protease inhibitor cocktail (Thermo Fisher Scientific) and incubated on ice for 15 min. NP-40 was added to a final concentration of 0.75% (v/v), and the tissue suspension was vortexed for 10 s and then incubated on ice for 4 min. Nuclear and cytoplasmic fractions were separated by centrifugation at 3,000 g for 4 min at 4°C. Nuclei were resuspended in high-salt buffer containing 20 mM HEPES, pH 7.8, 0.4 M NaCl, 1 mM EDTA, 1 mM Na 3 VO 4 , 1 mM DTT, and 1:500 phosphatase and protease inhibitors, sonicated on ice, and incubated on ice for 5 min. Nuclear extracts were cleared by centrifugation at 3,000 g for 5 min at 4°C. Proteins were separated by SDS-PAGE, transblotted to nitrocellulose, and incubated with primary antibodies as indicated in the figure legends, including rabbit polyclonal antibodies to histone H3-K9ac (1:1000; AbCam) and histone H3 (1:1000; Cell Signaling).
RNA analysis
RNA was extracted from liver tissues with TRIzol Reagent (15596-018, Ambion, Thermo Fisher Scientific). RNA (2 µg) was treated with RQ1 DNase (M6101, Promega) and converted by the High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific) to cDNA. SYBR Green PCR Master Mix (Thermo Fisher Scientific) was mixed with the Bio-Rad mouse primers gapdh (CED0027497) and fasn (CID0015083). Reactions were analyzed with the use of the CFX Connect Real-Time PCR Detection System (Bio-Rad) (40). Gene expression levels were calculated by the C t method and normalized to GAPDH expression.
Blood collection and analysis
Blood was collected from hepatic veins, and serum was obtained by centrifugation for 15 min at 1,500 g at 4°C. Serum alanine aminotransferase, aspartate aminotransferase, cholesterol, HDL, LDL, and triglycerides were measured by the Virginia Commonwealth University Hospital Department of Pathology.
Sphingolipid measurements
Lipids were extracted from liver tissue and whole blood, and sphingolipids were quantified by LC/ESI/MS/MS (AB Sciex 5500) as previously described (41).
Fig. 1.
Mice on a WD supplemented with SW develop obesity, fatty livers, inflammation, liver fibrosis, and steatosis. B6/129 female mice were fed a CD or WD with SW for 16 weeks. Mice were 12 weeks old at the beginning of feeding the WD + SW (n = 10), whereas the control mice group fed the CD consisted of 4 mice that were 10 weeks old and 6 that were 12 weeks old. A: Body weight. B: Liver weight. C: Hepatic triglycerides levels. D: Glucose tolerance tests. E: Histology scores for liver steatosis, inflammation, hepatocyte ballooning, NAFLD activity score, and fibrosis. F: Representative images of liver sections stained with H&E or Picrosirius red. Size bars: 10 m. Data are expressed as means ± SEMs, n = 8-10 mice per group. *P < 0.01 and **P < 0.001.
Statistics
Statistical significance was determined with unpaired two-tailed Student's t-tests for comparisons of two groups or by ANOVA followed by post hoc tests for multiple comparisons (GraphPad Prism). P < 0.05 was used to indicate statistical significance.
DIAMOND mice develop obesity, fatty liver, dyslipidemia, and steatohepatitis
Previous studies demonstrated that feeding B6/129 mice an HFD supplemented with SW mimics a typical WD (42) and triggers NASH development (26). In agreement, we observed that when female B6/129 mice were fed a WD with 42% calories from fat together with drinking water containing fructose and glucose (SW) for 16 weeks they rapidly gained weight (Fig. 1A), leading to increased liver weight (Fig. 1B) and accumulation of hepatic triglycerides (Fig. 1C) compared with mice fed a CD. Similar to previous studies (26), glucose tolerance tests showed that glucose levels were higher following intraperitoneal glucose administration in mice fed a WD with SW, although it was not statistically significant (Fig. 1D). As expected, liver histology indicated that these mice also developed fatty livers, with extensive macrovesicles and small droplets (Fig. 1F), indicative of steatosis (Fig. 1E) and consistent with the increased levels of hepatic triglycerides (Fig. 1C). Extensive steatohepatitis was also characterized by increased lobular inflammation and hepatocellular ballooning, all leading to an increased NAFLD activity score (Fig. 1E). Staining with PicoSirius Red to visualize collagen fibers (fibrosis) in liver sections revealed only a small pericellular sinusoidal fibrosis (Fig. 1E, F), suggesting that fibrosis had not yet developed significantly.
FTY720 improved glucose tolerance and reduced steatosis in DIAMOND mice
Previous studies have shown that FTY720 reduced muscle lipid accumulation and improved glucose tolerance in mice fed an HFD (43). However, the dose of FTY720 used in this study was too high to be clinically relevant in humans. Because our results support the notion that B6/129 mice fed a WD with SW for 16 weeks is indeed an excellent model for studying the early development of steatohepatitis (26), it was of interest to examine the effects of a lower, clinically relevant dose of FTY720 in DIAMOND mice. Orally administered 0.3 mg/kg FTY720 three times per week slightly reduced body weight ( Fig. 2A) and significantly reduced liver weight (Fig. 2B) without affecting spleen weight (data not shown) in mice fed an HFD with SW for 16 weeks. FTY720 improved glucose tolerance and decreased the area under the curve by 13.7% (Fig. 2C). However, DIAMOND mice remained insulin-sensitive at 16 weeks, as assessed with an insulin tolerance test, and FTY720 administration had no significant effects (Fig. 2C). Although there were no major changes in circulating levels of cholesterol or in HDL or LDL, there was a small but significant reduction in circulating triglyceride levels (Fig. 2D). The assessment of NAFLD severity in a blinded manner in these mice revealed that treatment with FTY720 also significantly reduced steatosis compared with mice treated with saline (vehicle) (Fig. 3A). However, there were no significant changes in hepatic inflammation, hepatocyte ballooning, NAFLD activity score, or fibrosis scoring after FTY720 treatment (Fig. 3A, B). Consistent with a 27% reduction in circulating triglycerides (Fig. 2D), FTY720 also reduced the size and number of lipid droplets in liver sections (Fig. 3B), and there was a small reduction in oil red staining used to visualize neutral triglycerides.
FTY720 treatment decreased hepatic sphingolipids
Because it has been suggested that ceramides and S1P may contribute to the development of NAFLD (13)(14)(15)44) and FTY720 can reduce sphingolipid biosynthesis in vivo (32,33,43), we next examined the effects of FTY720 administration on hepatic sphingolipids. MS analyses showed that FTY720 significantly decreased levels of all classes of liver sphingolipids examined, including ceramides, monohexosylceramides, and sphingomyelins (Fig. 4A). Moreover, dihydroceramides, monohexosyldihydroceramides, and dihydrosphingomyelins, which mainly come from de novo sphingolipid synthesis, were also lower in mice treated with FTY720 compared with mice treated with vehicle (Fig. 4B). These decreases were especially robust in C16:0 and C24:1 species, although almost all chain lengths were significantly reduced (Fig. 5). Interestingly, although levels of S1P as well as dihydro-S1P were also significantly decreased in livers from mice that received FTY720, no concomitant increases were noted in levels of their precursors sphingosine Fig. 3. FTY720 administration reduced steatosis in DIAMOND mice. Mice were fed and treated as described in Fig. 2. A: Histology scores for liver steatosis, inflammation, hepatocyte ballooning, NAFLD activity score, and fibrosis. B: Representative images of liver sections stained with H&E, Picrosirius red, and oil red. Size bars: 100 m. Data are expressed as means ± SEMs, n = 8 mice per group. *P < 0.05. or dihydrosphingosine (Fig. 6), probably due to the much lower relative levels of the phosphorylated bases.
Because previous studies have suggested that CerS5 (20) and/or CerS6 (18) are important in maintaining C16:0ceramide pools that contribute to the development of dietinduced obesity and glucose intolerance, we next examined the effect of FTY720 on hepatic CerS expression. However, despite significant decreases in liver sphingolipids and ceramides, hepatic mRNA expression of the six CerS isozymes involved in the biosynthesis of ceramides was not significantly altered by FTY720 administration (Fig. 7A).
In addition to inhibiting ceramide synthesis, it is well established that FTY720 is a prodrug that is phosphorylated in vivo by SphK2 to the active FTY720-P that binds to all of the S1PRs except S1PR2 and modulates their actions (28). Therefore, we examined the expression of these receptors in livers isolated from DIAMOND mice fed a WD + SW treated without or with FTY720. In agreement with a previous study (45), S1pr1-3 were predominantly expressed, while S1pr4 and S1pr5 levels were below detection limits (Fig. 7B). However, FTY720 administration did not significantly affect the expression of either the S1P receptors (S1pr1-3), nor did it affect the expression of Sphk1 or Sphk2 (Fig. 7B). As previous studies suggest that FTY720 reduces macrophage-associated liver inflammation (46), we also examined the effect of FTY720 on proinflammatory cytokine and chemokine expression. However, 16 weeks after feeding a WD + SW, metabolic perturbation predominated and lobular inflammation was just beginning (Fig. 1). FTY720 treatment decreased, albeit not statistically significantly, the proinflammatory cytokine TNF-, which has been shown to increase in the progression of NAFLD to inflammatory NASH and cirrhosis (47) (Fig. 7C). Yet there were no effects on the expression of another proinflammatory cytokine, IL-6. Moreover, although the hepatic expression of two key monocyte-attracting chemokines, CCL2, also known as monocyte chemotactic protein 1, and CCL3, macrophage inflammatory protein 1-, were not significantly altered by FTY720 administration, there were significant reductions in the expression of CXCL10, also known as interferon -induced protein 10, a known chemoattractant for monocytes/macrophages, T-cells, and dendritic cells (Fig. 7C). Likewise, the expression of CCL5, also known as RANTES (regulated on activation, normal T-cell expressed and secreted), that plays an active role in the recruitment of leukocytes was also reduced by FTY720 (Fig. 7C).
FTY720 administration decreased HFD-induced FASN expression in DIAMOND mice
To better understand the mechanism by which FTY720 suppresses the development of steatosis, liver levels of mRNA encoding key enzymes involved in lipogenesis and lipid metabolism were determined by quantitative PCR. We particularly focused on genes whose expression levels have been shown to be elevated in the early phase of steatosis induced in DIAMOND mice (48) and are also hallmarks of NAFLD in humans (48). Surprisingly, FTY720 treatment did not significantly affect the expression of many of these genes, including sterol regulatory element binding transcription factor 1 (Srebp-1c), a transcription factor involved in sterol biosynthesis; farnesoid X receptor, an essential regulator of cholesterol homeostasis; acetyl-CoA carboxylase , which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis; carnitine palmitoyltransferase 1A, which is involved in -oxidation of long-chain fatty acids; and peroxisome proliferator-activated receptor and its coactivator, which regulate genes involved in energy metabolism (Fig. 8A). However, mRNA of Fasn, a key enzyme of de novo fatty acid biosynthesis that catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA, was significantly reduced by FTY720 administration (Fig. 8A).
Fasn expression in NAFLD livers correlated with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes (49). Similar to previous reports (26,49,50), there was a significant increase in the expression of Fasn in DIAMOND mice fed a WD with SW compared with a CD (Fig. 8B). FTY720 did not affect Fasn mRNA levels in mice fed a CD; nevertheless, it prevented the increase induced by WD and SW (Fig. 8A, B). Consistent with the effects on mRNA expression, FASN protein levels were also decreased by FTY720 treatment (Fig. 8C). Fig. 6. FTY720 treatment markedly reduced hepatic S1P and dihydro-S1P. Mice were fed and treated as described in Fig. 2. Lipids were extracted, and sphingoid bases and their phosphorylated products were determined by LC/ESI/MS/MS. Data are shown as box and whisker plots, n = 6-11 mice per group. ***P < 0.005. We next sought to examine how FTY720 treatment can regulate Fasn expression. Although FASN is usually regulated by the Akt/mTORC1 signaling pathway and upregulation of SREBP-1c, which transcriptionally activates FASN (51,52), this pathway is unlikely to be involved in the effects of FTY720. First, FTY720 had no effect on the expression of Srebp-1c (Fig. 8A). Second, activation of Akt as determined by its phosphorylation was not altered by FTY720 (Fig. 8C). FASN expression can also be controlled by histone acetylation and epigenetic regulation (50,(53)(54)(55). Because histone deacetylase (HDAC) inhibitors reduce the expression of Fasn (53, 54) and we have found that the phosphorylated active form of FTY720, FTY720-P, which accumulates in the nucleus, similar to nuclear S1P (56), is a potent class I HDAC inhibitor (40,57), it was also of interest to examine this possibility. Concomitant with the decrease in the expression of FASN at both mRNA and the protein the levels (Fig. 8A-C), FTY720 treatment led to accumulation of FTY720-P in the liver (Fig. 8D). FTY720 administration also dramatically increased histone H3K9 acetylation in the livers of DIAMOND mice fed WD + SW (Fig. 8E), consistent with reduced HDAC activity. Taken together, our results suggest that in addition to decreasing hepatic sphingolipids, FTY720 treatment also decreases FASN expression.
DISCUSSION
NAFLD is a complex disease that is closely associated with obesity (6,58). In the prevailing "two-hit hypothesis" for the development of NAFLD pathogenesis (59), the "first hit" is due to obesity and insulin resistance, which then increases the susceptibility of the liver to various "second hits," such as oxidative stress and the production of inflammatory cytokines, and, more recently, to the accumulation of ceramides and S1P (12,13). The prevalence of NAFLD is increasing in the United States, and currently there is no pharmaceutical cure (6). Furthermore, NAFLD has emerged as a leading indication for liver transplants and has contributed to the rising incidence of liver cancer (2).
In this work, we examined the effects of the prodrug FTY720/fingolimod, now used for the treatment of multiple sclerosis, in a diet-induced mouse model of NAFLD Fig. 7. Effects of FTY720 on the expression of sphingolipid metabolic genes, S1PRs, cytokines, and chemokines. Mice were treated as described in Fig. 2, livers were isolated, and mRNA levels of Cers1-6 genes (A), Sphk1, Sphk2, and S1pr1-5 genes (B), and cytokine and chemokine genes were determined by quantitative PCR and normalized to Gapdh. Data are means ± SEMs, n = 5-6 mice per group. *P < 0/05. ND, not detected.
that mimics the hallmarks of the human disease (26). The oral administration of FTY720 to DIAMOND mice attenuated increases in overall body and liver weight, improved glucose tolerance, decreased hepatic triglycerides, and reduced steatosis. FTY720 is structurally similar to the fungal metabolite myriocin and has been shown to decrease de novo ceramide production by inhibiting CerS (29,30). Indeed, after treating DIAMOND mice with FTY720, there were also significant reductions of hepatic sphingolipids, including ceramides, monohexosylceramides, and sphingomyelins, their dihydro species, as well as S1P. In this regard, it is important to mention that many of these sphingolipids have been implicated in mice models of hepatic steatosis. For example, pharmacological inhibition of glucosylceramide synthase or the genetic depletion of acid sphingomyelinase reduce hepatic triglyceride levels in mice susceptible to the development of a fatty liver (60). Our findings are consistent with studies showing that the administration of myriocin, an inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in de novo ceramide biosynthesis, not only reduced elevation of hepatic ceramide but also suppressed steatosis in different animal models of NAFLD (61,62). However, while myriocin is highly toxic to humans, FTY720 is already approved for use in humans. As with any medication, FTY720 also has dose-dependent adverse side effects and toxicity. The immunosuppressive effects of FTY720 increase risks of infections. Bradycardia associated with its administration to multiple sclerosis patients is only observed after the first dose and is manageable. In general, only low rates of longterm drug-related adverse effects have been observed with FTY720 (63).
Combining our findings with previous work suggests that mitigating ceramide levels may serve as an effective method for improving NAFLD pathology. The molecular mechanisms by which increased ceramide contributes to NAFLD development are not completely clear, although several possibilities have been suggested. Utilizing CerS2-null mice that cannot synthesize very-long acyl-chain (C22-C24) ceramides, it was shown that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl-chain composition of sphingolipids (64). Additionally, ceramide perpetuates NAFLD development by increasing hepatic fatty acid levels, promoting inflammation and oxidative stress, along with triggering mitochondrial dysfunction (13,65). C16-ceramide also mitigates the insulin receptor/ Akt signaling pathway and thus decreases fat utilization as an energy source by -oxidation (17,66). Mechanistic studies revealed that increases in C16-ceramide caused impaired mitochondrial -oxidation resulting from the inactivation Fig. 8. FTY720 administration decreased HFD-induced FASN expression. B6/129 female mice fed a CD or WD with SW for 16 weeks were treated 3 times per week with vehicle or 0.3 mg/kg FTY720 by gavage as indicated. A, B: Liver mRNA levels of the indicated genes were determined by quantitative PCR and normalized to Gapdh. Data are means ± SEMs. *P < 0.05 versus vehicle-treated mice. C: Proteins in liver extracts were analyzed by immunoblot analysis with the indicated antibodies. Blots were stripped and reprobed with anti-actin antibody to show equal loading and transfer. Blots were quantitated by densitometry. Data are means ± SEMs, n = 5-6 mice per group. *P < 0.05. D: FTY720 and FTY720-P levels in livers from mice fed a WD with SW for 16 weeks and treated with FTY720 were measured by LC/ESI/MS/MS. Data are means ± SEMs. E: Histone H3K9 acetylation in nuclear extracts from livers was determined by immunoblotting with antibodies specific to H3K9 and with anti-histone H3 antibody. of electron transport chain components (19). In agreement, Bruning and colleagues identified CerS6 and C16ceramide as a negative regulator of -oxidative capacity in brown adipose tissue and liver. However, these effects appeared to be independent of increases in respiratory chain capacity in contrast to another report (67). In this regard, daily administration of a selective inhibitor of CerS1, the enzyme that produces C18-ceramide, to mice fed an HFD increased fatty acid oxidation in skeletal muscle and impeded increases in muscle triglycerides and adiposity but did not protect against HFD-induced insulin resistance (68).
In agreement with our work, it was previously reported that the administration of FTY720 during the last 2 weeks of feeding C57BL/6 male mice a diet high in fructose, saturated fat, and cholesterol for 22 weeks decreased steatosis and triglyceride accumulation (46). However, the reduction in liver injury was accompanied by reduced inflammation, likely due to a reduction in hepatic macrophage accumulation without affecting hepatic sphingolipids, whereas in our model, FTY720 only had a minimal effect on inflammation. Nevertheless, FTY720 reduced the expression of the chemokines CXCL10 and CCL5 that have been shown to play a role in liver fibrosis (69). Importantly, we also observed that FTY720 strongly decreased many sphingolipid species. Here, we also made the interesting observation that FTY720 administration to DIAMOND mice reduces their increased expression of FASN, the rate-limiting and last step in de novo fatty acid biosynthesis (70). The main function of FASN is to catalyze the synthesis of palmitate (16:0) that is used for both triglyceride synthesis and the de novo biosynthesis of ceramide. Thus, FASN mRNA and protein expression suppression by FTY720 may be responsible for the reduction in hepatic triglycerides and ceramides we observed in DIAMOND mice.
The transcriptional regulation of FASN induced by HFD has been extensively studied, and SREBP-1c and Sp1, which interact and mediate sterol-induced FASN expression synergistically, have been identified as key transcription factors, whereas carbohydrate responsive element binding protein appears to be the main regulator of glucoseinduced FASN expression (51,52,71). Nevertheless, we found that FTY720 did not affect SREBP-1c expression. Instead, in accord with the notion that FASN expression can also be controlled by histone acetylation and epigenetic regulation (50,(53)(54)(55)72), our results indicate that reduction of FASN by FTY720 could be due to increased histone acetylation. Consistent with our previous findings that treatment with FTY720 leads to increased accumulation of the phosphorylated active form of FTY720, FTY720-P, in the nucleus which inhibits class I HDACs (56,57,73), we observed that FTY720-P accumulated in the liver of FTY720treated DIAMOND mice and markedly increased histone H3K9 acetylation. Thus, like other HDAC inhibitors (53,54,74), reduction in FASN could be due to inhibition of HDACs by FTY720-P and increased H3K9 acetylation. These findings are in agreement with a recent report showing that the expression of Fasn in the liver is associated with increased H3K9 acetylation that modulates the binding of carbohydrate responsive element binding protein to the promoter/enhancer region of the Fasn gene (75).
Excess fatty acids in the liver promote both liver and muscle insulin resistance, which ultimately leads to more accumulation of fatty acids in the liver (76,77), resulting in progression to NAFLD (78). Patients with hepatic steatosis have elevated levels of FASN compared with healthy individuals (49,79). Similarly, mice with hepatic steatosis also have higher levels of FASN (26,49). Despite numerous studies linking the lipogenic pathway to NAFLD, the development of pharmacological inhibitors of FASN has been slow, and no compounds have moved past the preclinical phase (80). Our data suggest that FTY720 deserves consideration as a therapeutic approach that in association with lifestyle interventions would concurrently improve elevated lipogenesis and could have beneficial effects on fatty liver disease and NASH. | 2019-05-22T13:31:48.545Z | 2019-05-20T00:00:00.000 | {
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118602759 | pes2o/s2orc | v3-fos-license | dNLS Flow on Discrete Space Curves
The local induction equation, or the binormal flow on space curves is a well-known model of deformation of space curves as it describes the dynamics of vortex filaments, and the complex curvature is governed by the nonlinear Schr\"odinger equation (NLS). In this paper, we present its discrete analogue, namely, a model of deformation of discrete space curves by the discrete nonlinear Schr\"odinger equation (dNLS). We also present explicit formulas for both NLS and dNLS flows in terms of the $\tau$ function of the 2-component KP hierarchy.
Introduction
The local induction equation (LIE) ∂γ is one of the most important models of deformation of space curves, where γ(x, t) ∈ R 3 is a smooth space curve parametrized by the arc-length x and t is a deformation parameter [6,13,16]. In a physical setting, it describes the dynamics of vortex filaments driven by the self-induction in the inviscid fluid under the local induction approximation [6]. It is well-known that if γ obeys LIE, then the curvature and the torsion, or equivalently, the complex curvature of γ solves the nonlinear Schrödinger equation (NLS) which is one of the most typical equations in the integrable systems. To show this, we use the Frenet frame Φ = Φ(x, t) = [T (x, t), N(x, t), B(x, t)] ∈ SO(3), where T , N, B are the tangent, the normal, and the binormal vectors defined by respectively. Note that it follows that |T | = |γ | = 1 since x is the arc-length. Then we have the Frenet-Serret formula where κ = |γ | and λ = − B , N are the curvature and the torsion, respectively. In this setting, LIE (1) is expressed as the deformation by the binormal flow and the corresponding deformation equation of the Frenet frame is given by The compatibility condition of the system of linear partial differential equations (3) and (5) ∂L Introducing the complex curvature u = u(x, t) ∈ C by the Hasimoto transformation we see that u satisfies NLS Also, one can show that this deformation is isoperimetric, namely |γ | = 1 for all t.
Discretization of curves and their deformations preserving underlying integrable structures is an important problem in the discrete differential geometry. For example, the isoperimetric deformation of plane discrete curves described by the discrete mKdV equation (dmKdV) has been studied in [10,12,14]. For discrete space curves, the deformations by the discrete sine-Gordon equation (dsG) and dmKdV has been studied in [4,11,12], and the deformation by dNLS is formulated in [9,17].
The dsG and dmKdV describe torsion-preserving isoperimetric and equidistant deformation of the space discrete curves with constant torsion. However, formulation of discrete deformation of space discrete curves with varying torsion is a difficult problem. The only example so far is presented by Hoffmann [8,9], where he has claimed that composition of certain two isoperimetric equidistant deformations can be regarded as a discrete analogue of LIE. Also, it was used for numerical simulation of fluid flow [17,19]. This formulation uses quarternions and its geometric meaning is clear, but description of the deformation parameters in terms of the complex curvature, thus the relation to dNLS are rather indirect.
In this paper, we present a formulation of the dNLS flow on discrete space curves from different approach; the deformation of curves is expressed in terms of the discrete Frenet frame with the coefficients given by the curvature and torsion of the curves explicitly. In this approach, dNLS arises as the equation governing the complex curvature of curves, which is the same as the case of smooth curves. Based on this formulation, we present explicit formulas for the NLS flow for smooth curves and the dNLS flow to discrete curves in terms of τ functions of the two-component KP hierarchy by applying the theory of integrable systems. We expect that our dNLS flow can be an alternative to Hoffmann's formulation when it is used to simulate the dynamics of fluids. Also, explicit expression of the scheme and exact solutions may promote further development of theoretical studies of discrete dynamics of discrete curves from both mathematical and physical point of view.
dNLS flow on discrete space curves
Let γ n ∈ R 3 be a discrete space curve with where is a constant. We introduce the discrete Frenet frame Φ n = [T n , N n , B n ] ∈ SO(3) by Then it follows that the discrete Frenet frame satisfies the discrete Frenet-Serret formula where and ν n , κ n are defined by In order to formulate a "good" discrete deformation (discretization of time), we resort to the theory of discrete integrable systems to preserve integrable nature of the NLS flow (4). As a discrete analogue of NLS (9), we consider which we refer to as the discrete nonlinear Schrödinger equation (dNLS) [1,2,18]. Here, u m n ∈ C, Γ m n ∈ R, n is the space discrete variable which corresponds to the label of vertices of discrete curves, m is the discrete variable corresponding to the step of deformation, and δ are constants which are the lattice intervals of n and m, respectively. Moreover, u m n is the complex discrete curvature defined by We impose the boundary condition as Then one of the main statements of this paper is given as follows: For a fixed m, let γ m n ∈ R 3 be a discrete space curve satisfying Let u m n be a complex discrete curvature of γ m n . We determine u m+1 n by dNLS (15) under the boundary condition (17) and put u m+1 Suppose that Γ ∞ and Γ −∞ are either 1 or 1 + 4 δ 2 . Then, it follows that (1) |γ m+1 n+1 − γ m+1 n | = . Namely, γ m+1 n is an isoperimetric deformation of γ m n .
(1) The deformation (21) is not an equidistant deformation in contrast with the deformation described by dmKdV [11]. In fact, one can show that (2) Continuous limit with respect to time can be simply taken as t = mδ and δ → 0. Then dNLS (15) and corresponding deformation equation (20) and (21) yields the semi-discrete NLS equation or the Ablowitz-Ladik equation and the deformation equation of discrete space curves [3,7,15] The dNLS flow (20) and (21) where α m n , β m n ∈ C are given by respectively. Here, * means the complex conjugate. The Frenet-Serret formula (19) which is known as the Lax pair of dNLS [1,2]. In fact, one can verify that the compatibility condition L m n M m n+1 = M m n L m+1 n yields dNLS (15).
Outline of the proof of Theorem 2.1 The first statement may be verified directly in principle, by computing γ m+1 n+1 − γ m+1 n and its length from (20), (21) and the discrete Frenet-Serret formula (19) under the assumption that u m+1 n is determined by dNLS (15). However, this computation is hopelessly complicated to carry out. To make it feasible, we change the Frenet frame to a different frame used in [6,13], which we call the complex parallel frame in this paper. Let F m n = [T m n , U m n , U m n * ] ∈ U(3) be the complex parallel frame defined by Note that it is related to the discrete Frenet frame Φ m n as Then the complex curvature u m n naturally arises in this framework; the discrete Frenet-Serret formula (19) and the deformation of the discrete curve are rewritten in terms of u m n as and respectively. The following lemma plays a crucial role in the proof: Let γ m n ∈ R 3 be the family of discrete space curves given in Theorem 2.1. Then it follows that |α m n | 2 + |β m n | 2 = Γ m n+1 .
By using (32), we have after long but straightforward calculations
Explicit formulas
The formulation of NLS and dNLS flows in terms of the Frenet frame enables us to apply the theory of integrable systems. As an example, we here present explicit formulas of the NLS and dNLS flows in terms of the τ functions. For the case of plane curves, see [10]. These formulas are established based on the bilinear formalism in the theory of integrable systems by applying suitable reductions and imposing complex structure to τ functions of the 2-component KP hierarchy, but here we only show the results, leaving full derivations to the forthcoming publications. For any N ∈ N, we first introduce the following three determinants, a 2N × 2N determinant τ, two (2N + 1) × (2N + 1) determinants σ and ρ as where Ø is the empty block. Then the formulas for NLS flow on smooth curves and dNLS flow on discrete curves are obtained by choosing the entries of determinant as follows: NLS flow on smooth curves We choose the entries of determinants as so that we write τ = τ n (x, t; z), σ = σ n (x, t; z), ρ = ρ n (x, t; z). Here, n and z are regarded as auxiliary variables. Putting we have: Then u satisfies NLS (9).
Then γ satisfies the Frenet-Serret formula (3) and the deformation equation (4). 1 The N-soliton solution for the tangent vector has been constructed by using the bilinear formalism in [5]. dNLS flow on discrete curves We choose the entries of determinants as so that we write τ = τ m n (r, s; z), σ = σ m n (r, s; z), ρ = ρ m n (r, s; z) with r, s and z being auxiliary variables. Putting we have: Then u m n satisfies dNLS (15). | 2016-01-06T12:27:25.000Z | 2015-11-25T00:00:00.000 | {
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269031345 | pes2o/s2orc | v3-fos-license | Delayed gastric emptying after pancreatoduodenectomy: an analysis of risk factors
Background Delayed gastric emptying (DGE) is a frequent complication after pancreatoduodenectomy. Preoperative factors are limited and controversial. This study aims to identify associated factors related to this complication in the Colombian population. Methods A retrospective review of a prospectively collected database was conducted. All patients over 18 years of age who underwent pancreaticoduodenectomy were included. Associations with DGE syndrome were evaluated with logistic regression analysis, Odds ratio, and b-coefficient were provided when appropriate. Results 205 patients were included. Male patients constituted 54.15% (n = 111). 53 patients (25.85%) were diagnosed with DGE syndrome. Smoking habit (OR 17.58 p 0.00 95% CI 7.62–40.51), hydromorphone use > 0.6 mg/daily (OR 11.04 p 0.03 95% CI 1.26–96.66), bilirubin levels > 6 mg/dL (OR 2.51 p 0.02 95% CI 1.12–5.61), and pancreatic fistula type B (OR 2.72 p 0.02 CI 1.74–10.00). Discussion Smoking history, opioid use (hydromorphone > 0.6 mg/Daily), type B pancreatic fistula, and bilirubin levels > 6 mg/dL should be considered as risk factors for DGE.
Introduction
Pancreatoduodenectomy (PD) remains the milestone treatment in pancreatic cancer [1,2].The perioperative mortality of PD in specialized centers is around 0-5%.However, the morbidity remains around 30-50% [3,4].Delayed gastric emptying (DGE) is recognized as one of the most frequent complications, presenting between 15 and 40% of the cases [1,4].DGE was first described by Warshaw and Torchiana, but no standardized definition was defined until 2007 [5,6].The international study group on pancreatic surgery (ISGPS) classified the condition into mild, moderate, and severe based on the nasogastric tube necessity and time to tolerate food intake [7,8].In patients taken to PD, it is the leading cause of increased morbidity, prolonged hospital length of stay, and readmission, with an incidence of around 25-75% [9,10].
Although the pathophysiological mechanisms involved in developing DGE are not yet elucidated, hypotheses related to preoperative, intraoperative, and postoperative factors have been described [11,12].Some of the attributed mechanisms include the loss of the pylorus, damage to vagal fibers, local ischemia, or lack of motilin secreted in the duodenum [10][11][12].Therefore, several variations in techniques have been attempted to improve the unwavering incidence of DGE.Among the interventions with the promising results is the kind of enteric reconstruction (ante colic instead of retro colic) [13,14], the preservation or resection of the pylorus (classic Whipple vs. Pylorus preserving PD) or the pancreatic enteric anastomosis (pancreatic-jejunum vs. pancreaticgastric) [14,15].However, the results are mixed, and many of the studies are small cohorts with retrospective analyses that lack consistency in the definition of DGE [15,17].
Despite the large amount of data available on DGE, information about the clinical and preoperative factors associated with this complication and data from Latin America is scarce [17,18].Reliable preoperative predictors are lacking to improve patient outcomes and to create preventative management of this serious condition [18].This study aims to evaluate the DGE presentation rate, clinical features, and the associated factors with DGE in patients taken to classic Whipple surgery between 2014 and 2022 in a center for hepatobiliary and pancreatic surgery in Colombia.
Study population
A retrospective review of a prospectively collected database was conducted with the Institutional (Hospital Universitario Mayor Méderi) Review Board's approval and following Health Insurance Portability and Accountability Act (HIPAA) guidelines.All patients over 18 years of age who underwent pancreaticoduodenectomy for all causes were included.Patients with missing data were excluded.Perioperative variables included demographics, associated comorbidities, previous mal-nutrition state, pyloric obstruction, parenteral nutrition, and previous bile duct drainage.Some operative variables: operative time and blood loss.And postoperative variables: Surgical site infection, postoperative pancreatic fistula, and opioid use.Technical characteristics of the surgery were ruled out as variables because the surgical technique is standardized for all cases.Ethical compliance with the Helsinki Declaration, current legislation on research Res.008430-1993and Res. 2378-2008 (Colombia), and the International Committee of Medical Journal Editors (ICMJE) were ensured under our Ethics and Research Institutional Committee (IRB) approval.
Sample size
Sample size calculation was performed following Freeman's model for unconditional logistic regression.Considering the delayed gastric emptying rate of 27% of our population and with an estimated dropout of 10%, the total sample size calculation was 205 patients.
Data management: statistical analysis
Descriptive statistics were reported in terms of variable nature.Normality was evaluated using the Shapiro-Wilk test.Qualitative analysis was performed in terms of frequencies and percentages, while quantitative analysis was done in terms of mean and standard deviations of normally distributed data and medians and interquartile ranges (IQRs) for non-normally distributed data.Based on the coefficient of variation (CV), the homogeneity of the quantitative variables was evaluated; if CV < 10% was assumed to be homogeneous, 10-20% was moderately homogeneous, and > 20% was heterogeneous.The association between demographic, clinical, and preoperative variables was evaluated using the chi-squared Pearson test, likelihood ratio, or Fisher exact test (Expected values less than 0.2), and association strength was assessed with relative risk with a 95% confidence interval.For quantitative variables, the homogeneity of the variables was evaluated using the Bartlett-Box test, and in cases of normal distribution, Student t test for two independent groups was performed.Otherwise, a nonparametric Mann-Whitney test was used.The overall group associations with delayed gastric emptying syndrome were evaluated with logistic regression analysis, odds ratio, and b-Coefficient were provided when appropriate.Statistically significant value was accepted if P value < 0.05.Statistical analysis was performed in Stata version 18.0.
Surgical technique
As a standardized classic technique, pancreatoduodenectomy is performed clockwise and divided into three parts: dissection, resection, and reconstruction.for the dissection part, a Kocher maneuver is made until visualization of the left renal vein, hepatoduodenal dissection, and lymph node retrieval, the hepatic artery lymph node is resected and sent to pathology, dissection, and repair of the gastroduodenal artery, gastrocolic aperture, and pancreatic neck dissection until a tunnel is created between the pancreas and superior mesenteric vein.The resection phase is made in this order, cholecystectomy, bile duct section over cystic duct, gastroduodenal artery ligation (always checking for no replacement of the hepatic artery), gastric section at 2 cm above pylorus, pancreatic resection using cautery and hemostatic stitches, and finally jejunal resection using line stapler at 10-15 cm below Treitz Ligament.The resection is completed by transection of the fixation of the uncinate process to the mesenteric vasculature.The final phase is the reconstruction.A transmesocolic ascent of distal jejunum is performed for a pancreato-jejunostomy and hepatico-jejunostomy.The first one is performed in a duct-mucosa technique in 2 layers with a probe left inside the anastomosis.Hepatico-jejunostomy is performed by a modified Blumgart technique at 15-20 cm distal to the PJ anastomosis.We use polydioxanone 4.0 or 5.0 in both anastomoses.Finally, an ante colic gastro-jejunostomy is made in two planes by hand at 60 cm of ascending asa.
Pancreatic fistula risk assessment
A pancreatic fistula score measures the risk of pancreatic fistula and decides to leave an active drain.We use the Fistula Risk Score (FRS) to calculate the risk of pancreatic fistula, which is available in an app.This scale was defined by Callery et al. [18], based on a homogeneous series of patients, it uses four parameters that include consistency of the pancreatic gland, histopathology of the resected pancreas, the diameter of the Wirsung's duct and intraoperative bleeding volume.According to the multivariate analysis performed, only soft pancreatic parenchyma, the presence of ampullary, duodenal, cystic, or islet cell pathology, a pancreatic duct diameter equal to or less than 3 mm, and intraoperative bleeding greater than 1000 were significant (3-times increased risk of developing CR-POPF).
DGE diagnosis and assessment
DGE was diagnosed using the ISGPS classification [7].Grade A: the requirement of a nasogastric tube (NGT) for 4-7 days, or reinsertion after POD 3, unable to tolerate solid oral intake by POD 7, grade B: the requirement of a nasogastric tube (NGT) for 8-14 days, or reinsertion after POD 7, unable to tolerate solid oral intake by POD 14. Grade C: as the requirement of a nasogastric tube (NGT) for > 14 days, or reinsertion after POD 14, unable to tolerate solid oral intake by POD 21.For grades B and C, enteral or parenteral nutrition was used as part of the treatment [7,13,18].
Cohort
A total of 205 patients underwent pancreatoduodenectomy between 2014 and 2022, matching the inclusion criteria and being included in our study.A total of 53 patients (25.85%) were diagnosed with delayed gastric emptying syndrome independent of the grade.Grade A DGE was present in 16.59% (n = 34) of cases, grade B in 4.88% (n = 10), and 3 patients with DGE grade C.
In terms of preoperative variables, 46.83% (n = 96) of the patients were classified with preoperative malnutrition (defined as loss of greater than 5% of the corporal weight); 4.88% (n = 10) of the patients underwent pancreatoduodenectomy in the scenario of pyloric obstruction due to tumor infiltration.Malignancy was confirmed in 84.80% (n = 173) of the patients, and in this case, tumor size was classified in most of the population as greater than 2 cm in 78.54% of the population.Median serum bilirubin from the previous surgery was 4.64 mg/dL (IQR 1.04; 10.04), and in 37.56% of the cases, patients required bile duct drainage before surgery.The median operative time was 290 min (IQR 240; 330).Regarding the postoperative variables, 94.15% (n = 193) of the population requires more than 0.6 mg per day of opioids to control postoperative pain.Postoperative outcomes were included as variables; overall postoperative pancreatic fistula was 28.78% (n = 59); and in most of the patients was classified as a biochemical leak according to the international classification.10.24% (n = 21) of the patients were diagnosticated with intra-abdominal collections.The surgical site infection rate was 6.03%.The mean hospital length was 17.76 ± 12.2 days, and the readmission rate of any condition was 8.78% (n = 18).Summarized data are displayed in Table 1.
Differential analysis by population regarding the presence of delayed gastric emptying was performed; both groups were similar in demographic, clinical, and operative characteristics.With slight differences regarding the presence of pancreatic fistula.Also, in-hospital length of stay was notably higher in patients with DGE (23.03 vs. 15.9 days).Summarized data are displayed in Table 2.
Associated factors: statistical analysis
The initial analysis demonstrated that smoking habit was related with statistically significant value with any grade of delayed gastric emptying syndrome (Chi 57.86 p = 0.00) and arterial hypertension is associated as well (CHI 2.34 p = 0.12).The use of opioid therapy with more than 0.6 mg of hydromorphone daily shows a statistical relationship with DGE (Chi2 2.04 p = 0.15).Tumor size is also associated with a statistically significant value (Chi2 1.98 p = 0.15).The total bilirubin value was related to DGE (Z − 2.1 p = 0.02) and preoperative biliary drainage (Chi 1.65 p 0.19).The presence of pancreatic fistula in any grade shows a relationship with DGE (CHI 1.74 p = 0.18), and especially Type B pancreatic fistula (Chi 7.09 p ≤ 0.00), intra-abdominal collections are related with statistically significant value (Chi2 1.82 p = 0.17).Bilirubin levels were evaluated using an ROC Curve, identifying that a total bilirubin level greater than 6 mg/dL shows a positive likelihood ratio of 1.5.A secondary analysis categorizing the variable shows a statistically significant association (Chi2 4.58 p = 0.03).All of these variables contribute independently to the presentation of DGE.(Table 2).In a final multivariate analysis, variables that show statistically significant association with DGE were, Smoking habit (OR 17.58 p 0.00 95% CI 7.62-40.51),hydromorphone use greater than 0.6 mg/daily (OR 11.04 p = 0.03 95% CI 1.26-96.66),Bilirubin levels > 6 mg/dL (OR 2.51 p = 0.02 95% CI 1.12-5.61),and pancreatic fistula type B (OR 2.72 p = 0.02 CI 1.74-10.00),see Table 3.
Discussion
Although it is not a life-threatening complication, DGE has been associated with increased morbidity, prolonged hospitalization, and higher estimated public health costs of up to 10,000 USD in the United States in the presence of other complications [3,6,15,16].Overall, in this study, DGE was present in 25.86% of pancreatoduodenectomies performed.16.59% was classified as grade A, 4.88% as type B, and 1.46% as type C DGE (Table 4).
The anatomical configurations of reconstruction are also thought to be important for gastric emptying.Antecolic reconstruction seems to be a protective factor against DGE [23].A meta-analysis performed in the United States included 1067 patients undergoing ppPD, 504 patients underwent ante colic reconstruction (AC), and 563 underwent retro colic reconstruction (RC), revealing a lower incidence of DGE in AC reconstruction of 12.1% and 20.7% in RC reconstruction [22,24].Kurahara et al. found that the overall incidence of DGE in the ante colic group was lower than in the RC reconstruction group [24].However, multiple retrospective studies have not observed significant differences, requiring more studies [25,26].In our institution, there is a standardized technique that we can summarize in the following points: use of a duct-mucosa pancreatic enteric anastomosis and trans-stent, ante colic gastric-jejunum reconstruction and the section of the stomach 2 cm above the pylorus (to preserve gastric nerves).Despite being in line with the most accepted surgical techniques, the incidence is still a matter of concern.
Therefore, we believe in the value of identifying preoperative risk assessment.This facilitates counselling, treatment, and adjustment of modifiable risk factors.In this study, the most relevant preoperative factors associated with DGE were smoking habit, opioid daily use greater than 0.6 mg/dL, high bilirubin levels, and the presence of type B pancreatic fistula.
Regarding the relationship between smoking habit and the risk of DGE, the studies are inconclusive and limited [14,15,27].We identify an association between smoking habit and DGE in the analysis (OR 17.58, p ≤ 0.00 95%, CI 95% 7.62-40.51).Along the same lines, some authors identified that patients with a smoking history have a higher risk than nonsmokers [6,28].However, the outcomes were not clinically significant and had a low statistical correlation because smoking didn't affect gastric motility by itself.On the other hand, a recent retrospective study in Germany of 274 subjects showed that active smokers have a lower incidence of DGE and present a better tolerance to solid food, acting as a protective factor [15].Our results align with the hypothesis that its negative effect on the vessel might lead to reduced gastric vascularization and motility [29].
Another relevant fact in this study was the association between analgesic therapy and the development of DGE.Usually, the pre-, peri-and postoperative analgesic management of PD is based on the enhanced recovery after surgery (ERAS, fast-Trak, or Clinical Pathways), which are multimodal strategies that aim to attenuate the loss of and improve the restoration of functional capacity after surgery, emphasizing pain management as a critical parameter in optimal postoperative recovery [30].Thoracic epidural anesthesia (EDA) remains the gold standard method for major open abdominal surgery (PD) [31]; however, there are instances where patient-controlled intravenous analgesia (PCIA) with or without opioids is used [30,31].
According to ERAS [30], it has been documented that EDA allows attenuating metabolic and endocrine responses induced by stress; additionally, increased levels of perioperative hypotension have been described, leading to hemodynamic instability, compromising enteric anastomoses, intestinal perfusion, and gastrointestinal function, increasing risk of surgical complications due to the increased fluid demand and the use of vasopressor support, both of which are related to the development of DGE [32].Prat et al. [33] reported a 15% reduction in postoperative gastrointestinal complications when using EDA vs. PCIA, Wande et al. [34] described an incidence of DGE of 8% in patients with PCIA vs. 9% in patients with EDA, leading to higher use of parenteral nutrition in up to 16% of cases.Despite these postulated benefits, the influence of EDA on clinically relevant outcomes is not clear; most trials to date have not correlated analgesic treatment with surgical complications [30,33,34].
Opioids have been the first analgesic management described; however, they have been associated with multiple adverse effects, including postoperative complications such as DGE and postoperative ileus [35,36].Numerous studies have shown that the opioid-sparing strategy results in accelerated gastrointestinal recovery with decreased incidence of DGE and shorter hospital stays.In our cohort, epidural analgesia was not used frequently.According to our study, 94.15% of patients (n = 193) were dosed with more than 0.6 mg/day of hydromorphone, which was directly related to the development of DGE (OR 11.04, p = 0.03; 95% CI 1.26-96.66).
A study conducted in patients who developed DGE without other intraabdominal complications shows that a higher bilirubin level is a risk factor; notwithstanding, in patients with another intraabdominal condition (like a fistula or intraabdominal collection), the bilirubin is not a significant risk factor, but the preoperative biliary drainage behaves like a protective factor, according to different research studies [37,38,39], nonetheless, the evidence is inconclusive, and indications for the preoperative drainage are limited.Our study reported an association between the development of DGE with elevated bilirubin levels > 6 mg/dL (IQR 1.04-10.04)and a history of intra-abdominal drainage (Chi 1.65, p = 0.19).
The data regarding the Latin American population is scarce.Nevertheless, available evidence shows morbidity rates range between 25 and 60% [20,45] in most of the series due to the development of postoperative pancreatic fistula (POPF); also, delayed gastric emptying with a range between 12 and 44%; [44][45][46]47].Even though there is no clear relationship with mortality, one study shows an independent association between DGE and mortality (OR 3.25, 2.16-4.88,p < 0.001) [44], consequently, all efforts should be focused on its prevention and opportune treatment.
Our study has several limitations that should be mentioned, first a selection bias can occur due to the retrospective nature of the study.The population involved was treated in only an institution by a unique group and with a standardized technique, therefore the influence in other surgical strategies cannot be evaluated.
Conclusion
In conclusion, DGE is a condition associated with high morbidity.All efforts to prevent this complication should be assessed.According to our data, smoking history, opioid use (hydromorphone > 0.6 mg/Daily), type B pancreatic fistula, and bilirubin levels > 6 mg/dL should be considered risk factors for DGE, and some strategies can be added to reduce them.Nevertheless, further prospective studies with larger sample sizes must validate our results.
Table 1
Patient's characteristics
Table 3
Initial statistical analysis
Table 4
Final statistical analysis | 2024-04-11T06:17:56.376Z | 2024-04-10T00:00:00.000 | {
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119207768 | pes2o/s2orc | v3-fos-license | Brightening of dark excitons in a single quantum dot containing a single magnetic ion
A promising method to investigate dark exciton transitions in quantum dots is presented. The optical recombination of the dark exciton is allowed when the exciton state is coupled with an individual magnetic impurity (manganese ion). It is shown that the efficient radiative recombination is possible when the exchange interaction with the magnetic ion is accompanied by a mixing of the heavy-light hole states related to an in-plane anisotropy of the quantum dot. It is also shown that the dark exciton recombination is an efficient channel of manganese spin orientation.
A promising method to investigate dark exciton transitions in quantum dots is presented. The optical recombination of the dark exciton is allowed when the exciton state is coupled with an individual magnetic impurity (manganese ion). It is shown that the efficient radiative recombination is possible when the exchange interaction with the magnetic ion is accompanied by a mixing of the heavy-light hole states related to an in-plane anisotropy of the quantum dot. It is also shown that the dark exciton recombination is an efficient channel of manganese spin orientation. Semiconductor quantum dots (QDs) are among the most promising single-photon emitters [1][2][3][4]. They have potential applications in quantum information processing, and quantum telecommunications due to their seamless integration in semiconductor circuits, their robustness, and their relatively easy handling. Crucially, semiconductor QDs provide the possibility to integrate photonic properties with the spin of an individual magnetic impurity [5]. The magnetic spin can be selectively manipulated and used for information storage [6,7]. However, the use of semiconductor QDs in a realistic working device requires a reliable control of the excitation process as well as an understanding of the emission channels.
An important, but nevertheless, little investigated recombination channel is related to the dark exciton states i.e. states with total angular momentum equal to 2 [8]. Random transitions between dark and bright excitonic states lead to exciton decoherence [9] and a significant modification of the recombination dynamics which can result in the delayed emission of photons [10,11]. Despite their importance, dark exciton states are difficult to probe. The radiative recombination of dark excitons is forbidden so that they usually cannot be studied directly using spectroscopic techniques. Their properties can be accessed indirectly by a detailed analysis of the dynamics in time-resolved profiles of the bright exciton photoluminescence [10][11][12]. The other possibility is to measure the weak optical transitions under conditions in which the dark exciton recombination is partially allowed. This has been achieved either by the use of the in-plane magnetic field which mixes the heavy-light hole states [13,14] or by placing the QD in a micro-pillar which enhances the coupling of the exciton with light [15].
Here we present an investigation of dark exciton optical transitions which are allowed due to the simultaneous spin flip of coupled single magnetic impurity. We analyze the dark exciton wave function and show that the radiative recombination of dark excitons is efficient only when the exchange interaction with the magnetic ion is accom-panied by mixing of the heavy-light hole states, related to an in-plane anisotropy of the QD. To demonstrate the interplay of both mechanisms, high magnetic field spectroscopy has been employed. We determine all relevant parameters such as the dark exciton oscillator strength, the in-plane anisotropy, and the exchange interaction. Additionally, we show that the dark exciton recombination can be used as an efficient channel for controlling the orientation of the spin of the magnetic ion.
The sample, which was grown using molecular beam epitaxy, contains a single layer of self-assembled CdTe QDs with a low concentration of Mn 2+ ions, embedded in a ZnTe matrix. The Mn 2+ concentration was adjusted to obtain a significant number of QDs containing exactly one Mn 2+ ion [16]. For the measurements, the sample was placed in a micro-photoluminescence (µ-PL) setup and kept at the temperature of 4.2 K. The resistive magnet produced magnetic field up to 28 T. The field was applied in the Faraday configuration, parallel to the the growth axis of the sample. The PL of the QDs was excited either above the gap of the ZnTe barrier (at 532 nm) or using a tunable dye laser in the range 570 − 610 nm. Both the exciting and the collected light were transmitted though a monomode fiber coupled directly to the microscope objective. The use of the monomode fiber combined with polarization optics outside the cryostat permits the control of the circular polarization of both the exciting and detected light.
The diameter of both the excitation and detection spot was less than 2 µm, which allows us to select spectra of different single QDs containing a single Mn 2+ ion. Representative results for two selected QDs with different inplane anisotropy are shown in the insets of Fig. 1. Characteristic PL spectra contain a neutral exciton (X) lines split into sextuplets due to the X-Mn exchange interaction [5,17]. The total spin of the Mn 2+ ion is 5/2, and it has six possible projections onto the direction of highly anisotropic excitonic spin. Thus, each of the six possible spin states of the ion is related to a specific component of Emission energy (meV) the excitonic sextuplet for a given circular polarization.
As in our previous work [7], we have selected dots containing a single Mn 2+ ion which have a nonmagnetic QD as a close neighbor. The resonant excitation of this dot is followed by a spin conserving X transfer to the Mndoped QD [18]. This permits the efficient and selective excitation of the luminescent dot as well as the selection of the spin polarization of excitons injected into this dot. Figure 1(a-b) shows the evolution of neutral exciton PL spectra with magnetic field, measured without polarization resolution for the two selected QDs. To elucidate the most characteristic spectral features of this evolution we initially focus on Fig. 1(a) showing the QD with the smaller in-plane anisotropy. In the magnetic field, the exciton sextuplet splits into two distinct Zeeman branches corresponding to σ + and σ − circular polarizations for the upper and lower branch respectively. The lowest component of each branch corresponds to the Mn 2+ spin antiparallel to the exciton spin [5]. The resonant excitation with σ − polarization prevents the Mn 2+ spin from thermalizing in the magnetic field [7] due to the interaction of the Mn 2+ ion with spin-polarized carriers injected into the dot. Therefore all six lines are visible in both branches up to magnetic fields ∼ 15 T.
At a magnetic field of around 12 T, an anticrossing of the outermost lines of both branches is clearly visible ("A" in Fig 1(a)). These two lines correspond to the same −5/2 state of the Mn 2+ spin, but two opposite spin states of X. At this field, the excitonic Zeeman splitting exactly compensates the X-Mn exchange interaction. The splitting (anticrossing) of these two lines is then simply due to the in-plane anisotropy of the QD, acting via the anisotropic component of the electron-hole (e-h) ex-change interaction, as observed for excitonic lines in the absence of magnetic field for nonmagnetic QD [19]. As we have checked, the two split lines show linear polarization (presumably along the symmetry axes of the anisotropic dot), in contrast to the remaining sextuplet lines which are polarized circularly. The anisotropic exchange splitting, determined from the anticrossing, is equal to 60µeV for QD1 in Fig 1(a), and 230µeV for QD2 in Fig 1(b).
Our optical method to align the Mn 2+ spin against the action of the external magnetic field [7] becomes less efficient at magnetic fields above ∼ 15 T. This is due to the accelerated spin-lattice relaxation of the Mn 2+ spin at high magnetic fields [20]. As a result, the Mn 2+ spin orientation thermalizes and the excitonic lines related to the less populated spin states vanish.
Strikingly, the QD with the larger anisotropic exchange splitting value (Fig 1(b)) has an additional, albeit weaker, lower branch consisting of only five lines. To understand the origin of this branch one should notice that optical transitions are possible in two situations: (a) The projection of total angular momentum of the exciton on the quantization axis is equal to ±1 (bright exciton). In this case the transition is dipole allowed and the Mn 2+ spin projection is conserved during the X recombination. Branches with six lines are related to this kind of recombination. (b) The projection of total angular momentum of the exciton is equal to ±2 (dark exciton, X d ). Then, in the first approximation the optical transition is dipole forbidden. However, the valence band mixing and the exchange interaction with the Mn 2+ ion result in a mixing of the electron and hole spin states. As a result, the X d states have an admixture of the X states with the Mn 2+ spin projection different by 1. Thus, the X d recombination is possible when accompanied by the simultaneous spin-flip of the Mn 2+ ion. As there are only 5 possible transitions between the 6 Mn 2+ spin states, the PL lines related to X d present a fivefold spitting. The upper energy branch of X d is not clearly visible in our experiment because it overlaps with much stronger X transitions.
The quantum states of the exciton and Mn 2+ spin can be described in the basis given by three quantum numbers: |S z , σ z , j z indicating the Mn 2+ , electron and hole angular momentum projections onto the quantization axis parallel to the magnetic field. Using the available information for the g-factors of the carriers and the Mn 2+ ion [21], we attribute the X d low energy branch to the recombination of |S z , +1/2, +3/2 states. Therefore, to satisfy the selection rules for X d dipolar recombination the projection of the Mn 2+ spin must be increased by 1. This implies that each possible X d recombination is related to a spin-flip of the Mn 2+ ion towards the state polarized opposite to the thermalized state. There are three different admixtures of the X d states which make this process possible (see Fig. 2(a)). The first one is the state with the opposite spin projection of the electron (|S z + 1, −1/2, +3/2 ). It is mixed with the X d state The width of splitting between the | + 1.5, +1/2, +3/2 and | + 2.5, +1/2, −3/2 lines ("B" in Fig. 1(b)) calculated from the model vs. splitting between these lines determined directly from the PL for those QDs for which it was possible.
due to the e-Mn exchange interaction. The second admixture, caused by the h-Mn exchange interaction, consists of a light hole with a spin projection different by 1 from the heavy hole of the X d (|S z + 1, +1/2, +1/2 ). The last admixed state, |S z + 1, +1/2, −3/2 , consists of a heavy hole with a spin projection different by 3 from the spin projection of the original state. This admixture is induced by two interactions acting together: the heavy-light hole mixing, which mixes hole states with spin projection different by 2, and by the h-Mn exchange interaction. A direct experimental evidence for the presence of valence band mixing is provided by the anticrossing of the highest energy line of X d and the lowest energy line of X at a magnetic field ∼ 7 T (labeled "B" in Fig 1(b)). These lines correspond to | + 1.5, +1/2, +3/2 and | + 2.5, +1/2, −3/2 states, respectively. The radiative recombination related to the first two of these admixtures results in the emission of a photon with σ + polarization. The amplitudes of these admixtures to the X d state depend only on the e-Mn and h-Mn exchange constants, i.e. on the overlap of the Mn 2+ ion and the carrier wave functions. In contrast, the recombination related to the third admixture produces a σ − polarized photon. The amplitude of this admixture depends not only on the X-Mn exchange interaction, but also on the valence band mixing, which, similarly to the anisotropic exchange splitting, results from the in-plane anisotropy of the QD [14,[22][23][24]. The role of this anisotropy in determining the polarization of the X d lines is clearly visible in our experiment. Fig. 3 shows spectra of the two QDs shown in Fig 1 for similar excitation conditions and magnetic field near the anticrossing of the lines corresponding to | − 2.5, −1/2, +3/2 and | − 2.5, +1/2, −3/2 states ("A" in Fig 1(a-b) 3. Spectra of the two QDs shown in Fig. 1 taken under excitation with σ − polarized light with polarization resolved (σ + or σ − ) detection for magnetic fields near the anticrossing of lines corresponding to | − 2.5, −1/2, +3/2 and | − 2.5, +1/2, −3/2 states ("A" in Fig 1(a-b)).
polarized lines are much more pronounced with respect to the σ + polarized ones for a highly anisotropic QD2. As a quantitative measure of the X d oscillator strength for both circular polarizations we use the X d /X intensity ratio. Since this ratio depends on the excitation power [25], one should use identical excitation conditions to be able to compare its value for different QDs. Experimentally, this is achieved by choosing the same XX/X intensity ratio equal in our case to 1/3. The XX intensity increases roughly quadratically with the excitation power, while the X intensity follows linear power dependence ( [26,27]). Thus, this ratio gives a measure of the excitation efficiency.
A strong increase of the X d /X intensity ratio in σ + polarization with increasing X-Mn exchange constant is clearly visible in Fig. 2(b). It confirms the origin of the first two recombination channels described above. However, the X d oscillator strength for σ − polarization depends both on the X-Mn exchange interaction and the QD anisotropy. To elucidate the role of the anisotropy, we can use the circular polarization of the X d . Fig. 2(c) shows that the polarization of the X d evolves towards σ − as the anisotropic exchange splitting increases. Therefore, we conclude that a high in-plane anisotropy of the QD leads to a strong mixing of light and heavy holes, which combined with the presence of the Mn 2+ ion is the reason for the strong σ − polarization and high intensity of the X d spectrum. It is important to note that for nonmagnetic QDs showing similar anisotropy [14] the optical transitions of X d are not observed, except in the presence of a strong in-plane magnetic field.
As each X d recombination in the lower energy branch involves an increase of the Mn 2+ spin projection by 1, dark exciton recombination can play the role of an effective Mn 2+ spin orientation mechanism. In our experiment the X d /X intensity ratio for a highly anisotropic QD and σ − polarized excitation was as high as 10%. This is comparable to the probability of a spin-flip of the Mn 2+ ion per one recombination of the X in the QD which was estimated to be ∼ 10% in Ref. [7]. While this orientation mechanism should be present for both circular polarizations of excitation, only the σ − polarization which populates the low energy branch of X d is fully seen. The σ + polarized excitation decreases the Mn 2+ spin projection [7]. Under such conditions the high energy branch of X d should be populated and the low energy branch should be virtually invisible. The latter is, indeed, confirmed in our experiment. However, it is not possible to observe directly the high energy X d branch, since it occurs in the same energy region as the much stronger X lines.
A quantitative description of key features of the data in Fig. 1(a-b) is provided by a simple model with the initial state of the QD after excitation event given by the following Hamiltonian ( [13,14,[28][29][30]), where S, σ and j are the Mn 2+ , electron and hole spin operators, respectively, the first three terms represent the Zeeman energy of the Mn 2+ ion, the electron and the hole, I e and I h are the e-Mn and h-Mn exchange interaction constants, a i and b i are e-h spin-spin coupling constants, 2γ is the heavy-light hole splitting and β represents the strength of the heavy-light hole mixing. The first term is also the Hamiltonian of the final state of the system after the exciton recombination. We also introduced an additional, phenomenological term related to the excitonic diamagnetic shift to facilitate a comparison of the model and experimental data. The energies of the optical transitions versus magnetic field, calculated using this Hamiltonian for the two circular polarizations, are plotted in Fig. 1(c). The calculations clearly reproduce the key features of the experimental data in Fig. 1(a-b), such as for example the X-Mn exchange splitting and the anisotropic exchange splitting. All parameters in the Hamiltonian (except for γ assumed to be 15 meV [24]) can be extracted by fitting to the experimental data. In particular, the heavy-light hole mixing can be estimated using the degree of circular polarization of X d lines. Such an approach permits an estimation of the β parameter even for those QDs, for which the anticrossing between the | + 1.5, +1/2, +3/2 and | + 2.5, +1/2, −3/2 lines ("B" in Fig. 1(b)) is not clearly visible. As shown in the inset of Fig. 2(c) the obtained value of this anticrossing remains in very good agreement with the value estimated directly from PL data. This confirms the proposed mechanism of X d brightening.
To conclude, we have used a QD with a single magnetic impurity (Mn 2+ ) to investigate the dark exciton transitions. The X-Mn exchange interaction, when com-bined with a mixing of the heavy-light hole states induced by the QD in-plane anisotropy, allows dark exciton recombination accompanied by a simultaneous Mn 2+ spin flip. High magnetic fields have been used to spectrally separate the PL lines related to X and X d transitions and to extract the important QD parameters (e.g. the anisotropic exchange splitting). The excitation of the Mn-doped QD via a closely lying second QD permits the precise control of the spin of the carriers injected to the QD and therefore a control over the Mn 2+ spin orientation. The simple Hamiltonian, which describes this system, reproduces correctly all the key features of the PL spectra of the QD in high magnetic field. | 2010-06-08T08:57:49.000Z | 2010-06-08T00:00:00.000 | {
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81346353 | pes2o/s2orc | v3-fos-license | Janani Shishu Suraksha Karyakram services utilization among rural mothers: community perspective from southern areas of West Bengal
Over the years, maternal and child health has evolved through various stages of conceptual approach, technological advances and social prioritisation. Despite the current efforts, the health of mother and child still considered as one of the most serious health problems affecting the community, particularly in developing countries. 1,2 The main reason may be due to women’s disadvantaged position in many countries and cultures, and in the lack of attention to women’s health and life. 2 In order to increase accessibility of health care services for pregnant mothers and sick neonates in public health facilities and reduction in out of pocket expenditure, the Govt. of India launched Janani Shishu Suraksha Karyakram (JSSK), which currently provides entitlements (benefits) to both mothers and sick infants (Entitlements were extended to 1 year of birth subsequently). 3,4
INTRODUCTION
Over the years, maternal and child health has evolved through various stages of conceptual approach, technological advances and social prioritisation. Despite the current efforts, the health of mother and child still considered as one of the most serious health problems affecting the community, particularly in developing countries. 1,2 The main reason may be due to women's disadvantaged position in many countries and cultures, and in the lack of attention to women's health and life. 2 In order to increase accessibility of health care services for pregnant mothers and sick neonates in public health facilities and reduction in out of pocket expenditure, the Govt. of India launched Janani Shishu Suraksha Karyakram (JSSK), which currently provides entitlements (benefits) to both mothers and sick infants (Entitlements were extended to 1 year of birth subsequently). 3,4 and few poor socio-economic indicators as in northern states of India. Fewer remote sub centres have very high rate of home deliveries in the southern districts of West Bengal even after implementation of maternity benefit scheme like JSSK. 5 Here many factors interact in different ways to affect the utilization of health services in different sections of the society e.g. place of residence and social economic status, women's age, education, religion, culture, clinical need for care, decision-making power, and age at child birth, parity, poverty and ignorance, costs, location and quality of services. The present study was conducted to understand the utilization pattern of JSSK services among women who have recently delivered and the cost incurred thereof.
Study setting participants
A community based cross sectional study was conducted in the rural areas of South 24-Parganas district of West Bengal (which has both delta and non-delta region), between January, 2016 and May, 2016. Women, who gave birth between 1 st July 2015 to 31 st December, 2015 in the studied areas of the district and given consent for participation, were interviewed irrespective of the current living status of the child/children. The mothers who were mentally unfit for interview were excluded.
Selection of the participants
According to district data (2014-2015) of South 24-Parganas, percentage of home delivery is 37%. 5 Home delivery was taken as non-utilization of JSSK. The remaining 63% was considered to be having minimum utilization of the services. The number of women to be interviewed with an estimated minimum utilization rate of 63% and a precision of 10% with a design effect of 1.5 was used to calculate the overall sample required, which was found to be 338 ≈ 340.
Initially all blocks of the district were divided into delta and non-delta areas. 6 Then the non-delta blocks were further divided into "blocks within 50 km of Kolkata" and "blocks outside 50 km of Kolkata". Communications and facilities vary between these geographical distributions. It was also conceptually presumed that regions far from Kolkata would have difference in facilities and infrastructure compared to those closer. Thus among non-delta regions the blocks within 50 Kms from Kolkata were the blocks nearer and those outside 50 Kms were distant from Kolkata. One block from delta blocks was randomly selected using random number table. From each cluster in non-delta blocks, two blocks were randomly selected. A third stage of random sampling was done to select one sub-center from each chosen block. The sampling scheme is shown in Figure 1.
Study variables and analysis
The selected mothers were interviewed using a predesigned and pre-tested validated structured questionnaire. Available medical records of the mother and infants, hospital discharge certificate, doctor's prescription, mother and child protection card (MCPC), laboratory report etc., records and registers (RCH register, referral register etc.) of the sub-centers, computer database from block primary health center were also reviewed. Data collection was done through house to house visits and personal interview of mothers. Data regarding basic socio-demographic information, childbearing experiences (e.g. parity, ANC visits, type of delivery etc.), and different services related to maternal health (like transportation type, place of ANC visit, place of delivery etc.) and the utilization of JSSK services (e.g., free diagnostics, free drugs & consumables, free transportation etc.) were collected and entered in Epidata 3.1. Analysis was done by using Statistical Package for Social Sciences (SPSS) Version 21.0 with a comparative approach among the participants of delta and non-delta blocks.
Ethics
The institutional review board of Christian Medical College, Vellore approved the study protocol. Permission was also given by the Institutional Ethics Committee of Medical College and Hospital, Kolkata. District health department gave official permission to conduct the study in this area. The respondents were briefed prior to interview regarding the purpose of the study. Written informed consent was obtained prior to the interview.
Background information
The mean age of the mothers was 23.58 years (±4.36 years) and ranged from 15 to 38 years. Around 1.7% of them were below the age of 18. Of the respondents from delta regions 81.7% belonged to Islam. Other blocks also had majority Muslim respondents but lesser compared to delta block. Majority of the respondents (35.2%, 32.4% and 34.1% respectively in delta blocks, non-delta blocks more than 50 kms from Kolkata and less than 50 kms from Kolkata) had education up to primary level. More than 15% women from non-delta region had higher education as compared to 5% in delta region. Husbands of the respondents belonging to delta region had lower level of education compared to non-delta regions with majority having primary education (40.8%), whereas at least 40% men from non-delta blocks had education above secondary level. Majority, of the women (81.7%, 99.3%, 90.6% in Delta, Non-delta more than 50 kms, Non-delta within 50 kms respectively) were house-wives. Majority of the husbands (39.4%, 27%, and 24.6% respectively) were unskilled workers. In delta regions more than half of the families (52.1%) belong to below poverty line (BPL) category. Most of the families were nuclear (80.3%, 63.8%, 56.5% in Delta, Non-delta more than 50 kms, Non-delta within 50 kms respectively) in this region. None of the respondents from delta block stayed within 5 kms of any health facility with a doctor available, where as 62.3% respondents from non-delta block close to Kolkata (within 50 km) stayed within 5 km of any health facility with a doctor. Table 3 shows the utilization of JSSK entitlements. It was clearly observed that the utilization any of JSSK entitlements was comparatively lower among the respondents of delta regions. While 18.4% of the respondents from delta region utilized free diagnostic services, 16.9% utilized the free delivery services. These utilizations were higher (92% and 83.3% respectively) among those residing nearer to the state capital. However the utilization for free drugs and consumables were observed to be comparable among the participants from the three regions. Among those residing in the nearby non-delta blocks 81.8% had free food services. But the utilization of free transport facilities were found to be lower. In this case 46.1% of the delta residents utilized the services. Among the non-delta residents living far 32.1% and 39.6% of those living nearer to Kolkata utilized the free transportation services from their homes to health facility. A marginally higher utilization of free transportation among the respondents of non-delta region was observed for transport back to home.
Very few respondents of those who utilized the JSSK services reported paying for services provided free under JSSK. Diagnostics and drugs and consumables were paid for more often. Among those who paid for diagnostic services the cost incurred was below INR 500. For drugs & consumables it was within INR 3000 and below INR 700 for food during hospital stay.
DISCUSSION
The current article provides an insight regarding the utilization of different entitlements of JSSK in southern parts of West Bengal. In doing so it also looked into the difficulties faced in availing health facilities by women of different region especially those residing in the delta regions. Many factors such as poor road access, scattered habitation and distance from health facilities were assumed to play a role, though understanding these hardships were beyond the scope of the current article.
Non-delta regions and delta region greatly differ in the availability of and access to health care facilities. Nondelta regions on contrary to the delta blocks have more health facilities resulting in more women reporting a center within 5 kms of their residence. Accessibility is also a problem. Dhak (using DLHS-3, 2007-06 data) found that distance to the health facility was another important influential factor for adopting ANC services. 7 In both delta and non-delta regions most of the women go to sub-centers for antenatal check-ups. Visiting unqualified persons (quacks) is a known practice in these regions. They choose to go to hospitals only if their condition deteriorates. The present study found that half of the women from the delta region had less than 3 antenatal check-ups where as in the non-delta region the proportion was much smaller at 12%. Functionality issues and higher population burden for BPHCs and PHCs or sub-centres may be contributing to this burden of under-utilization of government services along with several other community and individual level factors.
Most of the respondents (74.6%) in delta regions did not get any diagnostic tests done during pregnancy. In nondelta blocks close to Kolkata (<50 km), large number of respondents (92.8%) used exclusively government hospitals for diagnostic tests. This may reflect on reliance on quality and easy access, and show distance from the capital is influencing utilization. Awareness may play an important role. Chatterjee et al found in their study that only 29.17% women in rural West Bengal were aware of availability of free diagnostic tests in a government facility. 8 The concurrent assessment study of JSSK showed that out of the total number of patients recommended for any lab tests, a large number of patients in Rajasthan (100%), Maharashtra (98%), HP (96%), Tripura (91%) and Kerala (70%) got their tests done in government facilities. 9 A few beneficiaries reported going to private other than in Kerala where in diagnostic services are provided through public-private partnership arrangements. An attempt may be made towards increasing awareness about the importance of diagnostic tests and its availability, free of cost at a government facility which appears to be pertinent in view of the findings reported by the current study.
Regarding the under-utilization of the transport services this study did not explore if women chose to deliver at home due to lack of transportation facility. This needs further investigations to assess the role of free ambulance services and poor road conditions affecting the family or individual decision to deliver at home. Poor utilization can also be due to lack of awareness, unavailability of ambulance when called over phone and very bad road condition. Chatterjee et al found in their study that 35.42% women were aware of both free transport services, from home to institution and back to home. 8 Concurrent assessment of impact of JSSK (NHSRC) in 2013-14 found that usage of private hired vehicle as a mode of transportation for drop back is predominant in the states evaluated. 9 However, increased proportion of patients reported using government free transport while going back to home in comparison to the number of beneficiaries who reported to have used government transport while coming to the facility.
The poor utilization of government or accredited health facility may well be attributed to the lack of knowledge about the accredited hospitals for maternal and child health care. Chatterjee et al found in their study that 18.75% women in the rural areas were aware about free delivery. 8 In delta regions most of the deliveries (78.8%) took place at home. In comparison, non-delta regions within 50 km from Kolkata had a similar proportion with institutional delivery and at a government facility (76.8%). It shows that as one move closer to capital, there is an increase in institutional deliveries. The place of delivery also influences the mode of delivery and Kolkata is no exception showing very high Caesarean section (C-section) rate (39%) among the women living close to the metro. While this is much higher than recommended, overall C-section rate for delta block works out to be only 4%. Out of the women who chose to deliver in government or accredited hospitals, more than 80% were provided drugs and consumables free of cost. There is not much difference in availing free drugs and consumables in delta and non-delta regions. It may be argued that once they are admitted in a hospital, there is no bias in services, however it is difficult to conclude that the opposite is true.
Concurrent assessment of impact of JSSK (NHSRC) in 2013-14, found out that transportation is one main component where out of pocket expenditure was incurred by the beneficiaries of all the states. 9 Apart from transportation, blood transfusion and diagnostic services are the major cost incurred in Rajasthan. Medicines and diagnostic services were considered to be major cost incurred in Kerala and Tripura. In the current article the major out of pocket expenditure was observed with drugs and consumables and also with diagnostic services.
In the present assessment of service utilization by mothers the possibility of recall bias exists along with the chance of conscious falsification regarding perceived sensitive information. The availability of certain facilities like the number of ambulances, number of beds in the post-natal wards was not objectively measured. Anecdotal information is available in this study suggesting that the health system is not geared to cater to all the needs of the people. Lack of data makes it difficult to comment objectively on the impact of availability of these facilities on the utilization rates of JSSK. The scheme as a whole with all the entitlements should be promoted. Stress on free provision of blood and diagnostics to be given during promotion. Issue of institutional delivery should be promoted vigorously. Delta blocks need to be given special attention where the PHCs may be the only health care facility manned by qualified health professionals available to the population. Along with promotion of the services generation of awareness, acceptance and also implementation of methods to reach out to people should be focused more. | 2019-03-18T14:03:24.157Z | 2018-09-24T00:00:00.000 | {
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268514083 | pes2o/s2orc | v3-fos-license | Right upper lobectomy with mediastinal dissection under uniportal video-assisted thoracoscopic surgery for lung cancer in a patient with a right-sided aortic arch: a case report
Background A right-sided aortic arch is a rare congenital vascular structure variation. Right lobectomy is not commonly performed on patients with such a condition. Further, there are no reports on lobectomy under uniportal video-assisted thoracoscopic surgery (VATS) in this patient group. Case presentation A 67-year-old man with a right-sided aortic arch and Kommerell diverticulum underwent right upper lobectomy with mediastinal lymph node dissection under uniportal VATS for primary lung cancer. Due to the right descending aorta, which narrows the space of the dorsal hilum, handling of the stapler for stapling the right upper lobe bronchus from the uniport in the 6th intercostal space at the medial axillary line can be challenging. This issue was resolved by manipulating the staple over the azygos vein toward the inferior margin of the aortic arch. Via mediastinal lymphadenectomy, we found that the right recurrent laryngeal nerve branched from the right vagus nerve and hooked around the right-sided aortic arch. Conclusions Right lobectomy with mediastinal lymph node dissection under uniportal VATS can be performed for lung cancer in patients with a right-sided aortic arch.
Background
A right-sided aortic arch is a rare congenital anomaly, with retention of the 4th branchial arch artery on the right side.The incidence of this condition is approximately 0.1% [1].In the past 15 years, only eight cases of a right-sided aortic arch have been reported among patients who had undergone lobectomy for right lung cancer [2].To the best of our knowledge, this is the first case report in which right upper lobectomy with mediastinal lymph node dissection (ND2a-1) was performed under uniportal video-assisted thoracoscopic surgery (VATS) in a patient with right lung cancer.Herein, we report a case of lung cancer with a right-sided aortic arch.
Case presentation
A 67-year-old man was monitored for a right-sided aortic arch and Kommerell diverticulum (Fig. 1a and b).There was no significant medical history other than the right-sided aortic arch.He had a smoking history of at least 50 pack-years and currently smoke.The patient was symptom-free, and there were no remarkable findings on physical examination.The tumor markers carcinoembryonic antigen, CYFRA, and pro gastrin releasing peptide and spirometry were within normal limits.Echocardiography revealed no cardiac abnormalities.The most recent chest computed tomography (CT) scan revealed a 20-mm × 18-mm solid nodule in the right S1 (Fig. 2a).The nodule was suspected to be malignant based on the increase in lesions over time on CT scans.A systemic CT, brain magnetic resonance imaging (MRI), and bone scintigraphy examination revealed no other lesions.There were no anatomic variations in the pulmonary vessels or bronchi (Fig. 2b).Right upper lobectomy with ND2a-1 under uniportal VATS was performed as stage IA2 right upper lobe lung cancer was suspected.Using a soft tissue retractor, a 4-cm incision in the 6th intercostal space at the medial axillary line was made as the main manipulation port.The operative findings confirmed the presence of a right descending aorta that runs on the right ventral side of the vertebral body and that narrows the space of the dorsal hilum.After dividing the interlobar fissure with a stapler (Endo-GIA Ultra Universal Staples; Medtronic plc., Dubin, Ireland), the V 1 + 2+3 , A 2b , and A 1 + 3 were detached and divided sequentially (Fig. 3a-d).
After dissecting the right upper lobe bronchus, due to the right descending aorta, handling of a stapler (Powered Echelon Flex GST System 45; ETHICON Endo-Surgery, inc., Cincinnati, OH, USA) used to staple the right upper lobe bronchus located on the dorsal hilum was challenging (Fig. 3e-1).This issue was resolved by manipulating the staple over the azygos vein toward the inferior margin of the aortic arch (Fig. 3e-2).The diagnosis of the intraoperative frozen section was carcinoma.During lymph node dissection of the upper mediastinum, the right vagus nerve was identified at the cranial side of the azygos vein (Fig. 4b).Next, dissection was performed in the caudal direction.The right recurrent laryngeal nerve was found to branch from the right vagus nerve just beneath the right-sided aortic arch (Fig. 4c), and the mediastinal lymph node was subsequently dissected (Fig. 4d).The operative time was 325 min, and the volume of blood loss was 59 mL.The patient was discharged on postoperative day 8 without any complications such as hoarseness.The pathological diagnosis was small cell lung cancer.The tumor had a diameter of 21 mm × 14 mm with histopathological features of pm0, pl0, ly0, and v1.Four #2R+#4R lymph nodes, one #11 lymph node, and six #12u lymph nodes were retrieved, but there was no #10R lymph node.No lymph node metastasis was detected.After 8 months of adjuvant chemotherapy for the stage IA3 tumor, no
Discussion and conclusions
Given how uncommon the present case is, it is crucial to determine the anatomical features of the right intrathoracic space in patients who had undergone for primary lung cancer and had a right-sided aortic arch.According to the classification of Edwards et al. [3], our case was type IIIB, which is characterized by the dilated stem of the left subclavian artery.This condition is referred to as a Kommerell diverticulum, which runs from the right to left side behind the esophagus.As a result, the right descending aorta after branching a Kommerell diverticulum runs more ventral to the vertebral body than the left descending aorta in a normal anatomy.Therefore, if the dorsal hilum is pushed more ventrally, the right upper lobe bronchus located on the dorsal hilum is more challenging to dissect.
Uniportal VATS lobectomy is an effective surgical procedure [4].The greatest strength of the technique is its prospective benefits for patients, including reduced postoperative pain in comparison to other multiportal approaches, speedy patient recovery, short postoperative hospital stay duration, and improved clinical cosmetic outcomes [5].However, compared with multiportal VATS, it can be more challenging to interfere with thoracoscopic instruments used to expand the operative field.The direction of insertion is essential for the stapler that is deployed in a fan shape from the uniport because it is necessary to secure a space behind the separation.During resection of the right upper lobe bronchus located on the dorsal hilum, the presence of the right descending aorta narrowing the space of the dorsal hilum prevented the stapler from advancing cranially from the uniport.Therefore, this issue was resolved by devising the orientation of the stapler.
Regarding lymph node dissection, multiple VATS has advantages in terms of the amount of the dissected lymph nodes [6].The direction of insertion from the uniport may limit the operative field of view for the surgeon.The reported uniport VATS lobectomy often uses the anterior approach [5], but the direction of insertion from the uniport may limit the operative field of the dorsal view of the hilum and superior vena cava.In right upper lobectomy and ND2a-1, the main manipulation port was made in the 6th intercostal space at the medial axillary line because this site can conveniently show the dorsal view of the hilum and superior vena cava, which is beneficial to lobectomy and lymph node dissection.In the present case, the direction of the insertion from the uniport might have allowed an easier dissection of the upper lobe bronchus located on the dorsal hilum and the dissection of the lymph nodes dorsal to superior vena cava.
The aortic arch is overhanging the trachea.Hence, dissection of the upper mediastinal lymph nodes in patients with a right-sided aortic arch differs from the routine dissection.Matsubara et al. hypothesized that the left and right upper mediastinal lymph nodes are separated by the aortic arch, not the trachea [7].In addition, the leftright asymmetry of the upper mediastinal lymph nodes is caused by the retention and regression of the left and right branchial arch artery.According to this hypothesis, the upper mediastinal lymph nodes are classified as periaortic lymph nodes on the side where the branchial arch artery retentions are located [8].Dissection of the upper mediastinum of the right aortic arch should be performed according to left upper mediastinum dissection.In a previous report, dissection of the upper mediastinum of the right aortic arch was performed according to left upper mediastinum dissection based on anatomical characteristics [2].Regarding the differences between the left upper mediastinal dissection in a normal anatomy and the right upper mediastinal dissection in cases of a right-sided aortic arch, the azygos vein and superior vena cave are observed and the right recurrent laryngeal nerve must be identified without the ductus arteriosus.Thus, in the present case, the dissection areas are to the right lateral side of the aortic arch between the upper and lower edges of the aortic arch and to the right lateral edge of the trachea between the lower edge of the aortic arch and the head of the right main pulmonary artery.The ventral side of the dissection border becomes the superior vena cava.
To the best of our knowledge, this is the first detailed report on right upper lobectomy with ND2a-1 under uniportal VATS for primary lung cancer in a patient with a right-sided aortic arch and Kommerell diverticulum.The surgical procedure was performed despite the potential risks caused by anatomical changes.
Fig. 1
Fig. 1 Three-dimensional computed tomography scan image of the right aortic arch (a) The anterior view shows the right aortic arch and the right descending aorta running on the right side of the vertebral body (b) The lateral view shows the so-called Kommerell diverticulum of the left subclavian artery AAO, ascending aorta; DAO, descending aorta; LCCA, left common carotid artery; LSA, left subclavian artery; RCCA, right common carotid artery; RSA, right subclavian artery; KD, Kommerell diverticulum
Fig. 4
Fig. 4 Operative findings of the right upper mediastinum (a) An intraoperative view before the upper mediastinal lymph node dissection (b) The right vagus nerve was identified at the cranial side of the azygos vein while dissecting the mediastinal lymph node from SVC. (c) The recurrent laryngeal nerve, which branched from the vagus nerve, was identified at the caudal side of the azygos vein while dissecting the mediastinal lymph node from the AA.(d) An intraoperative view after the upper mediastinal lymph node dissection PA, pulmonary artery; RLN, recurrent laryngeal nerve; VN, vagus nerve; Tr, trachea; AA, aortic arch; AV, azygos vein; RUB, right upper lobe bronchus; DAO, descending aorta; SVC, superior vena cava | 2024-03-19T13:11:12.282Z | 2024-03-19T00:00:00.000 | {
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252100216 | pes2o/s2orc | v3-fos-license | Risk Factors and Outcomes of Stem Cell Mobilization Failure in Multiple Myeloma Patients
Introduction Autologous hematopoietic stem cell transplantation (ASCT) is a well-established treatment for patients with multiple myeloma (MM), and adequate stem cell collection must be assured before ASCT. However, prediction of poor mobilizers (PMs) is still difficult despite several risk factors for mobilization failure having been identified. Methods We retrospectively analyzed MM patients at Taipei Veterans General Hospital in Taiwan who underwent stem cell collection between October 2006 and August 2020. A CD34+ cell collection of <1 × 106 cells/kg was defined as a mobilization failure. The primary endpoint was mobilization failure. The secondary endpoint was overall survival (OS). Odds ratios (ORs) and 95% confidence intervals (CIs) for mobilization failure were calculated using a logistic regression model. The cumulative incidence of mortality was estimated using the Kaplan-Meier method. Results In the multivariate analysis, absolute monocyte count <500/µL (adjusted OR 10.75, 95% CI: 1.82−63.57, p = 0.009), platelet count <150,000/µL (adjusted OR 12.49, 95% CI: 2.65−58.89, p = 0.001) before mobilization, and time interval from diagnosis to stem cell harvest ≥180 days (adjusted OR 7.69, 95% CI: 1.61−36.87, p = 0.011) were risk factors for PMs. PM patients had poorer OS compared to patients with successful stem cell collection in the univariate analysis (log-rank test p = 0.027). The predicted probability of PMs was estimated by the multiple logistic regression model with a sensitivity of 84.6% and a specificity of 84.0%. Conclusion Absolute monocyte count <500/µL, platelet count <150,000/µL, and treatment duration more than 180 days before stem cell mobilization are risk factors for unsuccessful stem cell collection. Our prediction models have high sensitivity and specificity for mobilization failure prediction and allow for early interventions for possible PMs.
Introduction
Autologous hematopoietic stem cell transplantation (ASCT) is a well-established treatment for patients with multiple myeloma (MM), and patients with MM receiving ASCT have better progression-free survival compared to those without ASCT [1,2]. Pretransplantation hematopoietic stem cell (HSC) mobilization is required for peripheral blood stem cell (PBSC) harvesting, and adequate stem cell collection must be assured before ASCT. However, several risk factors impact mobilization efficacy and lead to mobilization failure in poor mobilizers (PMs). Patients with mobilization failure have poorer outcomes compared to those with successful mobilization, partially owing to being incapable of receiving subsequent ASCT [3][4][5].
Early interventions for possible PMs are likely to increase the total number of stem cells collected, avoid futile apheresis procedures, and reduce costs for patients who may need a second PBSC harvesting [9,10]. Well-established risk factors include old age, previous radiotherapy, lenalidomide use, multiple chemotherapy cycles, and prior alkylating agent exposure [6,8,[11][12][13]. However, it is still difficult to predict mobilization failure in MM patients despite several risk factors having been identified. To date, there is no predictive model for PMs before mobilization in patients with MM.
The definition of PMs in MM and lymphoma patients was proposed by the Gruppo Italiano per il Trapianto di Midollo Osseo (GITMO) working group in 2012, and the criteria for prediction of PMs were made [14]. The GIT-MO criteria were further validated by an Italian group but showed no significance between PMs and good mobilizers. Additionally, only 31.3% of the patients (71 of 227) had MM [15]. Wu et al. [16] reported a decision-tree algorithm for prediction of PMs in a cohort with positive and negative predictive values of 92.3% and 70.0%, respectively. Nonetheless, only 19% of those patients had MM.
In this work, we performed a single-center retrospective study to investigate the risk factors of PMs in MM patients. We then tried to create a scoring system that could predict PMs before stem cell mobilization.
Study Population
This is a retrospective cohort study to analyze the risk factors and the impact of poor stem cell mobilization on MM patients who underwent PBSC harvesting. All patients who were diagnosed with MM according to the International Myeloma Working Group (IMWG) criteria [17] at Taipei Veterans General Hospital between October 2006 and August 2020 were included in the study. Patients diagnosed with amyloidosis, solitary plasmacytomas, monoclonal gammopathy of undetermined significance, or smoldering myeloma were not included. Only MM patients who underwent PBSC harvesting were proceeded for further analysis. The study was approved by the Taipei Veterans General Hospital's Institutional Review Board (No. 2021-03-006CC).
HSC Mobilization
Mobilization is defined as the release of HSCs from bone marrow into peripheral blood following treatment with cytokine, targeted agents, and/or chemotherapy [18]. All of our patients received chemotherapy and granulocyte colony-stimulating factor (G-CSF) for stem cell mobilization, except one received G-SCF alone as mobilization regimen. All patients received G-CSF for at least 4 days in the PBSC harvesting course as a single (7 of 181 patients) or split (174 of 181 patients) dose until the last day of apheresis. The median dose of G-CSF was 9.46 μg/kg/day (range, 3.85-16.57 μg/kg/day). The chemomobilization regimen depended on the treating physician [13,19]. The circulating stem cells were collected via leukapheresis for two consecutive apheresis days. Most patients who did not collect minimal stem cell dosage for an ASCT will proceed to the third apheresis session. Stem cell dosage collected between minimal and optimal number (5-6 × 10 6 CD34 + cells/kg) proceeded to the third apheresis session depending on the treating physician [13,20]. Patients with optimal CD34 + cells collected for the first 2 days received the third apheresis if tandem transplantation was planned or decided by the treating physician. A collection of less than 1 × 10 6 CD34 + cells/kg is defined as stem cell mobilization failure since the administration of stem cell doses <1 × 10 6 cells/kg has been associated with increased RBC transfusion requirement and engraftment failure in previous studies [21][22][23][24].
Leukapheresis, Stem Cell Freezing, and Thawing
Stem cells were collected by using the COBE Spectra apheresis system (version 6.1, COBE Laboratories) via 20 Fr intravenous catheters or central venous catheters according to the manufacturers' protocol. The total volume processed was 2-3 times of total blood volume. Acid-citrate-dextrose (ACD) was used as an anticoagulant with an ACD-to-whole blood ratio of 1:13 to 1:14 [25,26]. The leukapheresis began when peripheral blood hematopoietic progenitor cells reached 20 × 10 6 cells/L [16,27,28].
Stem cells were transferred into the bags suitable for freezing (CryoMACS Freezing Bags). Pre-cooling plasma and cryoprotectant DMSO were mixed gently and transferred to the bags, with final DMSO concentration of 10%. A small amount of the mixed product was withdrawn for microorganism culture and preserved in the spike port of the bags as controls. The procedures were performed in the Biological Safety Cabinet. According to manufacturers' guidance, the bags and control samples were cooled in the controlled rate freezer (Planer Kryo 560-16) to implement cryopreservation protocols. At last, the bags and controls were transferred into liquid nitrogen freezers (MVE HEco 1500Series Freezer) for preservation [25,26].
At the time of stem cell transplantation, the bags were transferred to the patient bedside and thawed in a sterilized thermostatic water bath (37°C). For quality control, the samples were sent for microorganism culture and cell viability analysis. The quality standard of thawed stem cells was microorganism culture-negative and cell viability >80% at our institution. (International Staging System [ISS]), disease type, comorbidities (heart failure, chronic pulmonary disease, diabetes mellitus, and hypertension), and pathology reports on bone marrow aspiration and biopsy. In addition, we collected laboratory data such as hemoglobin, platelet count (PLT), levels of serum albumin, corrected serum ionized calcium, serum creatinine, serum lactate dehydrogenase, serum β2-microglobulin, and free light chain ratio both at MM diagnosis and before stem cell mobilization. Furthermore, we collected information about the number of prior lines of chemotherapy, time interval from diagnosis to stem cell harvest, mobilization regimens, radiotherapy before stem mobilization, and treatment modalities.
Statistical Analysis
The baseline characteristics of MM patients who underwent PBSC harvesting are shown as the total number (n) and proportion (%). The primary endpoint of this study was failure to collect total CD34 + cells for ≥1 × 10 6 cells/kg after mobilization. We first investigated the risk of stem cell mobilization failure in relation to patient characteristics at diagnosis and before mobilization. Odds ratios (ORs) and 95% confidence intervals (CIs) for mobilization failure were calculated using a logistic regression model. Potential risk factors were selected using a univariate model, and those with p < 0.1 in the univariate model were included in a multivariate analysis. All independent risk factors identified in the multivariate analysis were then used to build a predictive model of poor mobilization. The β-coefficients of all significant risk factors in the multivariate logistic regression model were used to build a new riskscoring system. We also built a simplified score by assigning one point to each significant variable. We presented the receiver operating characteristic (ROC) curve and defined an optimal cutoff value by Youden's index method and the closest-to-(0, 1) corner method to maximize the sensitivity and specificity for predicting the probability of poor mobilization. Model discrimination was estimated by sensitivity, specificity, and area under the curve (AUC).
The secondary outcome was overall survival (OS). The study cohort was followed from the date of PBSC harvesting until the date of death, dropout, or the end of March 2021. The cumulative incidence of mortality was estimated using the Kaplan-Meier method.
Clinical Characteristics of the Study Population
This study identified 628 patients diagnosed with MM at Taipei Veterans General Hospital between October 2006 and August 2020. Of them, 440 patients who did not receive stem cell harvest and 7 patients who were initially misclassified as MM were excluded. The final study cohort included 181 MM patients who underwent PBSC harvesting (shown in Fig. 1). The median age of the study patients was 59, ranging from 23 to 74, and 55.3% of the patients were men. ISS stages I, II, and III were 33.7%, 38.7%, and 26.5%, respectively. The median number of collected CD34 + cells was 6.3 (interquartile range 3.3-10.2) × 10 6 cells/kg. Fourteen patients (7.7%) failed to provide sufficient CD34 + cells (<1 × 10 6 cells/kg). Total stem cells collected ≥1 × 10 6 CD34 + cells/kg for the first day was reported in 138 patients (76.2%), ≥1 × 10 6 CD34 + cells/kg for the first 2 days in 165 patients (91.2%). Total stem cells collected ≥2 × 10 6 CD34 + cells/kg for the first day was reported in 92 patients (50.8%), ≥2 × 10 6 CD34 + cells/kg for the first 2 days in 143 patients (79.0%). Total stem cells collected ≥5 × 10 6 CD34 + cells/kg for the first day was reported in 29 patients (16.0%), ≥5 × 10 6 CD34 + cells/kg for the first 2 days in 88 patients (48.6%) (online suppl. Table 1; for all online suppl. material, see www. karger.com/doi/10.1159/000525565). Demographics of the patients with and without sufficient CD34 + cells collection are listed in Table 1.
Risk Factors of Failure to Collect PBSCs after Mobilization
We used a logistic regression to identify potential risk factors of mobilization failure. In the univariate analysis, absolute monocyte count (AMC) <500/µL (OR 6.43), absolute neutrophil count (ANC) <3,000/µL (OR 3.99), >1 line of prior chemotherapy (OR 3.66), PLT <150,000/µL (OR 10.55), time interval from diagnosis to stem cell harvest ≥180 days (OR 9.69), and prior irradiation (OR 3.12) before mobilization were associated with failure to collect sufficient PBSCs (Table 2). In the multivariate analysis, AMC <500/µL (adjusted OR 10.75, 95% CI: 1.82-63.57, p = 0.009), PLT <150,000/µL (adjusted OR 12.49, 95% CI: Furthermore, a sensitivity analysis was performed to evaluate if the risk factors were still useful at different levels by using the threshold of 2 × 10 6 CD34 + cells/kg and 5 × 10 6 CD34 + cells/kg. PLT <150,000/µL (adjusted OR 5.97, 95% CI: 1.37-26.08, p = 0.018) and time interval from diagnosis to stem cell harvest ≥180 days (adjusted OR 6.56, 95% CI: 1.46-29.46, p = 0.014) were the risk factors for poor mobilization when the cutoff point for stem cell collection was defined as 2 × 10 6 CD34 + cells/kg. When we used the threshold of 5 × 10 6 CD34 + cells/kg, hypertension, PLT <150,000/µL, and time interval from diagnosis to stem cell harvest ≥180 days were the significant predictors of failure to collect PBSCs after mobilization (online suppl. Table 2). In summary, PLT <150,000/ µL and time interval from diagnosis to stem cell harvest ≥180 days were independent risk factors of insufficient stem cell collection in all three thresholds of mobilization failure.
OS of MM Patients with and without Sufficient Stem Cell Collection
We further studied the impact of insufficient stem cell collection on patient survival. During the 11-year study period, 55 patients with MM (30.4%) died. The survival curves of patients with and without sufficient stem cell collection are shown in Figure 2. MM patients with insufficient stem cell collection had poorer OS compared with patients with successful stem cell collection (log-rank test p = 0.027, shown in Fig. 2).
We investigated the risk factors for mortality in the univariate analysis by the logistic regression model, and those with p < 0.1 in the univariate model were included in a multivariate analysis. Stem cells collected <1 × 10 6 CD34 + cells/kg, platelet <150,000/µL before mobilization, the time interval from diagnosis to stem cell harvest ≥180 days, and serum albumin <35 g/L at diagnosis were included in the multivariate analysis. However, no risk factors in the multivariate analysis were statistically significant despite the trend of the factors was equal to that in the univariate analysis (online suppl. Table 3).
Predicting Models and Validation of Risk Score
Multivariate logistic regression analysis was performed on the significant factors extracted from the multivariate analysis in Table 2 to determine the relationship between each variable on poor mobilization. The final model contains three variables, including AMC <500/µL (adjusted OR 11.28, 95% CI: 2.10-60.50, p = 0.005), PLT <150,000/µL (adjusted OR 11.85, 95% CI: 2.70-51.90, p = 0.001), and time interval from diagnosis to stem cell harvest ≥180 days (adjusted OR 9.79, 95% CI: 2.20-43.50, p = 0.003) (online suppl. Table 4). The predicted probability of poor mobilization was estimated by the following multiple logistic regression model: where x 1 : AMC <500/µL, x 2 : PLT <150,000/µL, and x 3 : time interval from diagnosis to stem cell harvest ≥180 days. Using a ROC curve analysis based on the prognostic index, a cutoff point for prediction of poor mobilization is defined as a score at or above 0.20 (online suppl. Table 5A). The sensitivity of the equation is 84.6%, while the A simpler risk model may be easier to use in clinical practice. We defined a simplified prognostic model by assigning one point for each of the three independent predictors (AMC <500/µL, PLT <150,000/µL, and time interval from diagnosis to stem cell harvest ≥180 days). The result shows that patients with a score at or above 2 would predict mobilization failure with a sensitivity of 84.6% and a specificity of 84.0% (online suppl. Table 5B). Figure 3 demonstrates the estimates of the ROC curves. The AUC is 0.901 for predicting poor mobilization using the prognostic index and 0.896 for those using a simplified risk model. There was no significant difference seen between the AUCs of the two prognostic indexes (p = 0.631) ( Table 3). The calculator of the two models can be found on our Website. Please refer to https://wd.vghtpe.gov.tw/ hemaonco/Fpage.action?muid=15117&fid=13842 or QR code in online supplementary Figure 1.
Discussion
This is the largest study on prediction of PMs in patients with MM in Asia so far. Our study has identified that low AMC, low PLT, and a treatment duration of more than 180 days before stem cell harvest is associated with mobilization failure. We created a risk model using three risk factors identified in this work that can predict PMs before stem cell mobilization.
For the minimum stem cell target for an ASCT, although some studies recommended 2 × 10 6 CD34 + cells/ kg as the minimal requirement cell dosage for an ASCT, there was still much evidence supporting using 1-2 × 10 6 CD34 + cells/kg. The American Society for Blood and Bone Marrow Transplantation (ASBMT) recommended that using 1-2 × 10 6 CD34 + cells/kg for an ASCT was acceptable in patients who benefited from ASCT and can be considered case by case [13]. Shpall et al. [35] reviewed clinical studies on the impact of CD34 + cell doses infused and the outcomes for ASCT. They concluded that 1-2 × 10 6 CD34 + cells/kg was adequate for ASCT, but CD34 + cell doses of ≥5 × 10 6 cells/kg were considered an optimal target with reduced neutrophil and platelet engraftment period, the lower cost, and the reduced need for transfusion support. Pérez-Simón et al. [22] reported the relationship of long-term graft outcome and CD34 + cells infused in 100 cancer patients who received ASCT. They found CD34 + cell doses of more than 1.1 × 10 6 cells/kg were adequate for long-term graft function. Watts et al. [21] evaluated the predicting factors for blood cell engraftment in a cohort with 101 lymphoma patients and reported the minimal number of CD34 + cells infused was 1.0 × 10 6 cells/kg. A single-center study with 28 myeloma patients investigated the OS and CD34+ cells infused. Their data showed the minimal CD34 + cell doses were 1.0 × 10 6 cells/kg and patients who transplanted less than this threshold had a shorter survival period (80% patients survived at 80 months for >1.0 × 10 6 CD34 + cells/kg infused and 67% at 76 months for <1.0 × 10 6 CD34 + cells/kg infused) [24]. A large cohort with 810 patients who underwent ASCT aimed to re-evaluate the threshold for minimal CD34 + cell doses for engraftment reported that 1-2 × 10 6 CD34 + cells/kg was acceptable [36]. Recently, some investigators tried to redefine the minimal requirement CD34 + cell doses for an ASCT. The reason is that despite the guidelines recommending a minimal CD34 + cell dose of ≥2 × 10 6 cells/kg for ASCT, there was still much evidence supporting lower CD34 + cell doses [37]. Furthermore, the defined "minimal cell doses" were based on cryopreserved stem cells; the actual CD34 + cells infused may be lower than this threshold [38]. The improvement of the freezing and thawing technique can increase the infused stem cell dosage, lowering the minimum requirement target. Liu et al. [37] investigated the outcomes for 56 lymphoma patients who underwent ASCT with inadequate stem cell doses. They divided their patients into two groups. The first was the unfavorable HSC group, Corso et al. [11] reported in a retrospective study that low white cell count, low PLT, previous melphalan treatment, and the interval between diagnosis and stem cell mobilization >6 months were associated with a lower number of CD34 + cells collected, which is similar to our findings. Notably, the patients in their study were treated mainly with chemotherapy. Our patients were treated with both novel agent-and chemotherapy-based regimens, indicating that these risk factors could be applied to both chemotherapy-and novel agent-based treatment. A retrospective study by Musto et al. [39], which enrolled 1,348 newly diagnosed MM patients in five clinical trials, conducted by the GIMEMA -Multiple Myeloma Italian Network, identified four risk factors for poor stem cell mobilization. The risk factors were age >60 years, lenalidomide use, grade 3/4 hematological toxicity under induction therapy, and baseline cytopenia defined as ANC <1,000/µL, Hb < 10 g/dL, or PLT <100,000/µL. Two out of the four risk factors concerned blood cell count, revealing its importance in the prediction of unsuccessful PBSC collection. Furthermore, Baertsch et al. [40] investigated the relationship between low PLT and the need for plerixafor use in a retrospective study with 380 patients who underwent ASCT in Germany. They found that patients with low ANC (OR 0.843, 95% CI: 0.715-0.996, p = 0.044) and low PLT (OR 0.994, 95% CI: 0.989-0.998, p = 0.003) before mobilization were associated with a higher probability of plerixafor use, compared to patients with higher ANC and PLT. Yang et al. [28] reported low AMC before stem cell harvesting correlated with low CD34 + cell yield. They performed a ROC curve analysis and showed high sensitivity (73.9%) and specificity (89.9%) when the cutoff value of AMC was 1.455 × 10 9 /L. However, the study suggested that AMC could predict PMs only just before harvesting. Regarding the different thresholds, our study found that low PLT and treatment interval between diagnosis and stem cell mobilization more than 180 days before stem cell mobilization were independent risk factors of stem cell dose yield <2 × 10 6 CD34 + cells/kg and <5 × 10 6 CD34 + cells/kg. Abba C. Zubair et al. [41] evaluated predictive factors for PMs in 103 cases. The study enrolled plasma cell dyscrasias patients, lymphoma patients, and normal stem cell donors for allogeneic transplantation. They reported that a baseline PLT ≥151 × 10 9 /µL could be predictive for a more than 5 × 10 6 CD34 + cells/ kg yield in treated plasma cell dyscrasia patients with a sensitivity of 95% and a specificity of 14%. Collectively, low blood cell counts, especially PLT, are associated with poor stem cell mobilization, low stem cell yield and PLT may be a surrogate for bone marrow reserve.
The platelet production is regulated by negative feedback according to the interaction between PLT, the number of megakaryocytes, and the concentration of thrombopoietin (TPO). Platelets and megakaryocytes internalize TPO via their receptor, Mpl, and resulted in a relatively low TPO level when there was an adequate PLT and megakaryocyte count, which decreased the production of platelet and megakaryocyte and vice versa [13]. The receptor of TPO is located not only on megakaryocytes and platelet but also on HSCs. TPO can increase the cycling and differentiation of HSCs when binding to its receptor, resulting in myelopoiesis, erythropoiesis, megakaryocytopoiesis, and the expansion of CD34 + cells [42][43][44]. Bakeer et al. [45,46] hypothesized that the predictive ability of low PLT for PMs was because low PLT reflected the decreased number or functional impairment of megakaryocytes, compromising the supportive role of megakaryocytes on CD34 + cells expansion. However, low blood cell counts, PLT, and reduced megakaryocytes in MM patients may also be due to bone marrow pathology and treatment-related toxicity. Interestingly, several studies evaluated the influences of the addition of recombinant human TPO (rhTPO) on traditional mobilization regimens and showed an increased number of PBSC yields [47][48][49]. Nonetheless, the role of rhTPO on poor stem cell mobilization of MM patients has not been established. The dosage and timing for rhTPO administration are unclear, too. Further study for the application of rhTPO in MM patients with poor stem cell mobilization is needed.
Several previous studies have reported that the enumeration of peripheral blood CD34 + cell count could predict PMs before PBSC harvesting. Moreover, in combination with the rising white blood cell count and PLT from nadir, the enumeration of peripheral blood CD34 + cell count can also determine the timing of apheresis [50][51][52][53]. However, as there was no tool to accurately predict PMs before stem cell mobilization, the result was a delay in ear-Transfus Med Hemother 2023;50:39-50 DOI: 10.1159/000525565 ly intervention. Baertsch et al. [40] created a ROC curve by using pre-mobilization PLT and ANC, but with a relatively low predictive value (sensitivity 38%, specificity 87% for low PLT; sensitivity 57%, specificity 77% for low ANC). Jantunen et al. [54] created a chemotherapy scoring system to predict PMs according to treatment regimens before stem cell mobilization. However, no threshold of mobilization failure was detected on the ROC curve (area under the curve 0.541, 95% CI: 0.375-0.706) [54]. Wu et al. [16] created a predictive model for poor stem cell mobilization with a sensitivity of 93.5% and a specificity of 65.7%. However, some of the factors in the model can be collected only before PBSC harvesting, making the model difficult to use. In comparison, our models are easy to use and have a high sensitivity and specificity for predicting PMs. Moreover, the models can predict mobilization failure before stem cell mobilization and make early intervention for mobilization failure possible.
According to our result, patients with high PM score pose a very high risk for mobilization failure and probably require remobilization or the use of a mobilization regimen different from good mobilizers. In addition, all except one of our patients received G-CSF plus chemotherapy (G + C) as mobilization regimen. The regimen may be, therefore, not suitable for patients with high PM scores. Plerixafor with G-CSF (P + G) for mobilization after mobilization failure with G-CSF alone was proven effective [55]. Furthermore, the combination was also effective for patients failing with G + C for mobilization and had better cost-effectiveness [56,57]. Afifi et al. [58] compared front-line use of P + G to G-CSF plus cyclophosphamide as a mobilization regimen and reported stem cells to yield more than optimal dosage (5 × 10 6 CD34 + cells/kg) were higher in the P + G group (94% vs. 83%, p = 0.013). Attolico et al. [59] evaluated plerixafor added to G-CSF plus chemotherapy (P + G + C) for mobilization in predicted PMs and reported the combination was safe and effective. The ASBMT also recommended P + G or P + G + C for remobilization regimen or mobilization failure prevention in high-risk groups [13]. As a result, we recommended P + G or P + G + C as the primary mobilization regimen for patients with high PM scores. However, National Health Insurance (NHI) only reimbursed plerixafor in patients proved as PMs in Taiwan. Furthermore, the application for reimbursement took roughly 2 weeks before use. Patients who collected suboptimal or inadequate cell doses and need plerixafor as salvage management should use at their own expense. Only 2 patients in our cohort used plerixafor. One patient was used for salvage management, resulting in good stem mobilization. The other patient used chemotherapy with G-CSF and pre-emptive plerixafor as a mobilization regimen. Unfortunately, the CD34 + cells yield <1.0 × 10 6 cells/kg for the patient.
Our data showed that the higher dosage of G-CSF had a trend of lower PM possibility. According to the ASBMT guideline [13], the first-line mobilization regimen in MM can be G-SCF alone with a dosage of ≥10 μg/kg/day for patients who did not receive more than one line of treatment or more than four cycles of lenalidomide-containing regimen. However, the optimal G-CSF dosage in G + C was not clear and differed between studies, with 5-10 μg/ kg/day being most frequently used [60][61][62][63]. There was no head-to-head comparison of the mobilization effect between chemomobilization with a higher or lower dosage of G-CSF, either. We incorporated the G-CSF dosage of ≥10 μg/kg/day and <10 μg/kg/day into the analysis of risk factors of PMs and found it was not significant in the univariate analysis (OR 0.28, 95% CI: 0.06-1.28, p = 0.101) by the logistic regression model ( Table 2). The result may be due to the small sample size and need a more extensive study population to evaluate the impact of higher dosage G-CSF on PMs in patients mobilized with G + C.
Zhu et al. [63] conducted a study with the addition of rhTPO to G + C for mobilization in patients with relapsed or refectory non-Hodgkin's lymphoma. The study group was rhTPO plus G + C (n = 40) and the control group was G + C (n = 38). The result showed that the total CD34 + cells collected were significantly higher in the study group (6.35 × 10 6 cells/kg vs. 3.3 × 10 6 cells/kg, p = 0.0054). Moreover, stem cells harvested that reached minimal (100% vs. 86%, p = 0.035) and optimal target (72.9% vs. 41.3%, p = 0.021) were higher, also. rhTPO in combination with traditional mobilization may have a role in PMs, and more investigation was needed to evaluate the dosage and the timing of rhTPO.
Our study found that patients who underwent stem cell mobilization with a CD34 + cell yield of less than 1 × 10 6 cells/kg are correlated with poorer OS compared to those with a CD34 + cell yield of more than 1 × 10 6 cells/ kg in the univariate analysis. A possible explanation is that patients with MM may be old, immunocompromised, fragile, or heavily treated. These factors can compromise PBSC harvesting and result in poor stem mobilization. However, PMs and low PLT were not statistically significant in the multivariate analysis despite a trend of significance was found. A more extensive study was needed to answer the questions and evaluated the impact of PMs and low PLT on mortality. Besides, we found ASCT was not a risk factor for mortality in the univariate logistic regression. Dhakal et al. [2] conducted a metaanalysis with 4 phase 3 randomized clinical trials for conventional meta-analysis and 5 for network meta-analysis in patients with MM and reported that ASCT did not affect OS in MM patients in the era of novel agents. The result may be due to the high come-out rate of new postrelapse medications.
This study has several limitations. First, because this is a retrospective study, our patients did not have a uniform treatment regimen or mobilization regimen. However, these factors make our risk score more reliable and applicable in the real world and clinical practice. Second, our study did not include risk stratification for every patient, to identify the influence of high-risk molecular patterns on stem cell yield. Nevertheless, it proves that our model can be applied to both high-and low-risk populations. Third, lenalidomide use has been established as a risk factor of PM. The British Society for Haematology guideline and the Medical Scientific Advisory Group to Myeloma Australia guideline recommended that patients treated with a lenalidomide-containing regimen should not exceed four cycles before stem cell harvesting to prevent mobilization failure [64,65]. The induction therapy with a triple-combination regimen including a proteasome inhibitor, an immunomodulating agent, and a steroid was recommended across clinical guidelines. Lenalidomide with bortezomib and steroid (VRd) was the preferred option in transplant-eligible patients among guidelines and alternative lenalidomide to thalidomide (VTd) was recommended in areas where lenalidomide was not available [64][65][66][67]. In Taiwan, lenalidomide was not reimbursed by NHI as induction treatment in transplant-eligible patients. Most of our patients received VTd as the front-line treatment before undergoing ASCT. Only three cases of our cohort received lenalidomide-containing regimen as induction treatment. The effects of lenalidomide on PMs cannot be evaluated in our study. Finally, despite low PLT and PMs were found possible risk factors for mortality in the univariate analysis the multivariate analysis was not significant. This result may be due to inadequate statistical power, and larger sample size was needed to detect the effects of the risk factors on mortality.
Conclusion
In conclusion, we found that AMC <500/µL, PLT <150,000/µL, and a treatment duration of more than 180 days before stem cell mobilization are risk factors for unsuccessful stem cell collection. Furthermore, our prediction models have both high sensitivity and specificity for mobilization failure prediction. This is the largest study in an Asian population for prediction of PMs in MM patients, and our predicting models have the highest sensitivity and specificity in mobilization failure prediction among the published literature. Our model allows for pre-emptive plerixafor use or other early interventions for possible PMs and may reduce costs owing to mobilization failure.
Statement of Ethics
This study has been approved by the Institutional Review Board at Taipei Veterans General Hospital (no. 2021-03-006CC). For this type of study, formal consent is not required.
Conflict of Interest Statement
All authors declare no conflict of interest.
Funding Sources
This study was supported by grants from Taipei
Author Contributions
Chia-Jen Liu had full access to the study data and takes responsibility for data integrity and accuracy of data analysis and acts as guarantor and accepts responsibility for the integrity of the work as a whole. Te-Lin Hsu, Chun-Kuang Tsai, Chiu-Mei Yeh, and Chia-Jen Liu designed the study and provided the final interpretation of the results. Te-Lin Hsu, Chun-Kuang Tsai, Chiu-Mei Yeh, Fen-Lan Lin, and Chia-Jen Liu collected the data. Chiu-Mei Yeh and Chia-Jen Liu acquired the data and performed the statistical analysis and drafted the manuscript. Te-Lin Hsu, Chun-Yu Liu, Liang-Tsai Hsiao, Yao-Chung Liu, Hao-Yuan Wang, Po-Shen Ko, Ting-An Lin, and Wen-Chun Chen made critical revisions to the manuscript for important intellectual content. Po-Min Chen, Jin-Hwang Liu, and Jyh-Pyng Gau were the study supervisors. All authors have read and approved the final manuscript.
Data Availability Statement
The data of this study are not publicly available because the information can compromise the privacy of research participants, and the participants do not consent to public data release. Data requirements can direct to the corresponding author upon reasonable request. | 2022-09-07T15:09:37.404Z | 2022-09-05T00:00:00.000 | {
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264189313 | pes2o/s2orc | v3-fos-license | A Multi-Material Flame-Retarding System Based on Expandable Graphite for Glass-Fiber-Reinforced PA6
A synergistic multi-material flame retardant system based on expandable graphite (EG), aluminum diethylphosphinate (AlPi), melamine polyphosphate (MPP), and montmorillonite (MMT) has been studied in glass-fiber-reinforced polyamide 6 (PA6). Analytical evaluations and fire performances were evaluated using coupled thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR) as well as cone calorimetry, UL-94 fire testing, and limiting oxygen index (LOI). A combination of EG/AlPi/MPP/MMT has been shown to provide superior flame-retarding properties when integrated at 20 wt.% into glass-fiber-reinforced PA6 (25 wt.%), achieving UL-94 V0 classification and an oxygen index of 32%. Strong residue formation resulted in low heat development overall, with a peak heat release rate (pHRR) of 103 kW/m2, a maximum of average heat release rate (MAHRE) of 33 kW/m2, and deficient total smoke production (TSP) of 3.8 m2. Particularly remarkable was the structural stability of the char residue. The char residue could easily withstand an areal weight of 35 g/cm2, showing no visible deformation.
Introduction
Non-reinforced polyamide 6 (PA6) is known for strong burn dripping behavior, extinguishing the upward burning process in vertical UL-94 burning tests and thus resulting in a V2 classification.The integration of glass fibers changes two essential fire phenomena: (1) Due to an increased melt viscosity, melt dripping is substantially reduced, stabilizing the burning process in vertical UL-94 burning tests.Accordingly, flame spread continues upwards, resulting in a lack of a UL-94 classification.(2) Simultaneously, protruding glass fibers increase the surface area and provide improved thermal conductivity and pyrolysis supply due to the "wicking effect".Both effects substantially change the physical conditions and accelerate the burning process [1].Halogenated flame retardant additives (FR) have been proven to work efficiently for glass-fiber-reinforced PA6.However, due to rising environmental requirements and toxicity concerns, halogenated FRs are increasingly substituted [2].Many publications have been studying phosphorous and nitrogen-containing FR alternatives such as melamine polyphosphate (MPP) [3], melamine cyanurate (MC) [4,5], phosphonate oligomers PSA and POSC [6], aluminum diethyl phosphinate (AlPi) [5,7,8], zinc borate (ZB) [5,9], microencapsulated red phosphorus (RP) [10], and phosphorus oxynitride (PO) [11].Furthermore, many additives have been found to show superior fire retardant properties in glass fiber when synergistically combined in multi-material systems.
MPP is a commonly used flame retardant additive for glass-fiber-reinforced PA6 and PA6.6 systems.Research has demonstrated that the integration of MPP triggers the formation of a phosphorus-rich intermediate phase on the glass fiber surface.As a result, the limiting oxygen index (LOI) can be increased for PA6 to approximately 29 vol.%Polymers 2023, 15, 4100 2 of 15 (15 wt.% filling degree) and for PA6.6 to 38-40 vol.% (15 wt.% filling degree).The observed difference in flame retardancy performances can be attributed to two possible modes of action.While the incorporation of MPP in PA6.6 initiates strong crosslinking, PA6/MPP combinations instead result in catalytical depolymerization (a decrease in viscosity [12]).Earlier depolymerization affects the flammability behavior in two contrary phenomena.Caprolactam is released at lower temperatures, accelerating the early flame spread since it provides a steady fueling of combustible pyrolysis gases to the flame.On the contrary, a lower fuel supply maintains the low flame growth rate, substantially limiting the heat release rate potential.Both effects reduce and improve fire retardancy properties but differ in efficiency.Additionally, decomposition products often form phosphoric acid, promoting char formation on the polymer's surface offering insulating characteristics [13].
Metal salts of alkyl phosphinate, such as aluminum diethyl phosphinate (AlPi), have also been shown to provide excellent flame retardancy properties in non-reinforced PA6.6.Two mechanisms are reported: (1) the formation of aluminum phosphate in the condensed phase and (2) the release of diethylphosphinic acid in the gas phase [14].However, AlPi does not provide a V0 classification in a UL94 test when used as a single FR additive in glass-fiber-reinforced PA6 and PA66.The performance drop can be explained by a capillary-driven effect that accelerates the pyrolysis gas flow, suppressing the flame retardant efficiency of the condensed phase mechanisms [15,16].
Furthermore, synergistic or catalytic effects have been reported when metal salts and polyphosphate compounds, such as MPP and AlPi, are combined.While MPP primarily acts in the gas phase via flame inhibition, combining AlPi and MPP results in a changed reaction path primarily occurring in the condensed phase.The reaction is based on a crosslinking mechanism by forming polyphosphate esters [17].A synergistic effect of MPP and AlPi has been demonstrated for PA6.6 [18] and PA6 [19].
A well-known strategy to reduce the pHRR or improve the LOI and UL-94 fire testing performance in PA6 recipes is the combined utilization of condensed and gas phase flame retardancy effects [20,21].Nanofillers have been successfully tested as synergistic additives for a variety of phosphorous FRs.They are predominantly known to improve fire performance via structural char residue enhancement and by providing a labyrinth effect, but can also act catalytically during polymer decomposition [13].A recent study has reported that a combination of MPP with MMT (nanofiller) can synergistically enhance the flame retardancy properties of GF-reinforced PA6, resulting in an improved V0 UL94 (V) rating [4].MMT has been shown to reduce the pyrolysis rate through a labyrinth effect as well as residual formation due to clay surface migration.The latter results in a clay layer barrier that improves insulation properties against an external heat source and provides a long-term barrier, hindering the pyrolysis gas supply [20,22].Other studies have investigated the use of expandable graphite (EG) as an alternative for phosphorus flame retardant additives, which were shown to be very effective in non-reinforced PA6 [23,24] and only somewhat effective in glass-fiber-reinforced PA6 [25].EG acts physically via the formation of a voluminous, thermally stable residue, providing excellent long-term fire protection properties [23,25].Due to its solely physical effect, the usage of expandable graphite has been reported for various polymeric systems (e.g., PE [26,27], PP [28,29], PS [30], PVC [31], ABS [32], PA6 [33]).
Recent studies have shown that expandable graphite (EG) can provide a very effective, long-term heat barrier when integrated into GF-reinforced PA6.However, no sufficient UL-94 classification could be reached (V2) [25,34,35].Within this study, expandable graphite (EG), aluminum diethylphosphinate (AlPi), melamine polyphosphate (MPP), and montmorillonite (MMT) have been investigated as one flame retardant multi-material system for glass-fiber-reinforced PA6.The fundamental concept is based on a combination of a long-term barrier effect (EG), char residue formation, and low pyrolysis rates (AlPi/MPP/MMT), improving flammability and heat development properties.Flammability properties and burning behavior have been investigated using the limiting oxygen index (LOI), UL-94 (V), and cone calorimeter tests.Coupled thermogravimetric analysis The multi-material systems were produced using a co-rotating, twin-screw extruder DSE ZSE HP 27 from Leistritz GmbH (Nuremberg, Germany).Barrel temperatures were controlled between 240 and 220 • C; the screw speed was held constant at 100 rounds per minute (rpm) at a feed rate of 10 kg per hour (kg/h).The multi-material systems containing EG, AlPi, MPP, and MMT were premixed in a universal powder mixer (200 u/min; 15 min) before processing.Two gravimetrical side feeding units were used for glass fibers and the premixed multi-material additive mixture.The strand was drawn off via a water bath chipped to granulate, and dried afterward before further processing.Injection molding was used to mold plate geometries (100 × 100 × 2 and 4 mm 3 ), which were then further processed via sawing and milling to generate sample geometries following the individual testing standards.Formulations tested and discussed within this study are listed in Table 2.
Thermal and Gas Analytics
Thermogravimetric analysis (TGA) and coupled Fourier-transform infrared spectrometry (FTIR) were used to analyze changes in the decomposition behavior.TGA measurements were conducted at various heating rates of 2.5, 5, and 10 K/min under a nitrogen atmosphere, using constant temperature ramps between 50 and 800 • C. Therefore, an STA F3 Polymers 2023, 15, 4100 4 of 15 449 Jupiter from Netzsch GmbH (Selb, Germany) was used.Decomposition/pyrolysis gases were transferred from the TGA exhaust to the FTIR gas cell Tensor 2 from Bruker Corp. (Billerica, MA, USA) via a coupled and temperature-controlled transfer line (230 • C).Due to the transfer time between the TGA exhaust and FTIR measurement, a signal delay between TGA and FTIR of about 30 s had to be considered.Activation energies were calculated from non-isothermal TGA measurements using a method suggested by Ozawa and Vyazovkin [36][37][38].Calculations were performed using an open access calculation tool.The descriptions and download link can be found in [39].
Fire Testing
To study fire retardancy properties, limiting oxygen index (LOI), UL-94, and cone calorimeter (CC) tests were conducted.All testing devices were from Netzsch Taurus Instruments GmbH (Weimar, Germany).
The LOI fire test is particularly useful to track flammability properties occurring from slight formula changes within multi-material flame retardant systems.The testing setup includes a vertically clamped sample (125 × 10 × <10 mm 3 ), which is surrounded by a controlled N 2 /O 2 atmosphere.During the testing routine, a 50 watt propane flame is systematically applied to the sample in a candle-like setup from above.If the sample is ignited, flame propagation is observed, the propagation time and distance are measured, and the N 2 /O 2 atmosphere contents are systematically adjusted.The resulting key figure "limiting oxygen index" (LOI) represents the lowest atmospheric oxygen content needed to provide either 50 mm downward fire propagation or 3 min of burning time.Tests were conducted following the DIN EN ISO 4589-2 [40] standards.
UL-94 (vertical) fire testing setups are used to study the self-extinguishing properties and fire-dripping behavior of polymeric materials under normal atmospheric conditions.During the test, a 50 watt methane flame is applied from underneath the sample (125 × 13 × <13 mm 3 ) following a standardized routine.After the testing flame is removed, burning times and dripping behavior are observed.Testing results are then clustered into three classifications from best to worst: V0, V1, and V2.V0 represents instant selfextinguishing behavior with no burn dripping.V2 classifications allow burn dripping and, compared to V0 classifications, longer burning times.Tests were conducted in accordance with the standards.
The cone calorimeter testing method is a valuable tool to gain insights into the burning behavior under enforced flaming conditions.During the testing routine, 100 × 100 × < 50 mm 3 sample geometry is placed underneath the cone heater.Pyrolysis gases are ignited via piloted ignition and the burning process is monitored by tracking the heat development over time until complete combustion.The amount of heat released as well as the heat development characteristics provide important information about the functionality of the flame retardant formulation.Within this study, tests have been conducted using three heater capacities 35, 50, and 65 kW/m 2 in order to calculate the heat release parameter (HRP).The appropriate literature targeting the correct use and interpretation of important key figures can be found in [42][43][44][45].The sample geometries used were 100 × 100 × 4 mm 3 .All tests were repeated at least three times, and averaged curves are presented in the results section.Tests were conducted in accordance with the DIN ISO 5660-1 [46] standards.
Char Residue Analysis
Char residue analysis was conducted using a SEM Ultra Plus system from Zeiss (Oberkochen, Germany).Samples were taken via cone calorimeter testing after complete combustion and prepared using platin-palladium.
Thermal Analysis and Evolved Gas Analysis-TGA-FTIR
TGA measurements were taken to evaluate the decomposition behavior of all of the tested formulations.Tests were performed at heating rates of 2.5, 5, and 10 K/min under Polymers 2023, 15, 4100 5 of 15 a nitrogen atmosphere.The results presented in Figure 1 show TGA-FTIR results for a heating rate of 10 K/min without (A) and with in situ FTIR coupling (B).
(Oberkochen, Germany).Samples were taken via cone calorimeter testing after complete combustion and prepared using platin-palladium.
Thermal Analysis and Evolved Gas Analysis-TGA-FTIR
TGA measurements were taken to evaluate the decomposition behavior of all of the tested formulations.Tests were performed at heating rates of 2.5, 5, and 10 K/min under a nitrogen atmosphere.The results presented in Figure 1 show TGA-FTIR results for a heating rate of 10 K/min without (A) and with in situ FTIR coupling (B).
TGA analysis measured at a heating rate of 10 K/min under a nitrogen atmosphere.The mass loss tracked over a constant heating rate gives insights about the decomposition process of a polymeric system.(B) Corresponding FTIR evolved gas analysis at the DTG (mass derivative) peak temperature.
The TGA analysis of PA6 containing 25 wt.% glass fibers showed a single gravimetrical step with the DTG peak appearing at approximately 451 °C (Figure 1A).Once a temperature of 500 °C was reached, all polymer fractions had evaporated, leaving only the glass fiber residue (25 wt.%).The evolved gases found were caprolactam (with a lactam peak at 1715 cm −1 and a fingerprint pattern at 1305, 1352, and 1361 cm −1 ), CO2 (at 2360 and 671 cm −1 ), ammonia (at 930 and 965 cm −1 ), CH2 groups (at 2940, 2865, and 1140 cm −1 ), and some traces of CO (at 2114 and 2174 cm −1 ) (Figure 1B).These gas phase products have also been found in numerous other studies as part of the PA6 decomposition [47][48][49][50].When 3 wt.%MMT is integrated, no observable changes in the decomposition onset temperature can be identified (Figure 1A).However, the DTG peak appears to occur slightly earlier at around 445 °C (also found in [51]).This earlier gravimetrical decrease corresponds to a higher concentration of caprolactam in the gas phase (1715 cm −1 ), which can be observed through a relative comparison of the FTIR peak heights between caprolactam and CH2 (Figure 1B).Also, a lower amount of CH3 and CO2 seems to be evaporated, which indicates an additional formation of intermediate aromatic compounds or intermediate bonding with the MMT surface.Since the residual fraction measured at 500 °C is in accordance with the filler integration (GF + MMT) and no changes within the gas phase composition occurred, no alterations of the main decomposition path of PA6 can be observed after the integration of MMT.This effect has also been discussed in other studies [52,53].The TGA analysis of PA6 containing 25 wt.% glass fibers showed a single gravimetrical step with the DTG peak appearing at approximately 451 • C (Figure 1A).Once a temperature of 500 • C was reached, all polymer fractions had evaporated, leaving only the glass fiber residue (25 wt.%).The evolved gases found were caprolactam (with a lactam peak at 1715 cm −1 and a fingerprint pattern at 1305, 1352, and 1361 cm −1 ), CO 2 (at 2360 and 671 cm −1 ), ammonia (at 930 and 965 cm −1 ), CH 2 groups (at 2940, 2865, and 1140 cm −1 ), and some traces of CO (at 2114 and 2174 cm −1 ) (Figure 1B).These gas phase products have also been found in numerous other studies as part of the PA6 decomposition [47][48][49][50].When 3 wt.%MMT is integrated, no observable changes in the decomposition onset temperature can be identified (Figure 1A).However, the DTG peak appears to occur slightly earlier at around 445 • C (also found in [51]).This earlier gravimetrical decrease corresponds to a higher concentration of caprolactam in the gas phase (1715 cm −1 ), which can be observed through a relative comparison of the FTIR peak heights between caprolactam and CH 2 (Figure 1B).Also, a lower amount of CH 3 and CO 2 seems to be evaporated, which indicates an additional formation of intermediate aromatic compounds or intermediate bonding with the MMT surface.Since the residual fraction measured at 500 • C is in accordance with the filler integration (GF + MMT) and no changes within the gas phase composition occurred, no alterations of the main decomposition path of PA6 can be observed after the integration of MMT.This effect has also been discussed in other studies [52,53].
When EG is integrated into reinforced PA6, a second decomposition step between 270 and 330 • C can be observed, resulting in a slight gravimetrical decrease of approximately 2.5 wt.%.Once the temperature exceeds 330 • C, the decomposition process accelerates, with a DTG peak appearing at 437 • C. The residual fraction left at 500 • C of 44 ± 1 wt.% is in accordance with the expected range; thus, no additional char formation can be observed.The slightly lower onset temperature for decomposition can be attributed to the expansion mode of EG, causing the sulfuric blowing agent to decompose and gasify.Compared to PA6 GF formulations, FTIR analyses reveal no changes within the gas phase composition.Thus, no alterations of the main decomposition path are triggered by EG integration.When integrating EG (15 wt.%), AlPi (3 wt.%), and MPP (2 wt.%) as a multi-material system into glass-fiber-reinforced PA6, two gravimetrical decomposition steps can be observed (Figure 2A).Similar to recipes based on glass-fiber-reinforced PA6 and EG, the initial gravimetrical step occurs between 270 • C and 330 • C marking the start of the EG expansion process.Exceeding 330 • C, the decomposition process accelerates between 400 and 470 • C. The DTG peak occurs at 443 • C, which is slightly higher than that observed for reinforced PA6/EG formulations (437 • C).The residue formation of 41 ± 1 wt.% (500 • C) is in line with expectations.Similar results have been reported in previous studies for non-reinforced PA6 [23,25,35].mately 2.5 wt.%.Once the temperature exceeds 330 °C, the decomposition process accelerates, with a DTG peak appearing at 437 °C.The residual fraction left at 500 °C of 44 ± 1 wt.% is in accordance with the expected range; thus, no additional char formation can be observed.The slightly lower onset temperature for decomposition can be attributed to the expansion mode of EG, causing the sulfuric blowing agent to decompose and gasify.Compared to PA6 GF formulations, FTIR analyses reveal no changes within the gas phase composition.Thus, no alterations of the main decomposition path are triggered by EG integration.
When integrating EG (15 wt.%), AlPi (3 wt.%), and MPP (2 wt.%) as a multi-material system into glass-fiber-reinforced PA6, two gravimetrical decomposition steps can be observed (Figure 2A).Similar to recipes based on glass-fiber-reinforced PA6 and EG, the initial gravimetrical step occurs between 270 °C and 330 °C marking the start of the EG expansion process.Exceeding 330 °C, the decomposition process accelerates between 400 and 470 °C.The DTG peak occurs at 443 °C, which is slightly higher than that observed for reinforced PA6/EG formulations (437 °C).The residue formation of 41 ± 1 wt.% (500 °C) is in line with expectations.Similar results have been reported in previous studies for non-reinforced PA6 [23,25,35].When MMT is additionally added into the formulation with EG (15 wt.%), AlPi (2.4 wt.%), MPP (1.6 wt.%), and MMT (1 wt.%) as a multi-material system, only slight changes within the TGA measurements can be observed.The DTG peaks shift slightly to a lower temperature of around 429 °C.The char residue at 600 °C remains within the expected range of 40 ± 1 wt.%.FTIR gas phase analysis also indicates no changes in the gas phase composition for any of the tested multi-material formulations (Figure 2B).However, a shift in the gravimetrical decomposition step toward lower temperatures indicates an accelerated depolymerization effect as well as early evaporation of the EG intercalated blowing agent.All major TGA data are summarized in Table 3.When MMT is additionally added into the formulation with EG (15 wt.%), AlPi (2.4 wt.%), MPP (1.6 wt.%), and MMT (1 wt.%) as a multi-material system, only slight changes within the TGA measurements can be observed.The DTG peaks shift slightly to a lower temperature of around 429 • C. The char residue at 600 • C remains within the expected range of 40 ± 1 wt.%.FTIR gas phase analysis also indicates no changes in the gas phase composition for any of the tested multi-material formulations (Figure 2B).However, a shift in the gravimetrical decomposition step toward lower temperatures indicates an accelerated depolymerization effect as well as early evaporation of the EG intercalated blowing agent.All major TGA data are summarized in Table 3.
Burning Behavior-Cone Calorimeter
The cone calorimeter results of all glass-fiber-reinforced formulations tested within this study are discussed in the following section.The results are presented in Figures 3 and 4
Burning Behavior-Cone Calorimeter
The cone calorimeter results of all glass-fiber-reinforced formulations tested within this study are discussed in the following section.The results are presented in Figures 3 and 4.
Burning Behavior-Cone Calorimeter
The cone calorimeter results of all glass-fiber-reinforced formulations tested within this study are discussed in the following section.The results are presented in Figures 3 and 4. Non-modified glass-fiber-reinforced PA6 is usually characterized by a steep increase in the heat release rate (HRR) (Figure 3A).Since a large fraction of polymeric fuel had been burned by the time the peak heat release rate (pHRR) was reached, the subsequent steep HRR decrease could be attributed to a quick reduction in the pyrolysis gas flow rate.No plateau formation could be observed for the given sample thickness, indicating no stationary burning state.Thus, further acceleration of the heat development would likely occur for thicker samples.By incorporating MMT into the glass-fiber-reinforced PA6, a substantial change in the burning characteristics could be observed.Ignition occurred slightly earlier, followed by a steep increase in the HRR and a subsequent plateau formation.Since the heat development continuously decreased since then, decay of the pyrolysis gas evaporation could be assumed.As a result, the pHRR decreased by about 57% compared to the non-flame retardant PA6/GF.This behavior remained constant for various external heater capacities applied.When comparing the heat release parameter (HRP), which was calculated from cone calorimeter tests conducted at heater capacities of 35, 50, and 65 kW/m 2 , MMT-modified samples showed substantially lower pHRR development (19 ± 0.3 versus 7.1 ± 1.3).Given that only 3 wt.% of the polymer was substituted by MMT, the decrease in the pHRR by 35% compared to PA6/GF is remarkable.Accordingly, independent of the fire scenario, PA6/GF equipped with MMT showed superior fire behavior.This observation is in good agreement with findings in other studies, such as [51,52,54].Two principal phenomena have been attributed to the performance increase:
•
Labyrinth effect: Nanodispersed MMT platelets form a labyrinth-like structure in the composite, increasing the material viscosity due to stronger material-particle interaction and extending the path of pyrolysis gases into the gas phase.As a consequence, the rate of gasification drops, which reduces the amount of available fuel and, thus, the burning rate.It is furthermore assumed that, for some polymeric systems, the formation of intermediate aromatic structures is favored.Thus, the labyrinth effect results in a prolonged meso-phase retention time, which increases the probability of (intermediate) char formation.Similar findings have been reported for many authors for various nano-scale systems.The relevant literature can be found in [55,56], although it is not limited to these references.
•
During the cause of a fire, MMT starts to migrate to the burning surface and accumulates to increase the char yield.This enhances the barrier formation, which reduces heat re-radiation toward lower layers of non-decomposed polymer fractions.As a consequence, lower decomposition/evaporation rates limit the fuel supply and thus the heat development.Studies reporting similar observations can be found in [54,57], although they are not limited to these references.
When integrating 20 wt.% EG, EG/MMT, or EG/AlPi/MPP/MMT into glass-fiberreinforced PA6, a substantial polymer fraction is substituted by the flame retardant additive system (Figure 4A).Hence, generally, lower heat development is to be expected.Since EG is the major flame retardant additive component providing improved thermal conductivity properties, heat can penetrate and dissipate more quickly over the entire cross-section.As the EG expansion and barrier formation proceed, the thermal isolation performance increases, counteracting the external heat penetration, lowering the heat penetration depth, and thus reducing heat development over time.Since barrier formation is a time-consuming process and residue formation in early burning stages needs some time to develop a sufficient thermal barrier, the heat release rate of formulations solely containing EG is relatively higher than that found for formulations additionally containing MMT or AlPi/MPP/MMT.By integrating MMT into the formulation, the EG barrier effect is combined with the previously described labyrinth effect.However, since the observable effect on the heat development is rather long-term and no change in the pHRR can be detected, the labyrinth effect is assumed to be abrogated by the expansion process.Hence, in formulations containing PA6/GF/EG/MMT, no synergistical effect could be observed for MMT integration.
By integrating AlPi/MPP in addition to EG/MMT into glass-fiber-reinforced PA6, the pHRR and average HRR are substantially reduced compared to EG and EG/MMT formulations.AlPi/MPP has been reported to react mostly in the mesophase, forming an aluminum phosphate residue [35,58].When additionally combined with MMT, studies found a strong residue-solidifying effect [59], which could also be identified for formulations within this study.Furthermore, the decomposition mechanism of MPP partially evaporates melam and inert gases such as ammonia, providing an additional cooling and dilution effect [12].The combination of long-term (EG) and short-term (AlPi/MPP) residue formation, pyrolysis gas stream reduction (MMT; labyrinth effect), improved residual stability (AlPi/MPP/MMT), and gas phase dilution (MPP) is thus found to work synergistically as a multi-material flame-retarding system for glass-fiber-reinforced PA6.The positive effect is also maintained over a set of different external heat capacities, resulting in a lower HRP of 1.1 (Figure 4B), indicating excellent flame retardancy performance with low sensitivity to environmental conditions.The described results fit well with findings in previously published studies.MMT has been found to substantially decrease the burning rate in non-reinforced (e.g., [24,60]) and glass-fiber-reinforced (e.g., [54]) PA6, observable in a lower pHRR and prolonged burning times using cone calorimeter tests.Various combinations of PA6/AlPi/MMT (e.g., [7]) and PA6/AlPi/MPP/MMT (e.g., [59]) have been extensively investigated, particularly distinguishing phosphorus/MMT combinations to provide improved intumescent char formation.This effect is generally dedicated to a lower melt viscosity and nano-particle surface migration, accumulating and stabilizing residue formation on the polymer surface [61].Glass-fiber-reinforced formulations containing EG and EG/AlPi/MPP have also been investigated (e.g., [23,25,35]).Results showed the lowest pHRR for PA6/GF formulations solely containing EG (<<100 kW/m 2 ), whereas formulations based on EG/AlPi/MPP exceeded a pHRR rate of >100 kW/m 2 .Despite the apparently very low pHRR values, no UL-94 V0 classification could be achieved [25].
The multi-material formulation (EG/AlPi/MPP/MMT) also showed superior properties regarding the residue stability.Figure 5 shows images of samples before and after cone calorimeter testing.The residue stability is visualized with a one kilogram weight, representing an area load of 35 g/cm 2 .No penetration of the weight into the residue surface could be identified.
tion effect [12].The combination of long-term (EG) and short-term (AlPi/MPP) residue formation, pyrolysis gas stream reduction (MMT; labyrinth effect), improved residual stability (AlPi/MPP/MMT), and gas phase dilution (MPP) is thus found to work synergistically as a multi-material flame-retarding system for glass-fiber-reinforced PA6.The positive effect is also maintained over a set of different external heat capacities, resulting in a lower HRP of 1.1 (Figure 4B), indicating excellent flame retardancy performance with low sensitivity to environmental conditions.
The multi-material formulation (EG/AlPi/MPP/MMT) also showed superior properties regarding the residue stability.Figure 5 shows images of samples before and after cone calorimeter testing.The residue stability is visualized with a one kilogram weight, representing an area load of 35 g/cm 2 .No penetration of the weight into the residue surface could be identified.Smoke production is another important property in fire critical applications.The general tendency of a burning material to generate smoke was evaluated via the key figure total smoke production (TSP) and that measured during cone calorimeter tests.
The TSP measured for all of the tested formulations was in a range between 1.2 and 8 m 2 (Figure 6).Formulations containing only AlPi/MPP or MMT compared to non-modified PA6/GF resulted in a slightly lower TSP between 6.5 and 7, which can be attributed to an overall reduction in the burning rate.The lowest value was observed for formulations containing only EG, since the flame retardancy mechanism is solely physical and provides an overall very low burning rate through voluminous residue formation.Through the integration of AlPi/MPP and MMT, the decomposition path is slightly disturbed, which leads to more incomplete combustion than that found solely for EG integration.As a consequence, the smoke production increases to 4.3 m 2 .All major cone calorimeter results are listed in Table 4.
to an overall reduction in the burning rate.The lowest value was observed for formulations containing only EG, since the flame retardancy mechanism is solely physical and provides an overall very low burning rate through voluminous residue formation.Through the integration of AlPi/MPP and MMT, the decomposition path is slightly disturbed, which leads to more incomplete combustion than that found solely for EG integration.As a consequence, the smoke production increases to 4.3 m 2 .All major cone calorimeter results are listed in Table 4.In contrast to net PA6, which attains a V2 classification due to significant burn dripping, glass-fiber-reinforced PA6 did not achieve a UL-94 V classification.Burn dripping could not occur since the presence of glass fibers increased the melt viscosity.This also affected the LOI value, which reached 22% and was in good agreement with the literature In contrast to net PA6, which attains a V2 classification due to significant burn dripping, glass-fiber-reinforced PA6 did not achieve a UL-94 V classification.Burn dripping could not occur since the presence of glass fibers increased the melt viscosity.This also affected the LOI value, which reached 22% and was in good agreement with the literature values [62,63].PA6/GF formulations containing 20 wt.% EG exhibited slightly better results in UL-94 (V2 classification) and LOI tests (24%).EG acts exclusively as a physical barrier, restricting the rate of pyrolysis gas that fuels the flame.EG is generally known to exhibit weaker performance in flammability tests because the barrier layer formed during the early burning stages takes some time to achieve sufficient effectiveness in order to stop the ignition process [25].By integrating EG/AlPi/MPP as a multi-material system into PA6/GF formulations, the oxygen index could be improved to reach 28%.However, the UL-94 testing results did not demonstrate any improvement.This behavior aligns with the findings of a previously published study [25].
Contrary to the results discussed earlier in the cone calorimeter section, the integration of MMT nanocomposites into PA6/GF showed a deteriorating effect on the oxygen index, measured to be reaching 19%.No UL-94 classification could be achieved.This effect has also been reported in the literature [54,63].Nanofillers have been investigated in various material systems and have been found to perform equally or worse in LOI and UL-94, but they perform substantially better in cone calorimeter tests.The effect is attributed to a change in melt viscosity, which alters the melt and burn dripping behavior.UL-94 and LOI fire tests are sensitive to melt dripping, since combustible fuel is physically removed from the burning area.The higher melt viscosity given through nanofiller integration suppresses a strong melt flow, negatively affecting UL-94 and LOI fire testing results [54,63].However, when integrating MMT in the multi-material formulation PA6/GF/AlPi/MPP/MMT, a significant improvement in UL-94 and LOI fire testing results can be observed.Figure 7B shows the UL-94 and LOI fire testing results for two formulations, with filling degrees of 10 wt.% and 20 wt.%.Interestingly, formulations containing a lower MMT loading level (0.2 wt.%) perform better at a lower filling degree, but do not achieve a sufficient UL-94 V0 classification at a higher filling degree of 20 wt.%.Formulations containing 0.5 wt.% and 1 wt.%, on the other hand, do not provide a UL-94 classification for a filling degree of 10 wt.% but provide a UL-94 V0 classification when 20 wt.% of the multi-material mixture is added into the system.All data are summarized in Table 5.
Char Residue Analysis
Char residue analysis was conducted using SEM analysis of the residual fractions left after cone calorimeter testing.Figure 8 shows a selection of images taken from various formulations.Residual fractions of PA6/GF/MMT can be described as a non-connected glass fiber network (Figure 8A).MMT could also be visually identified as agglomerated white powder fractions, yet it cannot be optically presented due to image resolution boundaries.Additionally, integrating EG into the system provides more voluminous sponge-like residue characteristics, whereas a combination of crosslinked char residue and residual polymer fractions acts as glue between the three-dimensional glass fiber network (Figure 8B).This is further improved via the addition of AlPi/MPP (Figure 8C,D).The aluminum phosphate residue, which is a result of AlPi/MPP decomposition, increases the amount of charring between the glass fibers and expanded graphite particles.This results in a more char-like structure, which provides superior structural stability, as previously shown in Figure 5.
after cone calorimeter testing.Figure 8 shows a selection of images taken from various formulations.Residual fractions of PA6/GF/MMT can be described as a non-connected glass fiber network (Figure 8A).MMT could also be visually identified as agglomerated white powder fractions, yet it cannot be optically presented due to image resolution boundaries.Additionally, integrating EG into the system provides more voluminous sponge-like residue characteristics, whereas a combination of crosslinked char residue and residual polymer fractions acts as glue between the three-dimensional glass fiber network (Figure 8B).This is further improved via the addition of AlPi/MPP (Figure 8C,D).The aluminum phosphate residue, which is a result of AlPi/MPP decomposition, increases the amount of charring between the glass fibers and expanded graphite particles.This results in a more char-like structure, which provides superior structural stability, as previously shown in Figure 5.
Conclusions
This paper has investigated a multi-material flame retardant additive system based on EG, AlPi, MPP, and MMT in glass-fiber-reinforced PA6.This study was able to show
Conclusions
This paper has investigated a multi-material flame retardant additive system based on EG, AlPi, MPP, and MMT in glass-fiber-reinforced PA6.This study was able to show that the sole integration of EG, while exhibiting excellent flame retardant properties in cone calorimeter tests, did not achieve a satisfactory UL-94 classification at a filler content of 20 wt%.By additionally adding AlPi, MPP, and MMT, a synergistic effect could be achieved, resulting in a stable V0 classification and good cone calorimeter properties.The synergistic effect was attributed to a combination of functional thermal barrier formation (EG), improved char formation (AlPi/MPP), and the labyrinth effect (MMT).Consequently, pyrolysis gas flow rates decreased, leading to lower flammability overall and heat development characteristics.
Figure 1 .
Figure 1.(A) TGA analysis measured at a heating rate of 10 K/min under a nitrogen atmosphere.The mass loss tracked over a constant heating rate gives insights about the decomposition process of a polymeric system.(B) Corresponding FTIR evolved gas analysis at the DTG (mass derivative) peak temperature.
Figure 2 .
Figure 2. (A)TGA analysis measured at a heating rate of 10 K/min under nitrogen atmosphere.The mass loss tracked over a constant heating rate gives insights about the decomposition process of a polymeric system.(B) Corresponding FTIR evolved gas analysis.
Figure 2 .
Figure 2. (A)TGA analysis measured at a heating rate of 10 K/min under nitrogen atmosphere.The mass loss tracked over a constant heating rate gives insights about the decomposition process of a polymeric system.(B) Corresponding FTIR evolved gas analysis. .
Figure 5 .
Figure 5.Initial sample (reference) and residue formation/stability after cone calorimeter testing.Figure 5. Initial sample (reference) and residue formation/stability after cone calorimeter testing.
Figure 5 .
Figure 5.Initial sample (reference) and residue formation/stability after cone calorimeter testing.Figure 5. Initial sample (reference) and residue formation/stability after cone calorimeter testing.
Figure 6 .
Figure 6.Total smoke production (TSP) measured via cone calorimeter testing with a heater capacity of 50 kW/m 2 .
Figure 6 .
Figure 6.Total smoke production (TSP) measured via cone calorimeter testing with a heater capacity of 50 kW/m 2 .
Figure 7 .
Figure 7. LOI and UL-94 testing results conducted for a sample thickness of 2 mm.(A) Results for singular and multi-material flame retardant additive integration.(B) Results of different filling degrees for two EG/AlPi/MPP/MMT formulations.
Figure 7 .
Figure 7. LOI and UL-94 testing results conducted for a sample thickness of 2 mm.(A) Results for singular and multi-material flame retardant additive integration.(B) Results of different filling degrees for two EG/AlPi/MPP/MMT formulations.
Table 1 .
Polymers, fillers, and flame retardant additives used within this study.
Table 2 .
Overview of all polymeric material recipes prepared and tested within this study.
Table 3 .
TGA measurement summary of the most important key figures and activation energies calculated using the Ozawa-Flynn-Wall method, with TGA heating rates of 2.5, 5, and 10 K/min.
(B) Heat response parameters calculated from the cone calorimeter results at 35, 50, and 65 kW/m 2 . | 2023-10-18T15:12:03.844Z | 2023-10-01T00:00:00.000 | {
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195760579 | pes2o/s2orc | v3-fos-license | Stay fit or get bit – ethical issues in sharing health data with insurers’ apps
Summary In the framework of digital health, mobile applications with health-related content are increasing in number and importance. Many of these apps are targeted at the general public and, although they differ in features and purposes, their functions are often based on the promotion of health and the monitoring of customers’ lifestyle data. Apps of this type have also recently been developed by health insurance companies. In many cases, insurers’ apps do not simply offer health-related recommendations to the users, but also provide economic incentives to those customers who agree to share their behavioural data through the applications. Although such apps may contribute to the adoption of a health-conscious lifestyle, the fact that they require the sharing of dense individual data with the companies responsible for insurance coverage raises some relevant ethical issues. This paper investigates the phenomenon of insurers’ apps permitting customers to share their data in exchange for monetary rewards currently available in Switzerland. After describing the features and functioning of the apps, we present some ethically relevant aspects related to their use. More specifically, we discuss the issues of trans-parency of data-sharing purposes, potential discrimination amongst insured people, “quantification” of the users and, finally, the potential tension generated between solidarity and responsibility. We conclude by emphasising that these apps are becoming a new paradigm for insurers in many countries and that a thorough assessment of their ethical and societal implications is required.
Introduction
Digital health is drastically changing the context of medicine and healthcare.Although a shared and clear definition of this term is lacking [1], it has been argued that digital health encompasses mobile health, health information technologies, wearable devices, telehealth and telemedicine [2].Although quite diverse in nature, digital health solutions share the distinctive feature that they "provide digital and objective data accessible to both caregivers and patients [thus leading] to an equal level doctor-patient relationship with shared decision-making and the democratisation of care" [3].
Health apps constitute a relevant component of digital health solutions.In general, the term "health apps" refers to all mobile applications aimed at the promotion of health through supporting self-management, generating pre-diagnosis or producing other therapeutic effects [4].A recent study on the potential and risks associated with health apps supported by the German Ministry of Health reported that there are over 100,000 apps with health-related content [5].One reason why health apps are thriving is that a great many of them are not aimed at healthcare professionals or at patients with a specific disease, but are rather targeted at the general public [6].
In this context, health insurers have recently also begun to commercialise some health apps [7].Many of these apps simply allow customers to deal more efficiently with their documents by digitalising some previously paperbased processes.Others, however, belong more closely to the field of digital health because they include some functions that are strictly related to health promotion [8] and because they aim at directly engaging the users with respect to their health [9].Specifically, to promote the adoption of a healthier lifestyle, these apps allow users to share their behavioural health-related data with their insurer to better track their habits and provide them with personalised health tips [10].The insurer often offers monetary awards or similar bonuses as incentives to share data and to try to improve health-related behaviour [10].
Although insurers' data-sharing apps aim at promoting health-conscious behaviour and at giving financial bonuses to people with an active lifestyle, the fact that they are based on the monitoring of individual behaviour and the sharing of health-related personal information also raises some legal and ethical concerns.Indeed, one of these apps was recently criticised by the Federal Data Protection and Information Commissioner (FDPIC), who recommended the revision of a few of its features [11].In particular, the FDPIC criticised the processing of Basic Health Insurance (BHI) data together with Additional Health Insurance (AHI) data and disapproved of the fact that economic rewards were given also to BHI subscribers.However, the idea of using apps to share behavioural health-related data with insurance providers in exchange for monetary rewards was neither questioned in principle nor investigated in depth.
In this article, we first provide an overview of the features and the functioning of insurers' apps currently available in Switzerland that offer monetary incentives in exchange for data-sharing.Then, we discuss some of the ethical issues related to the sharing of lifestyle data with insurances through these apps.Our discussion is based on an adaptation of the casuistry approach, relying both on the analysis of the apps available on the Swiss market and on further literature review on the topic of digital health and the use of incentives to modify health-related behaviour.
Methodological approach
To further investigate the phenomenon of insurers' datasharing apps, we searched for all the apps of this kind offered on the Swiss market.For the scope of our search, we defined as "insurers' data-sharing apps" those mobile applications developed or offered by a health insurance company whereby the sharing of individual health-related behavioural data earns a monetary reward or similar compensation.We based our definition on the FDPIC's official communication analysing one of these apps.According to our definition, the distinctive feature of insurers' data-sharing apps is that they provide -on top of general incentives, such as daily challenges or health tips -a series of direct or indirect monetary bonuses as a reward for sharing behavioural health-related data.In this sense, these apps are different from other health-promotion tools such as smoking cessation programmes.In fact, with insurers' apps users are even more strongly motivated to share behavioural health-related data, since -on top of the promise of the long-term benefit of improving their health -they also receive a direct, readily available and more tangible monetary benefit.In this context, we use the term "sharing" to refer to the transfer of data between app users and insurers, and not between users.(We use the term "sharing" and not "collecting" since in most cases these apps simply rely on data collected by other means [for example through fitness trackers].However, this same process could also be described as "secondary collection", i.e. collection from a source that had already gathered data from the data subject.) In order to be comprehensive, we decided to investigate the whole Swiss market, thus including insurers' data-sharing apps offered in the framework of both BHI and AHI.We did not consider health insurance plans that simply allowed for reduction of premiums upon participation in specific health promotion activities.(For example, "KPT Krankenkasse AG" offers an insurance plan named "active plus", where customers can obtain premium reductions if they participate in certain health-promoting activities [e.g.yoga courses].To obtain the discount, customers need to provide evidence of participating in these activities [e.g.receipts], which they can also scan and submit via the insurance app used to submit other documents [e.g.hospital bills].In this case, however, a proper digital health component is missing, since the app is simply used as an alternative to paper transmission.One example is the ActivePlus Program [12].) To identify insurers' data-sharing apps, we checked insurers' websites as listed by the Federal Office of Public Health [13] to establish whether they offered apps that fitted our scope of inquiry.After having identified apps that fitted our definition, we analysed their characteristics by reading their description on the insurers' website and on the app stores (Google Play and Apple Store).We then read the terms and conditions (T and C) of the identified apps to better map their functioning.We finally discussed the features of insurers' data-sharing apps by continuing the investigation started by the FDPIC and using a casuistry approach [14][15][16].Casuistry, in contrast to principlism approaches in bioethical analysis, is based on a bottom-up procedure, whereby a specific and concrete situation is investigated to elaborate considerations, reflections and maxims of a more general validity and potentially applicable also to similar circumstances to the one analysed [17].
Insurers' data-sharing apps in Switzerland: features and functioning
We finalised our search in January 2019 and were able to identify seven different apps that could fit our definition.Upon further reading of the T and C of the identified apps, we excluded two of these for the following reasons: The app "Ignilife" [18] was indirectly sponsored by one insurance group, but it was specified that the app did not belong to the group and that the apps' producers would not share data with the insurer [19].The app "maxyourhealth" [20] was sponsored by one insurer, but did not explicitly include a reward programme allowing users to receive monetary incentives for sharing data [21].We thus included in our analysis the five remaining insurers' data-sharing apps, whose most relevant features are presented in table 1 and described in more depth below.
"Helsana+" permits users to either manually "feed" the app with data about health-related behaviours (e.g.photos of outdoor activities), or link the app directly to other healthmonitoring devices (such as fitness trackers).When linked to fitness trackers, the app then automatically collects data on the insured person's lifestyle.In exchange for sharing behavioural data, users are awarded bonus points.Bonus points can either be used to reduce the insurance premium, or they can be redeemed as vouchers spendable with selected marketing partners.According to the insurance company, sharing data through the app can lead to savings of over CHF 300 per year [22].It is available for both BHI and AHI subscribers, but the former's discount is capped at CHF 75 annually.
"MyCSS" allows users -amongst other services such as scanning insurance documents -to register for the "MyStep" functionality.Once activated, "MyStep" users are not allowed to manually feed data, but they can link their fitness trackers directly to the insurer's app.The app then automatically monitors how many steps the customer takes.As a reward, for every day that the insured person achieves specific step-thresholds (see table 1), he or she receives a cash-credit.These cash-credits are transferred to the insuree's personal account and can be redeemed as cash, for up to CHF 146 per year.MyStep is only available for AHI subscribers.
The "Active" app by Sanitas follows a very similar model to that of "Helsana+".Insured people can collect bonus points if they share individual health-related data with the app.Data-sharing can be performed either through manual feeding by the user (for example by uploading data about nutrition), or via automatic means, by linking the app to a fitness tracker.Once collected, insurees can then convert the bonus points into vouchers to spend on products offered by business partners.Everybody can register on the app, but only Sanitas insurees can collect bonus points.
"Benevita" is slightly different from the other apps.In order to obtain bonus points, users have to fill in a questionnaire and communicate data concerning their lifestyle habits.Depending on their questionnaires, a certain number of bonus points are assigned to customers.The number of bonus points determines the discount users receive on their AHI premiums.Automatic collection of behavioural health-related data through linking the app with fitness trackers is possible, but it does not allow collection of points and it is simply used to create health-related challenges for the user.
SanaHealth is similar to the "Active" app in its structure, but -being a pilot project limited to 1500 users -it is less developed.Customers can share their data with the app either automatically, by connecting the app to fitness trackers or manually, by providing individual pieces of information about lifestyle habits.Sharing gains users bonus points, which they can then use to buy products from an online shop.It is downloadable for free for all customers of Sanagate, the insurance company offering it.
Ethically relevant aspects of health-data sharing though insurers' apps in exchange for monetary rewards
The widespread collection and sharing of personal health data has the potential to revolutionise healthcare.Since life-habits and health outcome are linked [33], apps offering the chance to contribute to the adoption of responsible health-related behaviours may lead to health improvements and beneficially affect health interventions [34].Moreover, the use of financial incentives in this context could encourage responsible behaviour by providing a tangible and short-term benefit on top of the long-term expectations of positive health outcomes.
However, the constant monitoring of behaviour that insurers' data-sharing apps require also raises a number of ethical and legal questions.When analysing one of these apps, the FDPIC already observed that there were some issues related to the legitimacy of data-linkage, the provision of consent and the payment of economic bonuses.By expanding this analysis in light of the common features of all insurers' data-sharing apps available on the Swiss market, we have identified four further areas of ethical concern, which are presented here.
Transparency: what are the (true) purposes of datasharing?
One fundamental principle of legitimate data usage is that of clearly defining the purpose for which data is collected, shared and processed [35][36][37].Indeed, Swiss law explicitly limits -with regards to the extent and the objectives of the processing activities -private institutions processing personal data to those purposes that are disclosed at the moment when data is shared and consent by data subjects Coins can be redeemed as vouchers to be spent with one of partner companies.Collected credits cannot be exchanged for cash.
Insured people with additional insurance.
1. Automatically: data from a fitness tracker is synchronised with the app.2. Manually: a declaration can be completed online by answering to a series of health-related questions.
Bonus points: depending only on the declaration, a specific amount of points is granted.
Bonus points determine the status of the user and the entitlement to a premium discount up to 15%.
Insured people.
SanaHealth [ is obtained [38].The importance of clearly disclosing the purposes of data processing is especially fundamental with respect to sensitive information such as data concerning health [39].
In this regard, there seems to be an issue concerning transparency with respect to insurers' data-sharing apps, since the entire range of purposes for which users' data is processed is not equally disclosed.The common message that the insurers promoting their apps deliver is that the key purposes of data sharing are the following: (1) improve individual health; (2) help users save some money.For example, the webpage describing and promoting the "Active" app claims that "in the medium and long term you benefit from better health.And in the short term you benefit from the bonus system of our Active app!" [40].Similarly, "myStep" is advertised as having the objective of "Turn[ing] steps into a bonus", 2reward[ing] your physical activity" and "encourag[ing] people to take more exercise and inspir[ing] them to enjoy their health" [24].However, while these certainly represent some of the purposes of data-sharing and processing, they are certainly not the only reasons.In fact, the T and C customers must agree to in order to use these apps explicitly specify that the purposes of data collection also include more delicate aims, such as using data for marketing purposes and forwarding data to third parties.For example, users of "Helsana+" need to accept that the insurance company "is entitled to forward user data within the context of the above-mentioned processing purposes to all companies in the Helsana Group or to third parties who process the user data for Helsana on a contractual basis" [23].
It is true that by listing the purposes of data sharing and data processing in great detail within the T and C and by requiring explicit agreement therewith, the insurers may err on the side of caution, legally speaking.However, from an ethical perspective, there appears to be a problematic lack of transparency, particularly given that consumers often do not read the fine print closely [41] and that, when confronted with documents such as T and C, people often simply click "Agree", thus giving a type of "blind" consent [42].
In this sense, there is some imbalance concerning the weight given to different types of information by the insurers providing the apps.On the one hand, the beneficial purposes of sharing and processing the data from the perspective of potential users are the main focus of all related webpages and information sheets that describe the product and present the reasons of data sharing.On the other hand, the more delicate purposes of data sharing and data processing are remotely indicated in a document, the T and C, which -because of the nature of how consent is given for the purchase of apps -few users are likely to read.That the users are not adequately informed about the purposes data will be used for seems even more problematic as the data in question is often sensitive, behavioural health-related information.Insurers should be up-front about the use of data for marketing and third-party reuse on the main pages as well as in the small print.
Data-driven discrimination: is it ethical that lifestyle data collected through apps is used to personalise insurance?
Another ethically relevant aspect related to insurers' datasharing apps concerns whether favouring users who share behavioural health-related data is ethically acceptable.Some degree of differentiation amongst insured people has always been accepted in Switzerland both in the BHI framework and (even more) in the AHI market.In this sense, individual choices -such as deciding one's own franchise or participating in health promotion programmes -have always had an influence on the amount of money paid for insurance coverage.In consequence, it could be argued that the idea of furthering this tendency through the monitoring of users' behavioural data is acceptable as well, insofar as an active -and thus allegedly beneficiallifestyle entails a premium-reduction or another economic advantage.Moreover, this idea of enhancing insured people' responsibility for their own behaviour seems consistent with a recently developed definition of health as "the ability to adapt and to self-manage, in the face of social, physical and emotional challenges" [43].
In general, where to set the exact boundary between acceptable differentiations amongst insured people and illegitimate discrimination is a bone of contention [44,45].
With respect to Swiss healthcare, it is important to draw a clear distinction between the context of BHI and AHI [46].Despite being offered by private companies, BHI is mandatory and covers a broad range of healthcare services that are equal for everybody and defined by the law.Insurers are obliged to accept all applicants irrespective of their risk profile and -although they have some degree of freedom in setting the price for their premiums (e.g. they can offer lower premiums to people as old as 25) -they are not allowed to profit.AHI, on the contrary, is a market-based system, where insurers can make profit, define eligibility and establish prices and exclusion criteria.Since most of the insurers' data-sharing apps offer an economic advantage that is linked to AHI, the main issue at stake is whether -in the context of the market-oriented AHI -it is ethically acceptable for insurees to receive better economic arrangements (through discounts or rewards) if they share lifestyle data.
Even in market-oriented models of health insurance, different economic arrangements are often seen as ethically troubling if based on features upon which the insured person has no control, such as race, gender or genetic data [47].This is true also for Switzerland, where the law governs the disclosure of genetic data to insurers in a special way and insurers cannot -for example -ask applicants to undergo genetic testing as a condition of subscribing to AHI [48].In contrast, differentiation based upon factors that depend on individual choices does not seem to be fundamentally unfair, especially in the framework of AHI.From an ethical standpoint, this is because a difference is often drawn between voluntary and involuntary risk-seekers: whilst a person with inherited genetic features that determine a predisposition towards certain illnesses is seen as a "victim" who deserves support, an individual who, for example, smokes is considered "faulty" and deserving of (economic) disadvantages [49].In this framework, discriminating -by awarding economic benefits -in favour of those that share Review article: Biomedical intelligence data documenting their healthy lifestyle might therefore seem acceptable.
Yet there are three reasons that seem to make this form of "data-driven" discrimination ethically problematic, even in the framework of AHI.Firstly, the basic claim that discrimination based on lifestyle choices should generally be justifiable because behaviour is both voluntary and a matter of free choice is highly doubtful.Not only has it long been known that unhealthy behaviours are associated with socio-economic conditions over which the individual has little control [50], but behavioural economics has also more recently demonstrated that individuals are not rational decision-makers even with respect to important issues such as health [51].Secondly, this form of discrimination seems unfair because it interferes with the autonomy of those that have a healthy lifestyle, but refuse to share their data through insurers' apps, perhaps because of concerns related to data protection.Making economic benefits dependent on consent to share dense behavioural data through an (admittedly) fallible app seems to create pressure to share data, rather than merely incentivise healthy lifestyle habits.Positive behaviours could be monitored (and incentivised) in many other manners, either less privacy-invading -such as by asking insurees to provide receipts of fitness courses -or more reliable -such as through professional health check-ups.Thirdly and more importantly, this "data-driven" discrimination seems ethically unsound because the exact consequences of data sharing on individual coverage are not clearly outlined.
In fact, the T and C of most data-sharing apps say that lifestyle data can be processed for profiling purposes through behaviour analysis [23,27,29].Whereas risk-profiling per se is a normal task for insurance companies, it seems ethically problematic to perform it retrospectively (i.e. after being insured) and on the basis of lifestyle data.The piece of legislation governing AHI (Swiss Federal Law on Insurance) already requires individuals to truthfully disclose all relevant information related to their risk profile before they sign an insurance contract, and protects insurers from deceitful non-disclosure as -if the individual lies -the insurance contract can be voided [52].In this sense, it seems more ethically acceptable to perform the whole processes of profiling, risk-assessment and personalisation of coverage and premium costs upfront in a transparent and explicit fashion, rather than leaving them open to potential ex-post evaluation via lifestyle data.In order to be ethically sound, differentiation in economic arrangements and medical underwriting should be based on clear facts directly and consciously declared by individuals and not on complex profiling based on dense lifestyle data automatically shared through apps.
The quantified self: are users merely what their data say they are?
At a practical level, the principle underlying the idea of rewarding insured persons for sharing some health-related data is that adopting a responsible lifestyle contributes to improving health.In this sense, a discount on the insurance premium or other monetary bonuses are presented as incentives for adopting a healthy lifestyle.Moreover, there are also economic reasons for granting a monetary reward in exchange for data confirming the adoption of a responsible health-related behaviour.Just as with discounts grant-ed for the performance of regular health check-ups [53], the assumption is made that if insured people demonstrate they are trying to keep fit, they will be less likely to make use of healthcare services, thus reducing their consumption of healthcare resources and earning them an economic benefit.On the other hand, people who do not share data would not have access to discounts, because insurers assume that their undocumented lifestyle might be unhealthy, thus increasing the likelihood that they will be in need of healthcare services in the future.In this sense, insurers' data-sharing apps seem to foster the idea that individuals and their health status are quantifiable through the data they produce with their self-tracking devices and apps [54].
Quantification of individuals in the context of health has sparked a polarised ethical debate [55].On the one hand, advocates for self-quantification through tracking devices underline how this would serve the principle of beneficence.In fact, since tracking devices allegedly allow insurers to collect objective lifestyle health-related data, the latter can be used to personalise treatment and thus improve care.On the other hand, concerns have been raised with respect to autonomy and self-tracking has been described as disempowering, because it potentially allows the control and the surveillance of others who have access to the data of the tracked person.
In the context of insurers' data-sharing apps, promoting the idea that people are simply what their data say raises three issues.Firstly, it is disputable that the lifestyle data shared through the apps corresponds to the aspiration of truth and objectivity of self-tracking technology [56].In fact, because of such apps' inherent limitations, there is no guarantee that the few types of data shared are actually accurate.For example, the T and C of one app specify that the app "draws exercise data from third-party sources [e.g.fitness trackers] and assumes no liability for the accuracy and correctness of this information" [27].Secondly, as the amount of data that can be shared through these apps is limited (some relevant risks factors such as smoking are not included), their focus seems too narrow for them to be used to define a concept as complex as health status.For example, a person could reap substantial benefits from sharing data through these apps, but also be "hiding" other habits (such as heavy drinking), which could impose much heavier costs on the healthcare system than someone who merely does not share data or exercises less.Thirdly, the fact that these apps share data with insurers fuels the concerns that some form of surveillance could take place, since health insurers have a vested interested in analysing the health-related behaviour of their customers.
Tension with solidarity: is it ethical to stress the "moral" side of personal responsibility?One last remark must be made concerning the impact that insurers' data-sharing apps can have more broadly on some of the ethical foundations of healthcare as a component of the system of social security.Along with security and protection, the crucial pillars of healthcare systems in Europe are solidarity and responsibility [57,58].Solidarity dictates that people who are in specific situations of need or risk should be included in the protection given by the social security system, and that the costs of the disadvantages related to their condition should be shared amongst Review article: Biomedical intelligence all members of society, regardless of health status [59].Responsibility, on the contrary, stresses that social protection cannot be indiscriminate and that individual freedom cannot excessively go against the interests of the community.In this context, striking a socially accepted balance between individual responsibility and communal solidarity is a crucial challenge.In the Swiss healthcare system, this balance is achieved by combining the guarantee -thanks to mandatory BHI -of a large basic benefit basket for everybody, with the possibility of personalising this offer through the subscription of AHI or the selection of a BHI with higher deductibles or with a managed care model at a lower premium rate [60].Individual responsibility thus plays an important role, but it is anchored in personal preferences and objectively defined criteria, rather than in lifestyle and behaviour.
Because of their features, insurers' data-sharing apps can have a substantial impact on the delicate balance between personal responsibility and solidarity in Swiss healthcare.By rewarding those who accept being monitored, these apps "moralise" the idea of responsibility, since they introduce differences -albeit minimal, since rewards have a modest economic value for the moment -between "deserving" and "undeserving" agents [61].This latently reinforces the idea that insured persons are allowed to contribute less to the financing of healthcare because of their lifestyle.In other words, insurers' data-sharing apps stress the link between responsibility and lifestyle choices.In Switzerland, the debate the extent and the role of personal responsibility for health is an open one, and whether lifestyle should be considered as an important element to limit solidarity in healthcare is often discussed [62,63].In this respect, although increasing responsibility for health-relevant behaviours is not ethically problematic per se, whether this is the right direction for the future evolution of healthcare in Switzerland is something that ought to be collectively and openly discussed, rather than being subtly introduced through mobile apps that are not clinically validated.
Careful discussion before the delicate balance between solidarity and responsibility in healthcare is modified is particularly important from an economic standpoint.An argument for justifying premium reductions according to behavioural habits could be that people -by means of adopting a healthy lifestyle -still contribute positively to the insurance model, as the savings they produce through healthy habits would offset the small economic benefits they receive.However, whether this is the case is an empirical question and, before any such system is implemented, a reliable amount of evidence regarding the real economic consequences of following specific lifestyles should be collected.A recent study exploring the impact of digital health solutions on healthcare utilisation in the US demonstrated that these are not associated with any large shortterm increases or decreases in health care usage [64].More generally, empirical evidence seems to show that, from a purely economic perspective, the adoption of what could be regarded as an "active" lifestyle actually increases health expenditure, since active people have a longer life expectancy and are thus more likely to suffer from age-related chronic diseases [65].Moreover, as outlined above, the fact remains that people could "exploit" the system to obtain monetary benefits by providing biased data or by "hiding" other unhealthy habits that are not measured (e.g.smoking).This is even more likely given that these apps are not clinically validated and that T and C often underline that use of the apps is undertaken "entirely at the user's own risk" [23].
Conclusion
Insurers' data-sharing apps are a representative and relevant technological instrument in the field of digital health.The phenomenon of insurance apps permitting the transfer of health and lifestyle data in exchange for monetary rewards is thriving and apps of this kind are not exclusive to Switzerland.In 2016, Generali, the third largest insurance group in the EU [66], announced the launch of the "Vitality" programme [67], which is based on continuous monitoring of health-related behaviour of insured persons through, amongst other means, an app linked to fitness trackers.The "Vitality" programme is aimed at promoting a more active lifestyle amongst insured persons and offers several monetary rewards in exchange for the sharing of health-related data.The financial benefits include, for example, a 40% discount on the purchase of a fitness device and a definite amount of bonus points for every data-upload [68]."Vitality Health" in the UK also offers a similar programme, proposing tailored insurance plans with special discounts if the person accepts the transfer of personal health data through fitness tracker and regular visits [69].
The same model is offered by "Discovery" in South Africa, where sharing also entails the transfer of data concerning users' medication adherence [70].
From this article, it has emerged that insurers' data-sharing apps are presented as a way of incentivising the adoption of a healthier lifestyle, saving money and reducing the burden of those illnesses that are co-caused by some specific health-related behaviours.However, the use of these apps also raises a series of problematic ethical issues, particularly with regard to the issues of transparency, discrimination, the quantification of the self and the balance between responsibility and solidarity in healthcare.Moreover, in addition to some widespread scepticism concerning the use of monetary incentives to improve behaviour [71], at present decisive evidence demonstrating the beneficial impact of health-related mobile applications -either in terms of clinical effectiveness or in terms of healthcare-resources consumption -is lacking [64,[72][73][74][75][76].
As society ages and the impact of chronic illnesses grows, the pressure of increasing efficiency while decreasing healthcare costs will continue to rise.In this sense, the use of data and the implementation of digital health represent powerful ingredients to help reduce costs and better allocate scarce resources [77].However, new developments such as insurers' data-sharing apps must continue to be critically assessed and evaluated in order to protect citizens -both healthy and unhealthy -from potential discrimination and exposure to privacy risks.
Financial disclosure AM and BE acknowledge the financial support provided by the Swiss National Science Foundation (SNF NRP-74 Smarter Health Care, grant number 407440_167356).The funder had no role in the drafting of this manuscript and the views expressed therein are those of the authors and not necessarily those of the funder. | 2019-07-02T13:47:55.154Z | 2019-06-30T00:00:00.000 | {
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237898377 | pes2o/s2orc | v3-fos-license | Efficacy of single-site radiotherapy plus PD-1 inhibitors vs PD-1 inhibitors for oligometastatic non-small cell lung cancer
Purpose Growing numbers of clinical trials test the efficacy of radiotherapy (RT) plus immune checkpoint inhibitors (ICIs), but the number of irradiated sites is not uniform. We aimed to evaluate the efficacy of single-site RT plus immunotherapy in oligometastatic non-small cell lung cancer (NSCLC) with smaller disease burdens and low tumor heterogeneity. Methods We retrospectively identified oligometastatic NSCLC (< 4 metastatic sites) patients treated with PD-1 pathway inhibitors with or without RT to a single lesion in our institution between 2018 and 2020. The primary endpoints were the best objective response rate (ORR) and progression-free survival (PFS). Results Of the 152 patients enrolled, 93 and 59 were identified as the ICI alone group and the ICI plus RT group, respectively. The addition of RT to ICI therapy significantly increased the best ORR from 31.2% to 50.8% (p = 0.015). The out-of-field (abscopal effect) response rate could reach 41.3% (95%CI 26.5%–56.1%) in the ICI plus RT group. Median PFS was 8.9 months (95%CI 4.7–13.1 months) with ICI alone versus 13.8 months (95%CI 9.5–18.1 months) with ICI plus radiotherapy (hazard ratio [HR] 0.556; p = 0.035). In an exploratory subgroup analysis of PFS, the addition of RT brought greater benefits in patients aged < 65 years (p = 0.016), patients with ECOG PS = 0 (p = 0.048), and patients with 1–2 metastatic sites (p = 0.024). No unexpected adverse events or significantly increased toxicities were observed in the experimental arm. Conclusion Single-site RT plus anti-PD-1 inhibitors significantly increased systemic responses and improved survival outcomes in oligometastatic NSCLC patients. Supplementary Information The online version contains supplementary material available at 10.1007/s00432-021-03849-3.
Background
The introduction of immunotherapy has transformed the treatment paradigm for advanced non-small cell lung cancer (NSCLC). Immunotherapy mainly refers to checkpoint inhibitors, such as PD-1, PD-L1, and CTLA-4. Chemotherapy combined with PD-1/PD-L1 inhibitors immunotherapy or immunotherapy alone has been approved as the standard first-line treatment for advanced NSCLC based on the results of the KEYNOTE (010, 024, 042) (Herbst et al. 2016;Mok et al. 2019;Reck et al. 2016). Unfortunately, only approximately 20% of unselected NSCLC patients could benefit from immunotherapy, spurring efforts to explore combination strategies (Borghaei et al. 2015;Gandhi et al. 2018).
Radiotherapy (RT) increases the expression of tumorassociated antigens and causes tumor cell immunogenic death, which promotes migration of T lymphocytes to tumor sites, thereby enhancing the local antitumor effects (Deng et al. 2014;Formenti and Demaria 2013;Verbrugge et al. 2012). Furthermore, RT can cause the decrease or regression of tumor outside the irradiation field. This phenomenon called the abscopal effect is because local RT causes a systemic immune response Khalife et al. 2019;Sezen et al. 2021;Theelen et al. 2020;Zhuang 2020).
However, data on the efficacy of the anti-PD-1 treatment with RT among metastatic NSCLC patients generally do not show better results than those among patients who have received immune checkpoint.
Inhibitors (ICI) alone (Samuel et al. 2020;Theelen et al. 2019). The reason for the inconsistent outcomes may be the single-site irradiation in these studies. Given the larger tumor burden and non-equal immunogenicity in metastatic NSCLC, irradiating only a single lesion in patients with multiple metastases might not be sufficient to induce systemic responses (Brooks and Chang 2019).
In metastatic NSCLC patients, approximately 25-50% of patients presented with oligometastatic disease (Parikh et al., 2014). (Bauml et al. 2019) conducted a single-arm phase II trial specifically focusing on oligometastatic NSCLC patients treated with local ablative therapies at all sites, plus pembrolizumab. The results with a 19.1-month median PFS-P (from the start date of pembrolizumab use) were significantly better than the historical control, with a PFS of 6.6 months. Despite this success, the irradiation of a single lesion continues to be the cornerstone of current strategies designed to test the efficacy of RT in combination with immunotherapy. Moreover, no prospective studies have been conducted on this trial design for oligometastatic NSCLC.
Therefore, we conducted a retrospective study of immunotherapy with or without irradiation of a single lesion for oligometastatic NSCLC. This study evaluated whether single-site RT was sufficient to enhance the systemic response of immunotherapy.
Patient selection
Management of oligometastatic disease in patients with limited metastases is supported by relatively high-level evidence. In our institution, local therapies are recommended in combination with systemic therapy in well-selected patients. Gains in survival due to the ICIs therapy have further inspired research into the oligometastatic paradigm. We retrospectively reviewed the data of patients with oligometastatic NSCLC (defined as having < 4 metastases) (Bauml et al. 2019) treated in our institution (2018-2020) with immunotherapy (PD-1 checkpoint inhibitors) combined with or without RT (Fig. 1). The RT was limited to singlesite irradiation in this treatment phase. Other key eligibility criteria included: (1) At least 1 separate lesion was required, which was measurable according to the Response Evaluation Criteria in Solid Tumors version 1.1; (2) ≥ 2 cycles of anti-PD-1 treatment; and (3) no epidermal growth factor receptor (EGFR) and/or anaplastic lymphoma kinase (ALK) targetable mutations. Patients were ineligible if they had (1) prior treatment with immunotherapy and (2) no complete clinical and follow-up data. Patients were divided into two groups based on whether they received RT or not: ICI alone and ICI plus RT groups. The collected data included baseline demographics, ECOG performance status, prior systemic treatment, immunotherapy regimens and RT details, treatment-related toxicities, and follow-up data.
The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Research Ethics Board of Shandong Cancer Hospital, and individual consent was waived owing to its retrospective nature. The authors are accountable for all Fig. 1 The flow chart of patient selection aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Treatment and outcomes
Patients received one of the following anti-PD-1 agents every two or three weeks with or without chemotherapy: sintilimab (Innovent Biologics, China), toripalimab (Shanghai Merck & Co.), camrelizumab (Jiangsu Hengrui Medicine, China), nivolumab (Bristol-Myers Squibb, USA), or pembrolizumab (Merck & Co., USA) (Supplementary Table 1). In the anti-PD-1 plus RT group, the radiation sites included primary tumors and metastatic lesions. Tumor response was assessed using radiographic imaging by the investigators according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1), and adverse events (AEs) were evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.03, with causality to treatment recorded. In particular, the response of unirradiated lesions (out-offield) was also evaluated in the anti-PD-1 plus RT group.
The primary endpoints were the best objective response rate (ORR) and progression-free survival (PFS). Secondary endpoints included safety and disease control rates. PFS was defined as the time between the commencement of anti-PD-1 treatment to the date of progression or death, whichever occurred first.
Statistical analyses
Baseline characteristics and quality-of-life measures were summarized by descriptive statistics and compared using χ2 contingency analyses. The Kaplan-Meier method and logrank test were used to evaluate PFS. In the subgroup analyses, the effect on PFS of the addition of RT to immunotherapy was assessed among the subgroups using Cox proportional hazard models presented in a forest plot. Statistical significance was set at P < 0.05. Analyses were conducted using the Statistical Package for the Social Sciences software package, version 23.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism, version 7.00 for Windows (GraphPad Software).
Patient characteristics and disposition
Between July 2018 and March 2020, 152 eligible patients were retrospectively identified and assigned to the ICI alone group (n = 93) and the ICI plus RT group (n = 59). Figure 1 shows the selection of patients and patient characteristics are summarized in Table 1. The median age of these patients was 62 years (range, 34-81 years), and 128 (84%) were male. There were 39 (42.0%) patients in the ICI alone group and 21 (35.6%) patients in the ICI plus RT group receiving PD-1 inhibitor as first-line therapy. The PD-L1 status was collected in 50 patients. Patient demographics, including Fig. 2B). Among the 60 patients who have received PD-1 inhibitors as first-line treatment, the ORR observed were 38.5% in the anti-PD-1 alone group (n = 39) and 57.1% in the anti-PD-1 plus RT group (n = 21) (Fig. 3A). In the 92 patients receiving PD-1 inhibitors as second-or later-line treatment, the ORRs observed were 25.9% in the anti-PD-1 alone group (n = 54) and 47.3% in the anti-PD-1 plus RT group (n = 38) (Fig. 3B).
We performed an exploratory analysis to determine whether any feature was associated with PFS in the ICI plus RT group. As shown in Supplementary Table 2, we could not identify any clinical variables that were significantly associated with PFS in the univariate analysis; therefore, we did not perform multivariable analyses.
Safety
We conducted a safety evaluation of the ICI plus RT group (Table 3). The overall incidence of AEs was 97% (57 of 59), and most of the observed AEs were grade 1-2 (Table 3). Grade 3-5 treatment-related AEs occurred in nine patients (15%), and one patient died of severe pneumonia. These grade 3-4 AEs were pneumonia (four patients, 7%), bone marrow suppression (four patients, 7%), transaminitis (one patient, 2%), and headache and/ or dizziness (one patient, 2%). Most AEs were clinically manageable, with no new toxicity signals. Objective response rate (ORR) and progression-free survival (PFS) categories by primary tumor RT and brain RT (A) Best ORR in patients received ICI alone vs. primary tumor RT plus ICI. B Kaplan-Meier estimates of PFS in patients for the ICI alone vs. primary tumor RT plus ICI treatment comparison. C Best ORR in patients with brain metastasis received ICI alone vs. RT plus ICI. D Kaplan-Meier estimates of PFS in patients with brain metastases for the ICI alone versus brain RT plus ICI treatment comparison. ICI Immune checkpoint inhibitor 1 3
Discussion
This study reported the efficacy and safety of combining single-site RT and ICIs in patients with oligometastatic NSCLC. Our study showed that the addition of single-site RT to immunotherapy could improve ORR and PFS with acceptable AEs. Of note, this combination therapy enhances the occurrence of out-of-field (abscopal) response. The favorable clinical outcomes were also observed for patients with brain metastases. Subgroup analysis revealed that younger patients, patients with a better physical constitution, patients with fewer metastatic sites, and patients who received ICIs as first-line therapy benefited more from the combined approach.
Although no matched paired analysis was performed due to the relatively limited sample size, strict inclusion and exclusion criteria were followed to avoid potential bias. Our results showed that the clinical features were well balanced between the two groups. To further confirm this conclusion, we also performed a subgroup analysis (including brain metastases and primary tumors irradiation). Therefore, we believe our conclusion is interesting enough to warrant large-scale studies.
Oligometastasis with a small disease burden can be classified as an indolent state between the extensive and locally advanced stages. Despite having a relatively short follow-up time for oligometastatic NSCLC patients in the study, anti-PD-1 monotherapy achieved a median PFS of 8.9 months, with an ORR of 31.2%. This was significantly better than the results of the CheckMate 057 and KEY-NOTE-001 studies in a second-line setting and was also 6 (10) 6 (10) 0 higher than the median PFS of 6.4 months (KEYNOTE-407) in a first-line setting (Borghaei et al. 2021;Herbst et al. 2016;Paz-Ares et al. 2020). This result suggests that a smaller tumor burden might be necessary for increasing the response to immunotherapy. Local therapy for oligometastatic NSCLC has been shown to improve clinical outcomes in multiple clinical trials (Gomez et al. 2016;Qiu et al. 2017;Weickhardt et al. 2012). RT as a primary local treatment provides local control of the irradiated lesion, and when administered in combination with immunotherapy, enhances antitumor response far outside of the radiation, which is known as the abscopal effect. This phenomenon crucially determines the anti-tumor efficiency of the local RT and ICI combination strategy (Ngwa et al. 2018). However, current strategies designed to test the efficacy of the combination strategy cannot optimally achieve abscopal effects through single-site irradiation in metastatic tumors (Kwon et al. 2014;McBride et al. 2021). In our study, we observed a significant response rate of 50.8% and an out-of-field response rate of 41.3%, which was higher than the ICI alone group (31.2%). Such a trial design is selected for oligometastatic patients with a small disease burden and equally immunogenic tumors that may fully activate the patient's immune system. In our subgroup analysis, patients with 1-2 vs. 3-4 metastatic sites benefited more from anti-PD-1 treatment plus RT, which further supports the view of a small disease burden in favor of immune responses. In contrast to oligometastatic disease, the heterogeneity of polymetastases means that tumor-associated antigens exposed to RT might not be present at other unirradiated locations, or, if they are present, they might only be recognized in subgroups of the tumor lesion and not in the entire cellular population, making immune clearance at these other unirradiated locations impossible or greatly limited (Easwaran et al. 2014;Heppner and Shekhar 2014;Sharabi et al. 2015;Spiotto et al. 2016).
Undeniably, more biological and clinical evidence supports the use of comprehensive RT delivered to multiple lesions combined with immunotherapy. Irradiating multiple sites helps to increase the likelihood of exposure to both shared and exclusive tumor-associated antigens and promptly reduce tumor burden (Brooks and Chang 2019). A randomized clinical trial from MADCC assessing the effect of combining pembrolizumab with stereotactic body RT showed an out-of-field response rate of 38%, which was much higher than that of PD-1 monotherapy (Welsh et al. 2020). Overall, using multisite irradiation combined with immunotherapy could be beneficial to achieve better therapeutic outcomes. However, this approach is not being widely tested in clinical trials, most likely owing to the lack of official guidelines or fear of AEs.
RT is a local treatment that acts on both the tumor and the surrounding non-malignant tissues; therefore, the likelihood of a successful immunogenic event is also influenced by the tumor microenvironment, the surrounding tissue or organ, and the nodal characteristics of the irradiated site. For example, irradiation of liver metastases in NSCLC patients has resulted in stronger activation of antitumor immunity than the irradiation of pulmonary metastases (Tang et al. 2017). For liver metastases, our sample size is inadequate for accurate analysis. Additionally, the results of our subgroup analyses showed that patients who derived benefit had good prognostic factors, including young age, good body condition, and have received immunotherapy as first-line treatment. Certainly, these optimizations of the ICI combination with RT still need to be validated in prospective clinical trials.
Nevertheless, our study had several limitations. First, this was a retrospective, single-institution analysis with a small sample size, which may introduce selection bias. For example, the percentage of patients with brain metastases in this study was higher than that reported in other studies. Another weakness of our study is the heterogeneity of treatment, including administering different PD-1 pathway inhibitors and different RT regimens; this represents a significant confounding factor. Third, because of the relatively short followup period, the survival analysis is limited to PFS, and the conclusion should be interpreted with caution. Moreover, the PD-L1 status of most patients in our study was unknown, making comprehensive subgroup analysis difficult. Nonetheless, we still analyzed the available data, despite being limited.
Conclusion
In conclusion, our results support that combining single-site RT with PD-1 inhibitors increased responses significantly and improved clinical outcomes in oligometastatic NSCLC patients. This treatment approach warrants further prospective investigation in a randomized clinical trial. Sciences (2019RU071), the Academic Promotion Program of Shandong First Medical University (2019ZL002) and the foundation of National Natural Science Foundation of China (81972863, 81627901and 82030082).
Availability of data and material
The datasets used in this study are available from the corresponding author on reasonable request.
Conflict of interest
The authors have no confict of interest or potential fnancial disclosures.
Ethics approval This study have obtained appropriate institutional review board approval and have followed the principles outlined in the Declaration of Helsinki for all human investigations. As a retrospective study, formal consent is not required.
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/. | 2021-09-01T15:14:55.129Z | 2021-06-18T00:00:00.000 | {
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244081958 | pes2o/s2orc | v3-fos-license | The Mesoamerican mid-summer drought: the impact of its definition on occurrences and recent changes
The mid-summer drought, veranillo or canícula, is a phenomenon experienced in many areas, including Mexico, Central America, and the Caribbean. It generally is experienced as reduced rainfall in July–August, in the middle of the typical rainy season (May–September). Many past studies have attempted to quantify changes in mid-summer drought characteristics during the recent past or for future climate projections. To do this, objective definitions of a mid-summer drought’s occurrence, strength, and duration have been developed by many researchers. In this effort we adopt a recent set of definitions and examine the impact of varying these on the characterization of mid-summer droughts and the detected changes over the past 4 decades. We find the selection of a minimum intensity threshold has a dramatic effect on the results of both the area considered as experiencing a midsummer drought and the changes detected in the recent historical record. The intensity chosen can affect both the magnitude and direction of changes reported in the recent observed record. Further, we find that the typical mid-summer drought pattern may not be occurring during the time it has historically; whether examining past or future changes or developing improved seasonal forecasts, the non-stationarity of its timing should be accommodated.
Introduction
In many parts of Mexico and Central America (usually on the Pacific slope) there is a well-defined summer rainy season, often marked by early and late peak periods separated by a brief period of reduced rainfall. This reduced rainfall event, which typically persists for 2-4 weeks in July-August, is often referred to as the mid-summer drought (MSD) in the climate science community. In Central America it is referred to by locally distinct names, such as the veranillo or canícula (Magaña et al., 1999;Maldonado et al., 2016). Variability in different characteristics of the MSD is well established (García-Oliva and Pazos, 2021) and can have important agricultural and economic consequences for the region, especially in the area denoted as the Central American Dry Corridor (Hidalgo et al., 2019;Stewart et al., 2021).
While in specific locations an MSD definition may be defined historically using specific dates, such as 15 July-15 August, the regional variability in those dates and their inflexibility for representing change in MSD timing make their use in studies such as ours impractical (Alfaro, 2002;Curtis, 2004;Magaña et al., 1999). In many regions of Central America, the timing and magnitude of the early and late rainy periods are critical for a first and possible second planting season; subsistence farmers who mostly rely on rain-fed agricultural practices must time their planting and harvesting to anticipate the end of the MSD and the arrival of a second peak of rainfall. How the presence of an MSD pattern and its timing, intensity, and duration are affected by climate variability and change therefore is intimately tied to the agricultural cycle and farmer livelihoods.
Because of this regional importance, there have been many studies of the MSD, both examining the recent observational record to detect trends in its characteristics (e.g., Anderson et al., 2019) and looking toward the future to discern what a 1426 E. P. Maurer et al.: The Mesoamerican mid-summer drought: the impact of its definition disrupted climate might produce (e.g., Corrales-Suastegui et al., 2020;Maurer et al., 2017;Rauscher et al., 2008;. When considering either current or future MSD characteristics and metrics to evaluate these, most studies adopt at least some of the methods established by Karnauskas et al. (2013) using monthly gridded data or Alfaro (2014) using daily station data, including the timing, intensity, and duration of the MSD. However, the details in the definitions of what constitutes an MSD pattern and the quantification of MSD characteristics are less consistently defined. Some measures of past changes, as well as future projections, can be significantly affected by subtle changes in definitions of the MSD. For example, the definition of the timing when rainfall minima and maxima need to occur will affect whether a given year or location is counted as experiencing an MSD. In addition, temporal and spatial scales play a role in determining the existence of an MSD pattern. Zhao and Zhang (2021) found the existence of an MSD signal in some locations in Central America and Mexico dependent on whether a method used daily or monthly data.
An understanding of where in the study region an MSD pattern exists, and how it has been impacted by recent climate variability and change, has been elusive. This is at least partly due to limited mathematical descriptions of the phenomenon and to the lack of an exploration of the effects of variation of the parameters used for the determination of whether an MSD phenomenon is present. In addition, any assumption about how frequently an MSD pattern must be identified to declare a given area as being dominated by MSD is arbitrary yet will impact the area considered as having an MSD as well as the area where climate change might have affected the presence of characteristics of the MSD. Therefore, the impact of the mathematical definition as well as that of climatic change on MSD extent must be explored jointly.
A recent study (Anderson et al., 2019) used pentadal precipitation data from the quasi-global CHIRPS dataset, covering Guatemala, Honduras, Nicaragua, and El Salvador. For 1981-2018 they found significant trends in the duration of the MSD in many locations, but most other MSD characteristics did not show discernible trends. As Anderson et al. (2019) note, there may be a disconnect between statistically significant changes in objectively defined MSD conditions and the experience and understanding of the phenomenon by smallholder farmers, especially in the northern part of their domain in Guatemala and Mexico. The importance of extending a study domain of the MSD into more of Mexico is supported by recent studies characterizing its influence in the historical record (Perdigón-Morales et al., 2018) and potential changes in a disrupted climate in the northern, water-limited, and primarily agricultural regions of Mesoamerica (Corrales-Suastegui et al., 2020;Stewart et al., 2021). However, we are not aware of a study that has examined the sensitivity of the MSD spatial and temporal extent to its definition, and the impact the definition has on the presence of changes during the warming trends throughout Cen-tral America over the past 4 decades (on the order of 0.8 • C per decade, Stewart et al., 2021).
In this effort, we build on the past work to improve an objective, mathematical definition of the MSD that includes measures to evaluate the magnitude and timing of the phenomenon and to characterize the variability, trends, and changes in the spatial domain with an MSD pattern during the recent historical record. In particular, we (1) use an expanded domain, as compared to previous studies, that includes Central America and Mexico and that potentially exhibits MSD characteristics, (2) use daily data rather than monthly or pentadal aggregated data to characterize the MSD with finer precision, (3) build on past work to refine definitions of MSD characteristics and spatial extent, and (4) explore the effect of parameter variability in the MSD definition on the magnitude, direction, and changes during the recent observational record , which includes the warmest years in the observational record. Our work is motivated by the need for better understanding of past changes that align with smallholder experience, for seasonal forecasts of specific MSD features, and for projections on how the MSD may change through the 21st century.
Methods and data
We use precipitation-based definitions of the MSD, consistent with many past studies (Alfaro, 2014;Anderson et al., 2019;Karnauskas et al., 2013). The primary data source we use is the gridded daily precipitation product of the Climate Hazards group Infrared Precipitation with Stations (CHIRPS) v.2.0 dataset (Funk et al., 2015), aggregated to 0.25 • (approximately 25 km) as described by Stewart et al. (2021). The data were aggregated to reduce data volumes and facilitate exploration of the influence of different MSD definitions. To verify that this aggregation does not affect the results of this analysis, Fig. A1 and Table A1 show results for a reduced area in Central America using data at both the original CHIRPS resolution and the aggregated resolution, with consistent results at both scales.
CHIRPS is developed by the Climate Hazards Group at the University of California, Santa Barbara, and the US Geological Survey Earth Resources Observation and Science Center. Daily, monthly, and seasonal products are built around blending satellite cold cloud duration observations and improved interpolation techniques of high-resolution, long period-ofrecord precipitation estimates. CHIRPS forms the basis for the US Agency for International Development's Famine Early Warning Systems Network. We use the CHIRTS dataset for the limited temperature analysis in this paper (Funk et al., 2019).
In some recent studies, the inclusion of temperature in the analysis of the MSD has been recognized as important due to the vulnerability of the affected areas to soil moisture (Romero et al., 2020), reflecting the water deficit and warmer temperatures experienced by farmers. This was a motivation in one study for the use of a "hydrologic satisfaction" threshold (MAGFOR, 2010) to define the intensity of an MSD episode. For changes in the recent observed record in the study region, however, the influence of temperature variability on changes in drought indices is much smaller than that of precipitation changes (Stewart et al., 2021). For this reason, we only consider precipitation-based definitions for the MSD, though projections of future changes, when temperature changes will become more pronounced, should consider alternate definitions that include accounting for temperature increases.
To define whether an MSD occurs in any year and quantify its important features, we started with the method of Anderson et al. (2019) and modified it to work with our daily dataset. For each calendar year of daily precipitation data we follow these steps: (1) smooth the data using two passes of a 31 d triangular filter; (2) locate the minimum (which must be an inflection point) between 1 June and 31 August (window 1); (3) check that the minimum from step 2 is also the minimum between 1 May and 31 October (window 2); (4) locate the highest peak between 1 January and the minimum date; (5) locate the highest peak between the minimum date and 31 December; (6) if the two peaks from steps 4 and 5 are not within the 1 May to 31 October period, the year is not an MSD; (7) if those two peaks are not separated by a defined minimum duration (e.g., 15 d), the year is not an MSD; (8) if the average of the maxima minus minimum is not greater than a defined minimum intensity (e.g., 3 mm), the year is not classified as an MSD. The order in which these constraints are applied to the data and the magnitude of the parameter values matter. Figure 1 presents a flowchart illustrating these steps.
Finally, to define whether a location is classified as having an MSD, a threshold is defined for the percentage of years with an MSD according to the definition above. Anderson et al. (2019) set this at 33 out of 38 years (87 %), since they used a 38-year precipitation record . This study uses as a baseline that 80 % of the years must exhibit an MSD for it to be classified as an MSD cell.
Our "original" values for these characteristics are summarized in Table 1. These are designed to reproduce as closely as possible the methods of Anderson et al. (2019). These values are later varied to explore their influence on the extent These original values are adjusted to assess the influence of specific definitions on the determination of whether an MSD exists in a location and whether statistically significant changes have occurred over the recent historical record.
Statistical tests consist of comparing the proportions of MSD years in a 20-year period using Fisher's exact test (Mehta and Patel, 1983) and comparing the central tendency of statistics between two 20-year groups using a Wilcoxon (Mann-Whitney) signed-rank test (Helsel et al., 2020). Statistical significance is evaluated at a 5 % level (α = 0.05).
Study area
The Mesoamerican region of Central America and Mexico is a region with very distinct but spatially highly variable climatic patterns. Figure 2a shows the climatological precipitation pattern across the study domain. The CHIRPS data in this figure have been aggregated spatially and temporally (to monthly averages). Figure 2a illustrates the tremendous variability in climate that exists in the region, from wet tropical climates to cold arid regions in Mexico's highlands. Despite these differences, many regions are characterized by a highly seasonal climate, with a pronounced spring dry season followed by a summer (June-September) rainy season with monthly precipitation of 400-500 mm or more. A clear dip in July-August precipitation associated with an MSD pattern is apparent in many of the grid boxes of the domain. There are parts of Mexico and the Caribbean, regions excluded from many prior MSD studies, which have exhibited the canonical MSD pattern in the past (Perdigón-Morales et al., 2018). While warmer temperatures during the summer months prevail for the northern parts of the study area, the seasonal cycle of temperature is muted for areas closer to the Equator and/or to the coast, as would be expected, and with a few exceptions for high-elevation regions in the domain, temperatures remain well above freezing throughout the year. In Fig. 2b the precipitation changes between the early (1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000) and late (2001-2020) records vary widely across the domain. Particularly in Honduras, Nicaragua, and Costa Rica a decline is evident in precipitation in the wet season, including the July-August period of the MSD. Warming on the order of 1-2 • C has generally taken place throughout the domain. Observed temperature increases are variable month to month, though changes are broadly positive and statistically significant, both seasonally and annually. Less significant warming is observed in September-November ( Fig. 2b; Stewart et al., 2021).
Results
Applying our original definition of the MSD as defined in Table 1 and Fig. 1 yields Fig. 3.
In Fig. 3, two-thirds of the grid cells with statistically significant changes in the number of years with an MSD are locations that are dominated by an MSD in the early period Table 1. Aside from some areas on the Caribbean side of Mexico where MSD years have become more frequent, most of the significant changes are concentrated in the southern part of the domain (especially Panama), suggesting a change in precipitation seasonality in southern Central America. This is explored further below.
Summing MSD presence over the early and late periods of study results in Fig. 4, which (by design) closely resembles that of Anderson et al. (2019). Figure 4 shows that most of the pixels that are classified as experiencing an MSD pattern exhibit this throughout the past 4 decades. Expansion of the area with an MSD occurs in the northern part of the domain, mostly in Mexico's Yucatan Peninsula. Areas with MSD in the early period but not in the later period appear generally toward the southern part of the domain, especially evident in Panama. More intense drying of the northern part of Central America, with intensification of the MSD, has been identified as potentially indicative of a southern shift in the summer location of the Intertropical Convergence Zone (ITCZ) (Rauscher et al., 2011;Hidalgo et al., 2013), also an anticipated impact of climate disruption on this region (Rauscher et al., 2008). The regions classified as MSD in the late but not early period also coincide largely with the areas showing significant trends for greater MSD intensity (Fig. A2), due in part to an increase in the magnitude of the precipitation peaks, especially the first peak (Figs. A3 and A4), and a decline in the intervening minimum (Fig. A5).
While Mexico and Central America are often the focus of MSD studies, our analysis using consistent criteria demonstrates that the phenomenon is also widely present in the Caribbean, as shown by others (e.g., Almazroui et al., 2021), though the driving mechanisms for the MSD are distinct from the rest of the domain (Curtis and Gamble, 2008). For the portions of the Caribbean included in Fig. 4 there are few areas showing a tendency for increased MSD occurrence, (Table 1 and Fig. 1). Shading indicates pixels with an MSD for the early (1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000), late (2001-2020), or both periods. Also shown are specific points used in subsequent examples or discussion, selected to show a variety of MSD characteristics and different changes between the early and late periods. though many do see a continuing MSD classification for both the early and late periods.
For illustration of the types of details encountered by the classification scheme in any year, Fig. 5 shows for different locations (identified in Fig. 4) a sample of several time series of precipitation, after applying the smoothing described above. This also shows the outcome of applying the criteria as to whether an MSD exists in the year depicted. Figure 5 shows that, in most cases, even after smoothing the precipitation signal remains noisy. While the canonical (Table 1 and Fig. 1). Key time windows from Table 1 are indicated by vertical lines: blue lines correspond to window 1, in which a minimum is identified, red lines to window 2, in which peaks must occur. Red dots indicate first and second maxima and blue dots mark the minimum if they meet MSD criteria. Examples shown are (a) a canonical MSD pattern at point 4, (b) no minimum in window 1 at point 6, (c) a high minimum at point 5 but still an MSD, (d) insufficient intensity at point 5, (e) an early peak outside window 2 at point 6, (f) a second peak outside window 2 at point 6, (g) a lower minimum occurring in window 2 at point 5, and (h, i) a high variation in peaks at point 5.
MSD pattern (Fig. 5a) is what is often depicted in the literature (e.g., Anderson et al., 2019;Karnauskas et al., 2013), MSD years can have a wide variety of shapes in the precipitation record (Fig. 5c, h, and i). Years that might appear to be an MSD may fail one or more criteria (Fig. 5d, f, and g). These examples highlight the potential sensitivity of MSD classification to the details of its definition. Similar problems can arise when defining the MSD using alternative methods or even for the date of onset or demise of the rainy season. Several criteria have been developed for these two purposes, with advantages and drawbacks (e.g., Alfaro, 2014;Maldonado et al., 2016;Bombardi et al., 2017).
While Fig. 6 shows relatively subtle changes in average precipitation between the 20-year periods at all three points, underlying these are more systematic changes that affect the MSD classification. Point 1 does not show an MSD signal at all in the average precipitation for either period in Fig. 6a. This is because the typical pattern of precipitation has a single larger peak falling near the center of the 1 June-31 August window (in which a search is done for a minimum), with the minimum occurring closer to the extreme dates of this window and occurring with equal frequency before and after the larger peak, similarly to Fig. 5h and i. Point 1 shows an overall reduction in precipitation for the later period, especially from June through October. The declining precipitation produces smaller peaks resulting in a declining intensity (in years classified as an MSD), from 4.8 mm in 1981-2000 to 3.7 mm in 2001-2020, reducing the number of years satisfying the MSD criteria from 16 of 20 years in 1981-2000 to 9 in 2001-2020.
The average precipitation pattern for point 2 shows a more typical MSD pattern for both periods. Similarly to point 1, the 1981-2000 period is classified as having an MSD, while 2001-2020 is not. However, the changes are much more subtle, with the MSD years having nearly the same intensity for both periods (7.9 and 8.1 mm d −1 for the early and late periods, respectively). At point 2, Fig. 6b shows the shift of the second peak to slightly later in the season, which is the important change at this location. For 1981For -2000 of 20 years are classified as MSD, while 2021-2020 has 15 years of MSD, falling just below the threshold of 16 years required for an MSD location. In every case for both periods, the cause of a year not being an MSD is the second peak slightly outside the 1 October window required by the definition. Thus, at this location it is the timing of the second rainfall pulse that changes the MSD classification.
Similarly to point 2, point 3 (Fig. 6c) shows an average shift in the second precipitation peak to later in the season Table 2. Columns (a)-(c): total number of MSD grid cells (percent change from original in parentheses) with different values of criteria used to define an MSD for the early period (1981)(1982)(1983)(1984)(1985)(1986)(1987)(1988)(1989)(1990)(1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000), late period (2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020), and both periods. Changes in the total number of MSD pixels between the periods are shown in column (d). the dominant cause of failing to meet the MSD criteria is peak precipitation occurring outside of the established MSD windows, often in December-January. There is no evident reduction in average rainfall during December-January in Fig. 6c, nor is there a significant reduction at this location in December-February rainfall detected in a prior study (Stewart et al., 2021), so the effect is limited to peak events, but it has a strong impact on MSD classification. As the values of the different criteria for classifying an MSD vary, there are changes in the number of MSD grid cells and where the MSD occurs. Table 2 provides a summary of how the number of grid cells varies, and the following figures illustrate changes in the location of the MSD.
Since the precipitation values are smoothed with a 31 d filter, durations shorter than this have no effect on the classification of MSD grid cells. Thus, while we imposed a minimum 15 d duration as our original definition, changing this to 30 d would have no effect on results. Imposing a stricter requirement for longer durations reduces the number of MSD grid cells by about 12 % for durations of up to 50 d but does not substantially change the differences in MSD extent between the two periods from the original. The general insensitivity of MSD classification to the minimum duration is consistent with the majority of significant trends in duration being positive (Fig. A6) and focused on grid cells on the Pacific side that are classified as MSD cells for both periods. Duration definitions of 60 and more days unsurprisingly reduce the number of cells exhibiting an MSD pattern by 30 % and more. An MSD of that length is also inconsistent with the nature of the phenomenon as described by smallholder farmers and prior studies. Figure 7 shows the effect of varying the intensity criterion of the original MSD definition (3 mm) between 1 and 5 mm. Allowing the low intensity threshold for an MSD classifies nearly the entire domain as having an MSD, with the exceptions being only along the Caribbean side of Central America and most of Colombia. Requiring a more extreme 5 mm intensity for an MSD classification limits zones with MSD to a relatively thin band along the Pacific side of Central America. This larger intensity threshold excludes some areas, such as northern Nicaragua, where the MSD is a well-known phenomenon, indicating that a higher intensity threshold may not be appropriate. Figure 7 also shows the same spatial pattern of changing MSD grid cells as Fig. 4, with isolated areas in the northern part of the domain changing from not experiencing an MSD to being classified as an MSD in the latter half of the study period. Figure 8 highlights the changes between 1981-2000 and 2001-2020 in total MSD grid cells for the domain. The highest threshold isolates only those grid cells that experience the most intense MSD and also reveals an increase in MSD area. Thus, areas that have historically experienced lower-intensity MSD events have contracted in spatial extent over the last 4 decades. Conversely, areas with historically high-intensity MSD events have expanded.
As was illustrated in Fig. 5, peaks or minima can fall outside of the defined windows by only a day or two and cause a year to not be classified as an MSD. To explore this, we varied the dates of the windows by shifting them all uniformly 2 weeks earlier and 2 weeks later. The results are shown in Table 2 and Fig. 9. By shifting the dates earlier (Fig. 9a) there is a dramatic reduction in the area with an MSD, and the later period sees a steeper reduction, increasing the magnitude of the reduction in MSD area between the two periods. Shifting the dates later (Fig. 9b) increases the area classified as having an MSD for both periods, with 3 times the increase in area from the early to the later period compared to shifting the dates earlier. This indicates that a more extensive MSD exists later in the season in general and that there has been a shift in the last 40 years toward a later MSD.
Finally, the effect of modifying the number of years any grid cell must have an MSD to be classified as a location with an MSD is shown in Table 2 and Fig. 10. Adopting a looser (70 %) or stricter (90 %) requirement for an MSD grid cell changes the extent but has little effect on the spatial patterns during each period or the changes between the two periods.
Discussion and conclusions
The Mesoamerican MSD is typically defined by a set of precipitation characteristics. As studies explore the existence of historical trends or future projections of characteristics of the MSD, understanding the impact of decisions regarding the MSD definition is essential since these can affect results. We found that seasonal variability can cause individual years with detectable MSD signals to be indiscernible in a clima- tological average, highlighting the importance of assessing individual events for the presence of an MSD.
We examined the four precipitation characteristics defining the dry period between two peaks, centered in July and August: duration (the time between peaks), intensity (the level of decline between the two peaks), the timing (the dates defining windows within which the minimum and peaks must occur), and consistency (the percentage of years with a defined MSD occurring). Of these four, the two with the greatest impact on results were intensity and timing.
The application of a minimum intensity has a dramatic effect on the results of both the area considered as having an MSD and the changes in the recent historical record. Our results suggest that the intensity chosen can affect both the magnitude and direction of changes in the recent observed record. The regions with MSD of greatest intensity show a net increase in area, while areas with a characteristically lower-intensity MSD are decreasing in extent. This may reflect the increases in more extreme precipitation levels in the region, resulting in an intensified MSD, something projected as the climate continues to warm (Maloney et al., 2014;. The original timing established for defining the Mesoamerican MSD definition was that a minimum precipitation should occur in the 1 June-31 August window and that a peak inflection should exist on either side of it within the 1 May-31 October window. Shifting these dates 2 weeks earlier dramatically reduced the area with an MSD, and shifting them 2 weeks later increased the area. In addition, shifting the dates in either direction had a strong influence on the observed change in MSD extent between 1981-2000 and 2001-2020, suggesting a change in precipitation timing to later in the year, so the typical MSD pattern may not be occurring during the time it has historically. MSD timing, and its accurate prediction, is a challenge that could benefit socio-economic sectors throughout the study region (Alfaro et al., 2018). Thus, whether examining past or future changes in MSD or developing improved seasonal forecasts, the non-stationarity of MSD timing should be accommodated.
These results suggest that for studies of historical or future changes in MSD for this region, studies should be conducted for different levels of MSD intensity and timing to capture differing impacts as these characteristics vary across the domain. A greater understanding of the impact of the objective definition of the MSD on changes in the timing, intensity, and frequency of occurrence of the MSD pattern, and their relative importance to smallholder agriculture, may support assessments of climate change impacts connected to the MSD and the development of adaptation strategies.
Code and data availability. All code was written in the programming language R. A documented package with these functions is under development. All data sets used in this work are publicly available as detailed in the references cited in the Methods and Data section.
Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests.
Disclaimer. Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. | 2021-11-14T16:28:43.692Z | 2021-11-12T00:00:00.000 | {
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238715626 | pes2o/s2orc | v3-fos-license | Genetic propensity for obesity, socioeconomic position, and trajectories of body mass index in older adults
Identifying how socioeconomic positioning and genetic factors interact in the development of obesity is imperative for population-level obesity prevention strategies. The current study investigated whether social positioning, either independently or through interaction with a polygenic score for Body Mass Index (BMI-PGS), influences BMI trajectories across older adulthood. Data were analysed from 7,183 individuals from the English Longitudinal Study of Aging (ELSA). Interactions between the BMI-PGS and; lower educational attainment, self-perceived social status (SSS), and income, on BMI trajectories over 12 years across older adulthood were investigated through linear mixed effects models. Lower educational attainment, SSS and income were each associated with a higher baseline BMI for women, but not for men. There were interaction effects between BMI-PGS and social positioning such that men aged > 65 with a lower educational attainment (β = 0.62; 95%CI 0.00 – 1.24, p < 0.05), men aged ≤ 65 of a lower income (β = − 0.72, 95%CI − 1.21 - − 0.23, p < 0.01) and women aged ≤ 65 of lower SSS (β = − 1.41; 95%CI − 2.46 – 0.36, p < 0.01) showed stronger associations between the BMI-PGS and baseline BMI. There were few associations between markers of socioeconomic position and rate of change in BMI over the follow-up period. In sum, lower socioeconomic positioning showed adverse associations with women’s BMI in older adulthood. Moreover, the expression of the BMI-PGS, or extent to which it translates to a higher BMI, was subtly influenced by socioeconomic standing in both women and in men.
The prevalence of obesity, defined in adulthood by a body mass index (BMI) ≥ 30 1 , is associated with numerous adverse health implications in older age. These include an increased risk for diabetes 2 , cardiovascular diseases 3 , hypertension and even mortality 4 . This relationship is anticipated to further increase as the general population continues to age, with associated costs of obesity being estimated to reach £49.9 billion per year by 2050 5 .
The distribution of obesity is unequal across socioeconomic positions (SEP) 6 , with rates being higher among those with lower education 7 , income 8 or subjective social class 9 . Obesity rates demonstrate a strong gradient in the UK as result of a greater exposure to the obesogenic environment and more limited opportunities for adequate nutrition and physical activity across the lifespan 10 . While education, self-perceived social standing (SSS) and income are correlated, they reflect different aspects of SES at older ages 11 . Education is typically completed in early life and shapes occupational trajectories; while income is an indicator of economic resources that influences opportunities for mobility, food choice, and access to exercise facilities relevant to adiposity in later life 12 . Moreover, there is evidence to suggest that women's BMI in adulthood appears to be more adversely affected by lower socioeconomic settings than in men 6 . However, it is less well understood how BMI may be differentially influenced by specific markers of SEP between the sexes, potentially highlighting more specific and fruitful targets for obesity prevention efforts in older adulthood.
Nonetheless, obesity has a strong genetic basis, with an estimated heritability ranging from ~ 40 to 70% 13 . To uncover the molecular mechanisms underlying BMI, genome-wide association studies (GWAS) have been successful in identifying hundreds of significant loci associated with BMI, which together are shown to have a substantial additive influence on BMI 14 . These GWAS have led to the development of the polygenic score approach, which represents an aggregate measure of polygenic risk for BMI by exploiting all loci associated with Method Sample. Data were drawn from the English Longitudinal Study of Aging (ELSA), a nationally representative survey of English adults aged 50 years or older 23 . Measures of socioeconomic position and covariates were taken from wave 2 (2004-2005) for the core sample (82%), or waves 4 (2008-2009) and 6 (2012-2013) for the respective refreshment samples (18%). Baseline measures of BMI were taken from wave 2 (2004-2005) for participants who provided blood samples for genotyping at wave 2 (77%), or wave 4 for those who provided blood samples at wave 4 (23%). Follow-up BMI measures were obtained from waves 6 (2012-2013) and 8 (2016-2017). Ethical approval for each ELSA wave was granted by the National Research Ethics Service (London Multicentre Research Ethics Committee). All participants gave informed consent, and all experiments were performed in accordance with relevant guidelines and regulations. Study variables. Body mass index (BMI). BMI was calculated using standard formulae (weight in kilograms/height in square meters) 22 . Here, height and weight were measured during the nurse visit. Weight was measured using Tanita electronic scales to measure body weight without shoes and in light clothing. Height was determined by Stadiometer using the Frankfort plane on a ground level. At Wave 8, BMI was calculated using height measurements obtained from Wave 6, as no height measurements were obtained in 2016-2017.
Measures of socioeconomic position. Educational attainment. Educational Attainment was measured through self-reported highest educational qualification. Respondents were asked to self-report their highest educational qualification or attainment using computer-assisted interviewing, from a list response option including: degree level qualifications, teaching qualifications, nursing qualifications, A-levels or higher school certificate, O-level qualifications, GCSE level graded, NVQ qualifications, apprenticeship level, other qualifications, or none. Responses were derived into three categories: (1) Higher Qualification (undergraduate or postgraduate degree level), (2) Secondary Qualification (A/O or GSCE level or equivalent), (3) Primary Qualification (Below A/O/GSCE or no qualification).
Subjective social status (SSS). SSS was measured through the MacArthur Scale of Subjective Social Status 24 . This measure presents a drawing of a ladder with 10 rungs to respondents, representing where people stand in society, the higher up representing those with the most money, education, and jobs. Respondents were asked to place a single "X" on this ladder to rank their social standing, producing a score from 1 to 10, with 10 being the highest SSS. These raw scores were derived into tertiles: (1) Top Tertile, (2) Middle Tertile, (3) Lowest Tertile of SSS.
Income. Income was measured through self-reported household equivalised income, adjusted for variation in household size. Income was calculated from detailed assessments of a full range of earned and unearned sources of income. Earned sources of income included employment income, self-employment income, state pension income, and other benefit income; while unearned sources on income included income from assets, investments and financial transfers 25 . The income variable was divided into tertile to represent the groups of individuals with (1) High, (2) Intermediate and (3) Low Tertiles of Income.
Covariates. Demographic covariates included marital status (not currently married vs currently married) derived from a single item asking participants to disclose their current legal marital status as; single, married, remarried, legally separated, divorced, or widowed. Behavioural covariates included smoking status (not-cur- www.nature.com/scientificreports/ rent smoker vs current smoker) derived from one item asking participant whether they smoke cigarettes at all nowadays (yes vs no). Secondly, physical activity level (sedentary or low activity at least once a week, moderate activity at least once a week, vigorous activity at least once a week) was also included in the models, and derived through three separate items reporting on the frequency of either vigorous, moderate, or mildly energetic sports and activities; more than once a week, once a week, one to three times a month, or hardly ever or never 26 . These responses were categorised according to their highest level of activity reported at least once a week. Health related covariates included the presence of a longstanding illness (illness reported vs. no illness reported) reported through a single item; Do you have any long-standing illness, disability, or infirmity? (yes/no) and depressive symptomatology (current depressive symptoms vs no current depressive symptoms). Depressive symptoms were measured with an 8-item version of the Centre for Epidemiologic Studies Depression Scale 27 , which has comparable psychometric properties to the full 20-item scale; a score ≥ 4 was used to define participants with severe depressive symptoms 28 . Lastly, genetic ancestry measured with principal components (see below), was included as a covariate (four principal components) to account for any ancestry differences in genetic structures that could bias our results 29 .
Genetic data. The genome-wide genotyping was performed at University College London Genomics in 2013-2014 using the Illumina HumanOmni2.5 BeadChips (HumanOmni2.5-4v1, HumanOmni2.5-8v1.3), which measures ~ 2.5 million markers that capture the genomic variation down to 2.5% minor allele frequency (MAF). Samples were removed based on call rate (< 0.99), suspected non-European ancestry as identified though principal components analysis and self-identification, heterozygosity, and relatedness. Specifically, to investigate population structure, principal components analysis (PCA) 29 in PLINK 1.9 30 was conducted 21 . An inspection of PCA highlighted the presence of ancestral admixture in the 65 individuals. We removed these outliers and recalculated PCs using the updated samples; here, top 10 principal components were retained to account for any ancestry differences in genetic structures that could bias results 31,32 . Duplicated samples and cryptic relatedness between each pair of participants was evaluated using pairwise genome-wide estimates of three coefficients corresponding to the probabilities of sharing 0, 1 or 2 alleles between two individuals that are identical by descent 33 . We used the method of moments for estimating the identical by descent (IBD) probabilities 34 implemented in PLINK 30 1.9. IBD were estimated using autosomal SNPs where IBD = 1 highlights presence of duplicates or monozygotic twins, IBD = 0.5 shows that first-degree relatives are present in the sample, IBD = 0.25 and IBD = 0.125 highlights presence of second-degree and third-degree relatives, respectively 35 . We identified individuals with an IBD value of > 0.2 and excluded one of each pair at random 36 . Single Nucleotide polymorphisms (SNPs) were excluded if they were non-autosomal, the minor allele frequency was < 0.01%, if more than 2% of genotype data were missing and if the Hardy-Weinberg Equilibrium P < 10 −4 .
Polygenic score (PGS). To calculate PGS for BMI (BMI-PGS), we used summary statistics reported by the Genetic Investigation of Anthropometric Traits (GIANT) consortium (2018) 14 . BMI-PGS were calculated as a weighted sum of the allele dosages, summing over the markers abiding by the p-value threshold (P T ) (i.e., 0.001, 0.01, 0.05, 0.1, 0.3, and 1) weighted according to the strength of effect estimate were summed in a continuous score using PRSice. Using information on sample size (n), total number of independent markers in genotyping panel (m) and lower and upper Pvalues to select markers into polygenic score we estimated the predictive accuracy (R 2 ) P T = 0.001 (m = 255,091), P T = 0.01 (m = 114,862), P T = 0.05 (m = 62,583), P T = 0.1 (m = 194,940), P T = 0.3 (m = 412,954), and P T = 1 (m = 798,737) we estimated a predictive power of each PGS using Avenge me package implemented in R 37,38 .. Consequently, we estimated predictive accuracy for each PGS at P T = 0.001 (R 2 = 0.001, P = 0.004), P T = 0.01 (R 2 = 0.001, P = 0.035), P T = 0.1 (R 2 = 0.03, P = 3.83 × 10 -6 ), P T = 0.05 (R 2 = 0.002, P = 1.31 × 10 -5 ), P T = 0.03 (R 2 = 0.001, P = 0.003) and P T = 0.1 (R 2 = 0.001, P = 0.014) had sufficient, as indicated by significant P values, predictive accuracy to be employed in the analyses. As previously a large comparative study showed that a PGS at p value thresholds P T = 1 was the ultimate PGS to use in longitudinal studies 39,40 , we utilised PGS that was based on P T = 1 assuming all genetic markers contribute to trait development.
Statistical analyses.
To assess the interplay between BMI-PGS with socioeconomic position on BMI values at baseline and across the 12-year follow up period, we employed linear mixed effect models (LMMs) with maximum likelihood estimation 41 . LMMs with maximum likelihood estimation maximise the use of longitudinal data, adjust for the correlation between repeated measures, weight estimates for missing data between waves, and increase statistical power and precision 41 . Using Akaike Information Criterion and Bayesian Information Criterion, a quadratic model allowing for random intercepts and slopes was deemed most appropriate for our analyses. To test whether variation in BMI across SEP influenced the model results, heteroscedascity assumptions was examined, and where heteroscedascity was present, models used robust standard errors, using the vce(robust) command in STATA, relaxing the assumption that standard errors carry identical and equal distributions 42 . Interactions between BMI-PGS and all three measures of socioeconomic position were investigated using multiplicative models. Each analysis was stratified by gender and age group (i.e., < 65 years old vs > 65 years old). We used a significance level of 0.05 (two-tailed) for all analyses. All analyses were conducted in STATA release 16 (STATA Corp LP, USA) 43 .
Sensitivity analyses.
In the sensitivity analyses we repeated all analyses as described above but with missing values imputed for both socioeconomic position (educational attainment, SSS, and income) and all covariate measures using MissForest in RStudio version 44
Results
Sample characteristics. The total sample consisted of 7183 ELSA participants for whom the quality-controlled genome-wide genotyping and BMI during the follow-up were available; of these 46% (N = 3304) were men and 54% (N = 3879) were women. The baseline mean age for men was 64.40 (standard deviation (SD) = 9.15) and for women was 64.35 (SD = 9.56). A larger proportion of men (74.88%) than women (56.17%) reported a longstanding illness (x 2 = 6.11, P = 0.011); whereas a larger proportion of women (34.37%) than men (21.35%) showed elevated depressive symptoms (x 2 = 148.59, P < 0.001). Men and women differed further in terms of marital status, level of physical activity, income, and educational attainment all reported at baseline (Table 1).
Educational attainment and BMI-PGS in relation to BMI trajectories.
As compared to the group with a higher qualification, having a primary qualification was associated with higher BMI at baseline for women aged ≤ 65 years old (β = 1.25; 95%CI 0.64 -1.85) ( Table 2), and women aged > 65 years old (β = 1.04; 95%CI 0.35 -1.72) and men aged > 65 years old (β = 0.52; 95%CI 0.02 -1.07) ( Table 2). While having a secondary qualification was only associated with a higher BMI at baseline for women aged ≤ 65 years old (β = 1.02; 95%CI 0.45-1.60). Regarding interaction effects, a 1-SD increase in BMI-PGS was associated with a higher baseline BMI of 0.62 points in men aged > 65 of a secondary education as compared to those of a higher education (β = 0.62; 95% CI 0.00-1.24) (Fig. 1). For rate of change in BMI, in men aged ≤ 65 years, a secondary (β = 0.06; 95%CI 0.02 -0.10) and primary qualification (β = 0.06; 95%CI 0.01 -0.11) was associated with a steeper increase in BMI across the 12-year follow up than that found in the higher qualification group. (Fig. 2). In both men and women aged > 65 years old, there was no association between SSS and baseline BMI (Table 3). There was an interaction effect between BMI-PGS and SSS on baseline BMI for women aged ≤ 65 years old, such that a 1-SD increase in BMI-PGS was associated with lower There was an interaction effect between BMI-PGS and income for BMI at baseline for men aged ≤ 65, such that a 1-SD increase in BMI-PGS was associated with a lower baseline BMI value of 0.72 points for men in the lowest tertile of income but not in the highest (β = − 0.72, 95%CI − 1.21 to − 0.23) ( Table 4). There were no significant direct effects or interaction effects of income on the rate of change in BMI over the 12-year follow up for either men or women.
Sensitivity analyses.
After mutiple imputation for missing data, we observed that as compared to a higher qualification, men with a primary level education had a higher BMI at baseline in both the > 65 age group (β = 0.51; 95%CI 0.08-1.11) and ≤ 65 age group (β = 0.49; 95%CI 0.00-0.98) (Supplementary Table 3). For SSS, the interaction between BMI-PGS and the bottom tertile of SSS on rate of change in BMI in men aged > 65 uncovered in the main analyses was attenuated towards null. Moreover, women aged ≤ 65 and in the lowest tertile of SSS, no longer showed higher BMI at baseline (Supplementary Table 4). For income, women aged ≤ 65 Table 2. Adjusted longitudinal mixed models exploring the main effect of polygenic score for BMI (BMI-PGS) and educational attainment, and interactions between these two variables in relation to BMI trajectories during the 12-year follow-up period. The adjusted models were adjusted for 4 principal components to account for any ancestry differences in genetic structures that could bias the results, as well as; marital status, physical activity level, presence of longstanding limiting illness, self-reported health, depressive symptoms, and smoking status. CI, confidence intervals; PGS, polygenic score; BMI, body mass index. a The withinperson variance is the overall residual variance in cognition that is not explained by the model. www.nature.com/scientificreports/ www.nature.com/scientificreports/ and in the mid and lower income tertiles did not show higher BMI at baseline as in the main analyses (Supplementary Table 5).
Discussion
To our knowledge, the present study is the first to investigate GxE interactions between an aggregate measure of genetic risk for BMI and three dimensions of socioeconomic position the rate of change in BMI across older adulthood. Consistent with previous findings 15, 45 , our results showed that a higher BMI-PGS was associated with higher baseline BMI (~ 64 years of age) in both men and women. However, contrary to our second hypothesis, BMI-PGS was not significantly associated with a higher rate of change in BMI during the 12-year follow-up period. These results may imply that polygenic factors that contribute to BMI variation in mid-to-older adulthood may differ from those which influence BMI fluctuations at older ages. Consistent with previous findings, our results demonstrate that lower levels of educational attainment, SSS and income were associated with higher baseline BMI more so in women than in men 46 . Moreover, males with lower SSS and incomes even showed lower BMI values at baseline. Hence, women in mid-to-late adulthood may be more exposed to the limited opportunities for physical activity and lower quality diet present in lower socioeconomic settings than are men, and therefore show a stronger social gradient in BMI outcomes than men of the same social standing 47,48 . For instance, the well reported 'gender pension gap' or the tendency for women in the UK to enter retirement with a lower private pension wealth and income retirement than men across the social gradient 49 , may result in greater obesogenic exposures for women in later adulthood. With fewer resources and larger occupational demands in later life, women may be exposed to a greater risk of adiposity development across older adulthood than men of a similar social position 50 . Nonetheless, a reverse causal effect between obesity Table 3. Adjusted longitudinal mixed models exploring the main effect of polygenic score for BMI (BMI-PGS) and subjective social status, and interactions between these two variables in relation to BMI trajectories during the 12-year follow-up period. The adjusted models were adjusted for 4 principal components to account for any ancestry differences in genetic structures that could bias the results, as well as; marital status, physical activity level, presence of longstanding limiting illness, self-reported health, depressive symptoms, and smoking status. Adjusted models used robust standard errors to relax the assumption that standard errors carried identical and equal distributions, due to the presence of heteroscedascity. CI, confidence intervals; PGS, polygenic score; BMI, body mass index. a The within-person variance is the overall residual variance in cognition that is not explained by the model. The initial status variance component is the variance of individuals' intercepts about the intercept of the average person. The rate of change variance component is the variance of individual slopes about the slope of the average person. ×Represents an interaction between the two factors; interactions are presented based on multiplicative interaction model. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. www.nature.com/scientificreports/ and labour market outcomes may be present, as findings have consistently shown that developing obesity influences a women's labour market outcomes, and hence income and SSS, to a greater extent than men 51,52 . A novel finding was also that SSS was only associated with baseline BMI in adults aged 65 or less. It has been proposed that those who perceive themselves to have fewer social and economic lower resources may be more exposed and more susceptible to the obesogenic environment 20 . Hence, as younger populations have developed in the context of a more obesogenic environment, the influence of self-perceived resources might therefore be stronger in younger age groups 20 . This finding highlights how BMI inequalities may vary across specific age ranges, and future investigations may benefit from exploring social gradients across both gender, SEP measures, and varying stages of the life course. Three GxE interactions between socioeconomic positioning and BMI-PGS were observed. First, the BMI-PGS showed a stronger association with baseline BMI in men (aged ≤ 65) with a secondary qualification than those with a higher qualification. Hence, a lower educational attainment may accentuate genetic risk for BMI as less education may place individuals within more obesogenic environments where opportunities to express underlying genetic risk are more pervasive 17,21 . Similarly, we further observed that in men of a lower income (aged ≤ 65) a higher BMI-PGS scores were associated with higher baseline BMI values, as compared to those in a higher income group. Finally, for women aged ≤ 65 or younger, a higher BMI-PGS was associated with a lower baseline BMI only for those in the lowest SSS tertile. Together, these findings might suggest that women's expression of polygenic risk towards a higher BMI is more influenced by subjective measures of social standing than tangible levels of education or income. Nonetheless, while these findings provide evidence that the expression of polygenic predisposition may be sensitive to the socioeconomic environment, it is noteworthy that, similar Tyrell et al 21 , the present GxE interactions produced smaller effect sizes than the direct effects of socioeconomic status on BMI.
Strengths and limitations.
In the present study, we analysed a large population-based cohort who are representative of older adults in England. Confidence in these findings is also strengthened by using LMMs, which are an optimal way to describe the changes in continuous dependent variables over time taking into Table 4. Adjusted longitudinal mixed models exploring the main effect of polygenic score for BMI (BMI-PGS) and income, and interactions between these two variables in relation to BMI trajectories during the 12-year follow-up period. The adjusted models were adjusted for 4 principal components to account for any ancestry differences in genetic structures that could bias the results, as well as; marital status, physical activity level, presence of longstanding limiting illness, self-reported health, depressive symptoms, and smoking status. CI, confidence intervals; PGS, polygenic score; BMI, body mass index. a The within-person variance is the overall residual variance in cognition that is not explained by the model. The initial status variance component is the variance of individuals' intercepts about the intercept of the average person. The rate of change variance component is the variance of individual slopes about the slope of the average person. ×Represents an interaction between the two factors; interactions are presented based on multiplicative interaction model. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. www.nature.com/scientificreports/ account intra and inter-individual variation. Moreover, the sample utilised in the present study was appropriate for evaluating the stated hypotheses as it was substantially larger or similar in size to samples used in previous work 19,20 . Nonetheless, given the observational nature of this study, we cannot infer causality or eliminate the role of residual confounding. It is feasible that PGS utilised in the present study, having encompassed hundreds to thousands of common variants, may have accumulated noise which masks the true associations with changes in BMI over time 16 . Moreover, the poor generalizability of genetic studies across populations is noteworthy as PGSs are predominately based on in European participants 40 . Moreover, as GWASs do not, by design, capture other structural variants beyond SNPs such as rare variants, poorly tagged or multiple independent variants, G × G interaction, epigenetics and gene-environment correlation 53 . Moreover, to avoid overfitting the present GxE models we also were unable to adjust our analyses for interactions between the covariates and the present BMI-PGS and SEP variables, as advised by Keller et al. 53 Finally, the use of height data from Wave 6 (2012) to calculate BMI at wave 8 (2016) may have affected the validity of the final follow-up BMI measures.
Conclusion
The BMI-PGS was associated with higher BMI at baseline (~64 years old) but not with the rate of change in BMI over the 12-year follow-up period. Moreover, women's BMI appeared to be more adversely affected by lower education, lower SSS, and less income than men's BMI. Crucially, the current results highlight the potential for educational attainment, SSS, and income to influence BMI in adulthood through interaction with a BMI-PGS, although effect sizes were small. Taken together, lower socioeconomic positioning may adversely influence BMI in adulthood both independently and through accentuation of genetic risk. However, further research must clarify the extent to which cumulative measures of socioeconomic conditions may influence the expression of genetic propensity towards a higher BMI. www.nature.com/scientificreports/ | 2021-09-27T18:43:27.688Z | 2021-08-17T00:00:00.000 | {
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201172262 | pes2o/s2orc | v3-fos-license | YDJC Induces Epithelial-Mesenchymal Transition via Escaping from Interaction with CDC16 through Ubiquitination of PP2A
Lung cancer is the number 1 cause of cancer-related casualties in the world. Appropriate diagnostic markers and novel targets for lung cancer are needed. Chitooligosaccharide deacetylase homolog (YDJC) catalyzes the deacetylation of acetylated carbohydrates; however, the role of YDJC in lung cancer progression has yet to be studied. A549 lung cancer orthotopic mouse model was used for mice experiments. We found that YDJC overexpression contributes to lung cancer progression in an orthotopic mouse model. Long-term treatment (48 h) induces YDJC expression in sphingosylphosphorylcholine (SPC)-induced epithelial-mesenchymal transition (EMT). Gene silencing of YDJC (siYDJC) reduced N-cadherin expression and increased E-cadherin expression in SPC-induced EMT. Overexpression of YDJC reverses them but overexpression of the deacetylase deficient mutant YDJCD13A could not. Interestingly, overexpression of CDC16, a YDJC binding partner, suppressed EMT. ERK2 is activated in siCDC16-induced EMT. YDJC overexpression reduces expression of protein phosphatase 2A (PP2A), whereas CDC16 overexpression induces PP2A expression. YDJC overexpression induced ubiquitination of PP2A but YDJCD13A could not. CDC16 overexpression increased the ubiquitination of YDJC. These results suggest that YDJC contributes to the progression of lung cancer via enhancing EMT by inducing the ubiquitination of PP2A. Therefore, YDJC might be a new target for antitumor therapy against lung cancer.
Introduction
Lung cancer represents the highest mortality rate among all cancers worldwide [1]. Unmet needs for lung cancer are very high, but the lack of diagnostic markers and druggable targets for controlling the progression of lung cancer has become a bottleneck in the development of anticancer agents.
Chitooligosaccharide deacetylase homolog (YDJC) is a member of the YDJC family. YDJC carries out the deacetylation of the acetylated moiety in carbohydrates, an essential step in the decomposition of oligosaccharides although the exact substrate of YDJC in mammals has been identified [2]. Genetic approaches informed that the YDJC gene is related to several diseases such as ulcerative colitis, psoriasis, and Crohn's disease [3,4]. It was reported as one of the target genes induced by mTOR activation [5]. However, the function of YDJC in lung cancer progression is unknown although we published the first report about YDJC's role in keratin reorganization which is related to the changes of viscoelasticity in metastatic cancer cells [6].
Epithelial-mesenchymal transition (EMT) is considered the first step in lung cancer progression [7]. In the course of EMT, epithelial cancer cells in original tumor sites lose cell-cell contact and apicobasal polarity but gain the power for migration and invasion to adjacent tissues [8]. Although EMT facilitates cancer cell motility, EMT impacts the overall hallmarks of cancer, including proliferation, resistance to therapeutics, cancer initiation, survival, cancer metabolism, and immune evasion [8,9]. Recently, EMT is attracting the spotlight as a target to suppress immune evasion of cancer [10,11].
. . Cell Culture. The American Type Culture Collection (Manassa, VA, USA) supplied the A549, H838, and H1299 lung cancer cell lines. These cells were cultured at 37 ∘ C in 95% air and 5% CO 2 in an RPMI-1640 media added to a 10% heatinactivated FBS and PS solution (100 U/mL P, and 100 mg/mL S).
. . Western Blot and Quantitation of Band Density. Western blot was done, as mentioned before [6,12]. After termination of the cell culture, the lung cancer cells were rinsed 2 times with ice-chilled PBS and broken in RIPA buffer containing Xpert phosphatase and protease inhibitor cocktails from GenDEPOT Inc. (Austin, TX, USA) on ice for 30 min. Lung cancer cell lysates were separated by centrifugation at 15,000 rpm for 15 min at 4 ∘ C. The protein amounts were measured using the Bradford method. Proteins (20-30 g) in the lung cancer cell lysate were resolved by 8-12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane in the transfer buffer consisting of 192 mM glycine, 25 mM Tris-HCl (pH 8.8), and 10% v/v MeOH [6]. After blocking nonspecific sites with 5% nonfat dry milk, the membrane was treated with the primary antibody in 3% bovine serum albumin (BSA) PBS solution at 4 ∘ C overnight and then incubated for 1 h in a peroxidase-conjugated secondary antibody (1:5,000, Santa Cruz Biotechnology Inc.) PBS solution at room temperature (RT). Immunoreactive proteins were found by PowerOpti-ECL detection reagent from Animal Genetics Inc. (Gyeonggi, Korea) [6].
Determination of the Western band's density was achieved by ImageJ 1.52a (National Institutes of Health) and normalized to loading control. The results are representative data from 3 independent experiments.
. . Reverse Transcription-PCR (RT-PCR) and qRT-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen) as described by the manufacturer, and RT-PCR was performed according to the instructions provided by Access RT-PCR Systems (Promega, Madison, WI, USA). To quantify the RNA expression levels, qRT-PCR was performed in a 384-well plate on a LightCycler5 480 II real-time PCR system (Roche Diagnostics, Mannheim, Germany) using SYBR Premix and Ex Taq from Takara Bio (Shiga, Japan) as described by the manufacturer and the data were normalized to expression of a control gene GAPDH. The cycle number at which the product level exceeded an arbitrarily selected threshold (CT) was decided for each target sequence, and the amount of RNA relative to control RNA was depicted using the formula 2 −ΔCT , where ΔCT is the difference between the crossing point thresholds of a target gene versus GAPDH. The following primers were applied for qRT-PCR: YDJC, 5-GGA-GACGTGGATTTGCCTCA -3 and 5 -TAAAGCGACCCC-ATAGGC-3 ; CDC , 5 -ATGACCTCGACCAGCAACAG-3 and 5 -GCTGCAAGGTAACGACATGC-3 ; PP A-C, 5 -ACGGAATGGCTAATCTGT-3 and 5 -GATTGCTCT-CCTCCACTAA-3 ; GAPDH, 5 -GAAGGT GAAGGTCGG-AGTC-3 and 5 -GAAGATGGTGATGGGATTTC-3 . [6]. Genes were knockdown in the lung cancer cells with each siRNA using Lipofectamine6 2000 (Invitrogen) according to the manufacturer's protocol. The ratio of siRNA to Lipofectamine reagent was 1: MD, USA), as mentioned before [6,13]. Lung cancer cells (1×10 6 cells/mL) resuspended in serum-free medium were put to the upper chamber of the insert. Each lower chamber was filled with a medium containing 10% FBS [6]. After 16 h incubation, cells that failed to invade were removed off the upper surface of the membrane. Lung cancer cells that had invaded to the lower surface were stained by Hema 3 from Fisher Scientific Inc. (Houston, TX, USA) and photographed, and the cell numbers in four randomly chosen fields were computed at 20× magnification [6]. All the experiments were repeated at least 3 times with 2 replicates each [6].
. . Assay of Lung Cancer Cell Migration. The lung cancer cell migration experiment was carried out in 10 g/mL fibronectin-coated Transwell inserts [6]. Lung cancer cells (1×10 6 cells/mL) resuspended in serum-free medium were loaded to the upper chamber of each insert as mentioned before [14]. Each lower chamber was filled with a medium containing 3% FBS. After 6 h incubation, nonmigrating lung cancer cells were removed from the upper surface of the membrane. Lung cancer cells that migrated to the lower surface were detected with Diff-quick reagent. Migrated lung cancer cells in four randomly selected fields were counted at 20× magnification [6]. All the experiments were done at least 3 times with 2 replicates each [6].
. . Immunofluorescence Staining. Immunofluorescence staining was done, as described previously [6,15]. A549 cells were cultured on coverslips and fixed in cold MeOH for 10 min at RT. Then the A549 cells were permeabilized with a 10 min rinse in 0.1% Triton X-100 solution, also at RT, followed by rinsing several times in PBS containing 0.1% Tween-20. Next, the A549 cells were blocked with 3% BSA in PBS at RT for 1 h [6]. E-cadherin and N-cadherin primary antibodies were incubated with cells grown on coverslips overnight at 4 ∘ C, after which the antibody was rinsed out 4 times in PBS containing 0.1% Tween-20. Species-specific secondary antibodies conjugated in goat anti-mouse IgG antibody (Alexa Fluor 488, 1:500; Molecular Probes) and chicken anti-rabbit IgG antibody (Alexa Fluor 594, 1:500; Molecular probes) were incubated with the cells on the coverslips for 1 h at RT followed by 4 rinses in PBS with 0.1% Tween-20. The final samples were analyzed on slides using confocal microscopy (Nanoscope, Daejeon, Korea).
. . Immunoprecipitation. Immunoprecipitation (IP) was done, as mentioned before [6,16]. Lysates of A549 cells were treated with mouse monoclonal anti-PP2A, rabbit polyclonal anti-YDJC, or mouse polyclonal anti-CDC16 antibodies overnight at 4 ∘ C, respectively [6]. Protein A/G magnetic beads from Pierce (Rockville, IL, USA) were treated to each sample tube and reacted for 1 h at RT according to protocol. 50 L of each eluted sample was separated by SDS-PAGE and detected with Western blotting.
. . Construction of A549 YDJC , A549 YDJC-D A , and A549 shYDJC Cell Lines. A549 YDJC and A549 YDJC-D13A cell lines were established using plasmid DNA containing YDJC or YDJC D13A , respectively. pcDNA3.1YDJC and pcDNA3.1YDJC D13A were transfected into the A549 cells, and stable cell lines were chosen in 1 mg/mL G418 disulphate (Duchefa, Haarlem, Netherlands). A549 Con cells were established with the empty vector containing only the Neomycin resistance gene. A549 shYDJC was obtained using lentiviral shRNA for YDJC transfected into the A549 lung cancer cells using Puromycin as a selection marker.
. . Histology and Immunohistochemistry (IHC). At the time of sacrificing, lung tissues were fixed with 10 % formalin overnight and dehydrated in ethanol, embedded in paraffin, and sectioned at 4 m using Thermo Scientific Rotary Microtome Microm HM355S (Thermo Fischer Scientific, Germany) followed by staining with hematoxylin and eosin. Each slide was treated with 0.3% H 2 O 2 solution to inactivate endogenous peroxidase for 30 min at RT. Tissues were blocked in 5 % Goat Serum in PBS; after blocking, the mounted sections were immunostained with primary antibodies N-cadherin (1:100; ab18203; abcam), E-cadherin (1:100; 610181; BD Biosciences), PP2A (1:100; 610555; BD Biosciences), phospho-ERK1/2 (1:100; 9106; Cell Signalling Technology), and ERK1/2 (1:100; 9102; Cell Signalling Technology) in PBS containing 5% Goat Serum. The cells were incubated with a biotinylated secondary antibody solution for 1 h at RT. Detection of antibodies was performed by the avidin-biotin complex (ABC) method using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Serial sections were stained with the DAB peroxidase substrate kit from Vector Laboratories (Burlingame, CA) and hematoxylin. Sections were dehydrated and coverslipped with mounting solution from Vector Laboratories. Images were obtained with a Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany). For quantification analysis, three fields were randomly chosen on each slide. Each tissue section was semiquantitatively scored according to the percentage of positive cells. Positive cells were quantified using NIH ImageJ software (http://rsb.info.nih.gov/ij/) . . Orthotopic Mouse Model. A549 YDJC and A549 Con cells were directly introduced into the left lung of NOD/SCID mice (male; n=7/group) as depicted elsewhere [16]. A549 cells (10 6 ) were mixed with 50 L PBS containing Matrigel (1:1) and put into the left lateral thorax, approximately 1.5 cm above the lower rib line just below the inferior border of the scapula. The mice were euthanized 8 weeks later; the lungs were isolated and fixed in 10% neutrally buffered formalin, followed by histological examination. The Institutional Animal Care and Use Committee of the Korean National Cancer Center inspected all the experimental animal procedures.
. . Statistical Analysis. Data are represented as the mean ± standard deviation (SD) of at least 3 independent experiments done in triplicate. Analysis of the data was done using Student's t-test. Significance levels were * p<0.05, * * p<0.01, and * * * p<0.001.
. . Overexpression of YDJC Promotes Lung Cancer Progression in an Orthotopic Mouse
Model. From our previous study, YDJC plays a significant role in SPC-induced keratin phosphorylation and reorganization through ERK activation [6]. However, in the prior report, we could not prove the role of YDJC in the in vivo mice models. Therefore, we examined the involvement of YDJC in lung cancer progression in an orthotopic mouse model. A549 YDJC and A549 Con cells were introduced into the lungs of NOD/SCID mice by injection. We found that YDJC overexpression increased the tumor area by 2.9 folds (P=0.044) in mice injected with A549 YDJC cells (Figures 1(a), 1(b), and 1(c)). Based on the previous report about the role of YDJC, we examined pERK and PP2A expression by IHC. pERK expression significantly increased, and PP2A expression reduced in the A549 YDJC cellinjected mice group compared with A549 Con cell-injected mice ( Figure 1(d)).
Continuous phosphorylation and reorganization of keratin results in loss of keratin [17]. Interestingly, a recent study reported that loss of keratin 8 (K8), one of the epithelial cell-specific intermediate filament proteins, is one of the hallmarks of EMT [18]. So, we examined the expression of EMT markers, including E-cadherin (an epithelial marker) and N-cadherin (mesenchymal marker) in lung cancer tissues obtained from the orthotopic mouse model. E-cadherin expression was decreased more in tumor tissues from A549 YDJC than A549 Con injected groups (Figure 1(e)). By contrast, N-cadherin expression was induced higher in tumor tissues from A549 YDJC than A549 WT injected groups ( Figure 1(e)). These results imply that YDJC expression might be related to EMT, which contributes to tumor progression [7].
. . YDJC Is Induced in SPC-Induced EMT of Lung Cancer
Cells. Loss of E-cadherin expression and gain of N-cadherin expression were observed in tumor tissues from the A549 YDJC injected groups (Figure 1(e)). Therefore, we were interested in the role of YDJC expression in EMT and investigated the link between YDJC and EMT via long-term SPC treatment in lung cancer cells. First, we determined whether SPC induces EMT. SPC (5 M) induced downregulation of E-cadherin and upregulated expression of N-cadherin and YDJC in a timedependent manner (Figure 2(a)). This was also confirmed by confocal microscopy (Figure 2(b)). SPC-induced changes in EMT markers were also observed in H838 and H1299 lung cancer cells (Figure 2(c)). From the previous report, we know that 1 h SPC pretreatment can induce migration and invasion of lung cancer cells. We wonder whether 48 h SPC treatment also induces migration and invasion of lung cancer cells. 48 h SPC treatment also increased migration and invasion of A549 cells (Figure 2(d)). YDJC expression was also induced in TGF-1-induced EMT (Figure 2(e)).
. . YDJC Is Involved in SPC-Induced EMT and the Deacetylase Activity of YDJC Is Essential in YDJC-Involved EMT.
Next, to clarify the role of YDJC in SPC-evoked EMT, we YDJC is a member of the YDJC superfamily and has carbohydrate deacetylase activity, but no substrate has been identified in mammals so far [2,19]. It is very mysterious how YDJC, a carbohydrate deacetylase, plays a role in EMT. First, we investigated whether YDJC is involved in EMT through enzymatic activity or interaction with other proteins regardless of enzyme activity. Luckily, aspartic acid at position 13 (D13) is considered a critical amino acid as a vital proton acceptor of deacetylase activity in YDJC [6,19]. So, aspartic acid at position 13 (D13) of YDJC was changed into alanine (D13A) as the dominant negative form (YDJC D13A ). The expressions of EMT markers such as Ecadherin and N-cadherin were changed by YDJC (wild type) overexpression even without SPC treatment. In contrast, YDJC D13A overexpression failed to evoke changes in the expression of EMT markers (Figure 3(f), Fig. S4). These results were confirmed by confocal microscopy (Figure 3(g)).
Overexpression of YDJC D13A was unable to promote SPCpromoted migration and invasion (Figure 3(h)). These results propose that YDJC's deacetylase activity is a critical factor in SPC-induced EMT.
. . CDC Suppresses YDJC-Induced EMT. Previously, we found that CDC16 directly binds YDJC and suppresses its action [6]. First, we investigated the effects of CDC16 expression in YDJC-induced EMT. CDC16 overexpression increased expression of E-cadherin and decreased Ncadherin expression in A549 YDJC cells (Figure 4(a), Fig. S5). These results were proven by confocal microscopy (Figure 4(b)) and suggested that CDC16 blocked the action of YDJC on EMT in lung cancer cells. Then, we examined the effects of CDC16 siRNA on SPC-induced EMT. Interestingly, CDC16 gene silencing reduced E-cadherin expression and induced N-cadherin expression even without SPC treatment (Figure 4(c), Fig. S6). However, overexpression of CDC16 reduced N-cadherin levels in the presence of SPC (Figure 4(c)). These results were also proven by confocal microscopy ( Figure 4(d)). Furthermore, siCDC16 induced migration and invasion (Figure 4(e)). However, CDC16 overexpression mitigated SPC-induced migration and invasion (Figure 4(e)). Remarkably, in the YDJC stably knockdown A549 cell line, CDC16 siRNA failed to change the expression of EMT markers (Figure 4(f), Fig. S7). These were confirmed by confocal microscopic analysis (Figure 4(g)). These data imply that CDC16 suppresses YDJC-induced EMT.
. . CDC Induces but YDJC Reduces Protein Phosphatase A (PP A) Expression.
In the previous report, we confirmed that YDJC binds to CDC16 which inhibits YDJC in SPC-induced K8 phosphorylation and reorganization via the ERK pathway [6]. However, we could not explain why ERK is activated in SPC or YDJC-evoked K8 phosphorylation and reorganization. Several research papers, including ours, suggest that PP2A negatively regulates the ERK pathway. Especially SPC-induced loss of EMP2 results in PP2A degradation through interaction with alpha4 leading to ERK activation [20,21]. Therefore, we investigated the effects of YDJC and CDC16 on PP2A expression. By Western blot and qRT-PCR, we found that CDC16 induces but YDJC reduces PP2A expression (Figure 6(a), Fig. S10). In contrast, gene silencing of CDC16 reduces PP2A levels, whereas gene silencing of YDJC induces PP2A levels (Figure 6(a)). Interestingly, in the A549 cell line with a stably knockdown of YDJC, the PP2A level was increased regardless of CDC16 siRNA (Figure 6(b), Fig. S11).
We speculated that YDJC or CDC16 might affect ubiquitination of PP2A since CDC16 comprises a ubiquitin ligase in the APC/C complex [22]. So first, we examined whether CDC16 or YDJC directly binds to PP2A and found that YDJC and CDC16 bind to PP2A (Figure 6(c), Fig. S12). Remarkably, YDJC WT overexpression increased PP2A ubiquitination compared with control or YDJC D13A overexpression (Figure 6(d)), whereas CDC16 overexpression did not induce ubiquitination of PP2A (Figure 6(e)). These results suggest that PP2A ubiquitination is affected by YDJC but not CDC16 overexpression. Interestingly, YDJC was ubiquitinylated in CDC16 stably expressed A549 cells (Figure 6(f)). These results suggest that YDJC might induce ubiquitination of PP2A, but CDC16 might maintain PP2A expression by inducing ubiquitination of YDJC.
Discussion
In this study, we investigated whether SPC-induced YDJC expression promotes the progression of lung cancer via EMT. We found that YDJC induced EMT via ERK2 activation by PP2A downregulation through ubiquitination leading to the promotion of the progression of lung cancer and CDC16 reversed this by directly interacting with YDJC leading to ubiquitination. We previously reported that SPC or SPCinduced YDJC induces K8 phosphorylation and reorganization leading to migration and invasion of lung cancer cells [6]. These phenomena are involved in the change of viscoelasticity in cancer cells [17,23,24]. However, in the previous report, we could not confirm the role of YDJC in the progression of lung cancer in an in vivo mouse model. So we confirmed the involvement of YDJC in lung cancer progression using a lung cancer orthotopic mouse model (Figure 1). We also confirmed the activation of ERK2 and the possible involvement of PP2A by IHC staining (Figure 1(d)). Recent studies reported that continuous keratin phosphorylation and reorganization lead to loss of keratin [17], which is the hallmark of EMT [18]. So, we examined the possibility of EMT by IHC staining of EMT markers such as E-cadherin and N-cadherin (Figure 1(e)).
In this regard, we speculated that the EMT might be induced by long-term SPC treatment (48 h). In SPC-treated lung cancer cells, E-cadherin levels were downregulated, whereas N-cadherin and YDJC levels were upregulated (Figure 2), which was followed by increased migration and invasion (Figure 2(d)). These results strongly indicate that SPC promotes EMT in lung cancer cells.
From Figures 2 and 3, we showed that YDJC is induced and involved in SPC or TGF-1-induced EMT. The involvement of YDJC in EMT induced by TGF-, which is a wellknown typical EMT inducer, suggests that involvement of YDJC in EMT might be a common mechanism of action of EMT induced by other EMT inducers including TGF-1 and SPC.
How does YDJC drive EMT? Our data suggest that YDJC deacetylase activity is critical in SPC-induced EMT of human lung cancer cells although we do not know the exact mechanism of action of YDJC in EMT (Figures 3(f), 3(g), and 3(h)). Accordingly, it has been reported that the deacetylase activity of other proteins occurs in EMT. In the case of human renal epithelial cells, histone deacetylase activity modulated TGF-1-induced EMT [25]. Besides, the H3K9 deacetylase activity of SIRT6 mediates EMT and metastasis in malignant human colon carcinoma [26]. Moreover, the blocking of histone deacetylase suppressed TGF-1-induced EMT in hepatocytes [27]. These reports suggest that deacetylase activity plays an essential role in EMT. However, it is unknown whether this deacetylase activity in YDJC is limited to the removal of the carbohydrates' N-acetyl group or expands to the deacetylation of N-acetylated lysine in proteins.
We found that CDC16 might be a binding partner of YDJC [28]. Besides, we have previously shown that YDJC binds to CDC16, which suppressed YDJC's function [6]. Thus, we clarified the role of CDC16 on YDJC-induced EMT. Curiously, CDC16 gene silencing induced EMT, migration, and invasion, even without SPC treatment in lung cancer cell lines (Figure 4). Further, overexpression of CDC16 inhibited EMT, migration, and invasion ( Figure 4). Moreover, in an YDJC stably knockdown A549 cell line, CDC16 gene silencing did not induce EMT in the absence of YDJC (Figure 4(f)). These results indicate that YDJC might mediate siCDC16-induced EMT and CDC16 may be an upstream suppressor of YDJC-induced EMT. Still, we do not know the mechanism of CDC16 involved in the suppression of YDJC expression. CDC16 might promote ubiquitination of YDJC since CDC16 comprises a ubiquitin ligase in the APC/C complex [22]. However, it is unknown whether the APC/C complex ubiquitinylates YDJC, although lysine 92 in human YDJC is ubiquitinylated [29]. As shown in a previous report, the ERK pathway mediates YDJC-induced keratin phosphorylation and reorganization [6]. Moreover, activated ERKs are involved in EMT [30,31]. We found that CDC16, located upstream of YDJC, inhibits YDJC-induced EMT. We investigated whether ERK activation mediates CDC16 gene silencing-induced EMT and found that mitogen-activated protein kinase (MAPK) is involved in siCDC16-induced EMT. PD98059, which inhibits ERK phosphorylation acting as a MEK inhibitor, and SP600125, as a JNK inhibitor, prohibited siCDC16-evoked EMT, migration, and invasion of A549 cells ( Figure 5). In particular, ERK signalling mediates siCDC16-induced EMT ( Figure 5). Thus, subsequent studies need to focus on how CDC16 and YDJC regulate the activation of ERK. We found that ERK2 but not ERK1 mediates siCDC16-induced EMT ( Figures 5(d), 5(e), and 5(f)). Several other studies about ERK2 involvement in EMT also support our findings [12,[32][33][34].
PP2A induces inactivation of phospho-ERK [16,35,36]. Overexpression of CDC16 induced PP2A expression, whereas overexpression of YDJC reduced PP2A expression (Figure 6(a)). In contrast, gene silencing of CDC16 reduced PP2A expression, whereas gene silencing of YDJC induced PP2A expression (Figure 6(a)). Remarkably, in the Mesenchymal-like Epithelial-like (g) Figure 6: YDJC downregulates PP2A expression, and CDC16 reverses it. (a) CDC16 induces and YDJC suppresses PP2A expression. The expression levels of PP2A and PP A-C mRNA levels in the Control (siCon) or YDJC-knockdown (siYDJC) or CDC16-knockdown (siCDC16) or YDJC-overexpressing (YDJC) or CDC16-overexpressing (CDC16)-A549 cells were analyzed using western blotting or qRT-PCR. (b) Effects of siCDC16 on PP2A expression in YDJC stably silenced A549 cell lines. The expression levels of PP2A and PP A-C mRNA levels in the Control (siCon) or CDC16-knockdown (siCDC16)-YDJC stably silenced A549 cells were analyzed using western blotting or qRT-PCR. Mean± SD; n=3. All P-values were determined based on two-tailed Student's t test. In (a) and (b), A549 cells were transfected with the plasmid consisting of CDC16, YDJC, or an empty vector (4 g), respectively. Gene silencing of CDC16 or YDJC was carried out by transfecting the A549 cells with the indicated amounts of siCDC16, siYDJC, or control siRNA. Cell lysates were prepared, and the protein level was resolved to 8-12% SDS-PAGE. The expression level of PP2A was determined by western blotting; -actin was used as an internal control. qRT-PCR analysis of PP A expression in cDNA derived from the indicated cells. GAPDH is included as a loading control. (c) Binding of CDC16 to PP2A and YDJC. Immunoprecipitation with the CDC16 antibody (IP: CDC16) was carried out with the cell lysates of A549 cells overexpressed with CDC16 plasmids. The resulting immunocomplexes were analyzed with Western blotting with PP2A, YDJC, or CDC16 antibodies, respectively. (d) Stimulatory effects of YDJC on ubiquitination of PP2A. Immunoprecipitation with the PP2A antibody (IP: PP2A) was done using cell lysates of A549 cells overexpressed with wild-type YDJC WT or YDJC D13A plasmids. (e) Effects of CDC16 overexpression on PP2A ubiquitination. Immunoprecipitation with the PP2A antibody (IP: PP2A) was done using the cell lysates of A549 cells transfected with CDC16 plasmids. In d and e, the resulting immunocomplexes were detected by Western blotting with PP2A or ubiquitin (Ub) antibodies. (f) Effects of stably expressed CDC16 on YDJC ubiquitination in A549 cell lines. Immunoprecipitation with the YDJC antibody (IP: YDJC) was carried out using the cell lysates of A549 cells stably transfected with plasmids containing CDC16. The resulting immunocomplexes were detected by Western blotting with YDJC and Ub antibodies, respectively. (g) The proposed scheme of YDJC's mechanism of action.
How does YDJC increase the ubiquitination of PP2A? We do not know the relevant mechanism of action in YDJC's involvement in the ubiquitination of PP2A. However, we speculated the possible mechanism. Usually, acetylation of lysine prevents ubiquitination in the protein's lysine residue [37]. Therefore, if YDJC acts as a deacetylase of protein removal of the acetyl group of a lysine residue (lysine 41) in PP2A, YDJC might induce ubiquitination of lysine 41 in PP2A (Figure 6(g)). Based on these speculations, YDJC might increase the chance of the deacetylated lysine 41 in PP2A to be exposed to ubiquitinylation (Figure 6(d)). PP2A was ubiquitinylated by several ubiquitin ligases such as CRL4-DCAF1 and EDD E3 ubiquitin E3 ligases [38,39]. Especially, progestin-induced EDD E3 ubiquitin ligase ubiquitinylates the PP2A catalytic subunit by interaction with alpha 4 which was already described in the degradation of PP2A during the SPC-induced loss of EMP2 [20,21,39].
In summary, we showed that YDJC promotes the progression of lung cancer in the orthotopic mouse model of lung cancer via EMT (Figure 1). We also proposed the novel mechanism of CDC16/YDJC/PP2A/ERK2 in lung cancer progression although it is necessary to confirm the proposed mechanism of the scheme (Figure 6(g)). Our data also suggest that YDJC might become an unusual target to prohibit lung cancer progression, although currently there are no reports of any compounds that inhibit YDJC.
Data Availability
The data supporting the findings of our study are shown within this article. | 2019-08-23T02:03:33.835Z | 2019-08-07T00:00:00.000 | {
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8317971 | pes2o/s2orc | v3-fos-license | Kmerlight: fast and accurate k-mer abundance estimation
k-mers (nucleotide strings of length k) form the basis of several algorithms in computational genomics. In particular, k-mer abundance information in sequence data is useful in read error correction, parameter estimation for genome assembly, digital normalization etc. We give a streaming algorithm Kmerlight for computing the k-mer abundance histogram from sequence data. Our algorithm is fast and uses very small memory footprint. We provide analytical bounds on the error guarantees of our algorithm. Kmerlight can efficiently process genome scale and metagenome scale data using standard desktop machines. Few applications of abundance histograms computed by Kmerlight are also shown. We use abundance histogram for de novo estimation of repetitiveness in the genome based on a simple probabilistic model that we propose. We also show estimation of k-mer error rate in the sampling using abundance histogram. Our algorithm can also be used for abundance estimation in a general streaming setting. The Kmerlight tool is written in C++ and is available for download and use from https://github.com/nsivad/kmerlight.
I. INTRODUCTION
k-mers (nucleotide strings of length k) play a fundamental role in computational genomics. k-mers form the basis of many assembly and alignment algorithms. Understanding abundance of k-mers in sequence reads have applications in read quality estimation, read error correction [1], [2], [3], parameter estimation for genome assembly [4] and digital normalization [5], etc. We consider the problem of computing k-mer abundance in a sequence or in a read collection. Specifically, the goal is to compute the count of distinct k-mers occurring in the input as well as counts of distinct k-mers occurring in the input with given multiplicity (frequency) values. The histogram of such k-mer counts for different multiplicity values is referred to as k-mer abundance histogram.
k-mer abundance computation has several applications in genome analysis. In high throughput sequencing, k-mer abundance helps in assessing quality of sequence reads and in identifying k-mers originating from erroneous reads. This approach is typically used in spectral alignment techniques for read error detection and correction [6], [1], [2], [3]. de Bruijn graphs of k-mers are used in several assembly algorithms. Quality of de Bruijn based assemblers crucially depend on the value of k-mer size and k-mer abundance histogram is helpful in choosing appropriate k-mer size [4]. k-mer abundance computation also finds application in estimation of k-mer error rate in the reads, which helps in understanding sampling error rate [7]. Understanding abundance of k-mers in the genome provides insights into the sequence repetitiveness in the genome. Variations in shape of k-mer abundance histogram of reads, in particular the positions of peaks in the histogram, have relations to sequencing bias and to the presence of highly polymorphic genomes that contain large number of heterozygous locations in their haplotypes [8].
There are several existing techniques for k-mer counting and k-mer abundance computation. In k-mer counting, count of each k-mer present in the input is computed. Algorithms for k-mer counting can be used for computing k-mer abundance. Existing k-mer counting tools include Tallymer [9], Jellyfish [10], KMC2 [11], MSPKmerCounter [12], DSK [13], BFCounter [14], Khmer [15], KAnalyze [16], KmerGenie [4] and Turtle [17]. These tools perform either exact counting or approximate counting. Exact counting techniques rely on large main memory or on a combination of external memory and main memory to handle massive memory requirement. BFCounter uses Bloom filter as a pre-filter to reduce memory requirement. Kmergenie uses random sampling to do approximate counting with reduced memory and compute requirements [4]. It is however known that distinct (k-mer) count estimates computed by sampling based approaches are known to have large variance in general unless the sample size is close to the input size [18]. Khmer uses CountMin sketch [15] for approximate counting, for which error margins could be large in general. KmerStream [7] is a streaming algorithm for in-memory estimation of the number of distinct k-mers and the number of unique k-mers. Their approach is an extension of streaming algorithms for estimation of distinct count [19]. Estimation of k-mer counts with larger multiplicities was left as an open problem in [7].
A. Our Contribution
We present a streaming algorithm Kmerlight to estimate the total number of distinct k-mers in the input, denoted as F 0 , as well as the histogram of total number of k-mers with multiplicity i, denoted as f i . Our algorithm is an extension of streaming algorithms for count distinct problem [19] and for counting unique k-mers [7]. Streaming algorithm for computing provable estimates for f i for i > 1 was left as an open problem in [7] and we solve this problem. Kmerlight is very fast and it uses small in-memory data structures to compute estimates for F 0 and f i values with high accuracy. It uses logarithmic space and runs in linear time. We also provide analytical bounds on the error margins achieved by Kmerlight. To the best of our knowledge, our algorithm is the first streaming algorithm to efficiently compute F 0 as well as f i values with analytical guarantees.
We conducted several experiments to measure the accuracy and performance of Kmerlight. For instance, with less than 500 MB RAM (and no disk space), Kmerlight achieved 2% relative error. We provide a multi-threaded C++ implementation of our tool which is available for download and use. Kmerlight can be run on a standard desktop machine and it scales well to genome scale and metagenome scale data. Resource frugal nature of Kmerlight allows simultaneous computation of abundance histograms with different values of k, which for instance is required in parameter estimation for genome assembly.
We also exhibit few applications of Kmerlight. We use Kmerlight for de novo estimation of k-mer repetitiveness in the underlying genome from reads. We propose a simple probabilistic model for k-mer abundance histogram and use it for the estimation. Analyzing k-mer repetitiveness helps in understanding sequence repetitiveness in the genome. We also use Kmerlight for estimation of k-mer error rate in the reads and for estimation of the genome size.
Our algorithm can be used for abundance estimation in a general setting. To the best of our knowledge, our algorithm is the first streaming algorithm to solve this problem using only sublinear space and query time and with analytical bounds. This we believe is of independent theoretical and practical interest.
II. METHODS
Let F 0 denote the total number of distinct k-mers in the input. For i ≥ 1, let f i denote the total number of k-mers each occurring with multiplicity (frequency) exactly i in the input.
The histogram of f i values constitutes the k-mer abundance histogram.
A. Algorithm
We present a streaming algorithm Kmerlight to estimate F 0 and f i for i ≥ 1. Our algorithm maintains a 'sketch' of the input seen so far. The space used for this sketch is sublinear in the input size. Experiments show that the sketch size in the range of 500 MB to 1 GB RAM can provide high accuracy estimates. The sketch is updated upon seeing each k-mer in the input. At any stage, estimates for F 0 and f i values are computed from the sketch.
Our algorithm is an extension of streaming algorithms for count distinct problem [19] and for counting unique kmers [7]. We first give a brief overview of the KmerStream algorithm of [7] for estimation of F 0 and f 1 . Their algorithm is an adaptation of the F 0 estimation algorithm in [19]. The general idea is to perform multi-level sampling of the input k-mer stream. Each distinct k-mer is sampled exclusively by one of the levels and all occurrences of this k-mer is assigned to the same level. Sampling is such that level j samples all occurrences of roughly 1/2 j fraction of all distinct k-mers in the input. At each sampling level, an array of counters is maintained. When a k-mer is assigned to a level, it is further hashed to one of the counters in the counter array and the destination counter is incremented by one.
The number of counters with value zero is used to estimate F 0 . Estimated value of F 0 along with number of counters containing value 1 is used to estimate f 1 . Consider a level w ≥ 1 and let t 0 denote the number of counters at level w that contains value zero. Let r denote the total number of counters at level w. Under the assumption that ideal hash functions are used, multi-level sampling has following properties. The expected number of distinct k-mers sampled in level w is given by N w = F 0 /2 w . Furthermore, the expected number of counters with value zero at level w is given by r(1 − 1/r) Nw . Using t 0 as an estimate for this, estimateF 0 can be computed asF Finally a 'good' level w for estimating F 0 is chosen with the property that value of t 0 at this level is close to r/2. It was shown in [19] that this approach yieldsF 0 with the property that (1 − ǫ)F 0 ≤F 0 ≤ (1 + ǫ)F 0 with probability at least 1 − δ. The algorithm has O(1) update time and uses O( 1 Let t i be the number of counters at level w each with value i and is collision free. That is, no two distinct k-mers hash to any of these counters. The expected value of t i is given Knowing the value of t i , we can thus estimate f i asf Determining t 0 and t 1 are easy as counters holding values either 0 or 1 are collision free by definition. On the other hand, determining t i for i ≥ 2 is difficult because several of the counters with value i could have collisions. Furthermore, for larger values of i, there are several collision possibilities that results in a counter value of i and analyzing each of these possibilities to bound the error is extremely complicated. This makes the algorithm in [7] inadequate to estimate f i for i ≥ 2. In [7], authors prove that if In the following we discuss our Kmerlight algorithm for estimating f i for i ≥ 2 also in addition to F 0 and f 1 and with similar theoretical guarantees. Kmerlight sketch has t instances, where t is a parameter, and each instance has the following structure. An instance has M arrays T 1 , . . . , T M where each of these M arrays correspond to M different sampling levels. We choose M = 64 in our implementation, which is adequate for counting up to 2 64 distinct k-mers. Each of these M arrays have r counters, where r is the second parameter for the algorithm. Each counter of the array is a tuple of the form v, p where v ≥ 0 is the counter value and p is a number from {0, . . . , u−1}, where u is the third parameter for our algorithm. We use a special counter value '−1' to indicate that the counter is 'dirty'. For counter T w [i], we use .v and T w [i].p to indicate its v and p values respectively. Initially, all T w [i].v values are set to zero and all T w [i].p values are set to 'undefined'. Sampling level w ∈ {1, . . . , M }, for a k-mer is computed using the same approach as in [19], [7], where a k-mer is first hashed using a pairwise independent hash function h(). The number of trailing bits in the binary representation of the hashed value starting from the least significant '1' bit is used as its sampling level. For the special case of hashed value being 0, the k-mer is assigned level M . Each instance uses an independent hash functions. Let z be the hashed value of a k-mer and w be the sampling level assigned to it. Let x = z/2 w . We map the k-mer to a pair of values (c, j) ∈ {0, . . . , r − 1} × {0, . . . , u − 1} given by where c is the index of the counter at level w and j is the auxiliary information. .v = −1. As a consequence, 'dirty' counters are discarded from future updates. Note that every occurrence of the same k-mer is mapped to the same (c, j) pair. A counter is marked 'dirty' when a collision is detected. The T w [c].p values help in detecting collisions. Clearly not all collisions can be detected because two different k-mers could map to the same (c, j) pair at the same level. A false positive counter is a counter that remains non dirty in spite of collisions. We analytically show that the level w * chosen by the algorithm for estimation is a 'good' level such that the set of non dirty counters at level w * each holding value i have only few false positives. Hence its cardinality is a good estimate for t i . Moreover, the t i value at this level can be used to estimate f i with high accuracy. In order to reduce the error probability further, we maintain t independent instances of the above sketch, where t is a parameter, such that all t instances are updated for every input k-mer. The f i estimates are computed from each of these instances and their median value is used as the final estimatê f i .
Details of the update and estimation methods are given in Algorithm 1. Error bounds achieved by our algorithm are given in Theorem 1. The proof is given in the supplementary material (Section D) and it assumes truly random hash functions. Our algorithm uses logarithmic space, logarithmic time for query and O(1) time per update (for fixed λ and δ). We note that the time and space complexities given in Theorem 1 is for the simultaneous estimation of all f i s with f i ≥ F 0 /λ. We refer the reader to the supplementary material for more discussions on the time and space complexities. as follows: 15:
B. Repetitive Regions in the Genome
We show applications of k-mer abundance histograms computed by Kmerlight. In this section, we apply k-mer abundance histogram of reads for de novo estimation of k-mer repetitiveness in the underlying genome. If segments in the genome are repetitive, then k-mers from these segments would have correspondingly scaled multiplicity in the reads under uniform read coverage. Conversely, large fraction of k-mers having higher multiplicity values in the read collection is indicative of sizable repetitive regions in the genome. Thus, understanding k-mer repetitiveness in the genome for varying values of k can provide useful insights into the repetitive nature of the genome. Studying with multiple values of k is useful because large values of k would help in excluding short repeats (length less than k) from the analysis.
For a fixed k-mer length k, we define the multiplicity (repetitiveness) of any given location s in the genome as the number of times the k-mer starting at location s is present across the whole genome. Let g t denote the total number of positions each with multiplicity t in the genome and let g denote the genome size. Clearly, g = t g t . Thus, g t /g is the fraction of the genome locations each with repetitiveness t. In other words, g t /g is the fraction of the genome locations such that the k-sized fragment occurring at any of these locations occur in total t times in the genome. In the following, we propose a method to estimate g t values from reads. For this, we propose a simple probabilistic model for k-mer abundance histogram of reads. The model parameters include g t values, which can thus be inferred from observed histogram.
Generative Model for k-mer Abundance Histogram
We propose a simple probabilistic model for the k-mer abundance histogram of sequence reads. For this we follow the generative model proposed in [7] where the model assumes uniform coverage of the genome and that the k-mers are generated from each position of the genome are Poisson distributed as P oi(λ). Furthermore, the true k-mers are generated at each position as P oi(λ ′ ) and the erroneous k-mers are generated as P oi(λ − λ ′ ).
With these assumptions, we give a simple model for the abundance histogram of true k-mers. Parameter λ is related to total number of k-mers N from the reads as λ = N/g, where g is the genome size. The k-mer error rate is given by λ− λ ′ . We use the notations c for the read coverage, l for read length, n for total number of reads, N for total number of kmers and g for the genome size. Clearly N = n(l − k + 1). Since c = nl/g, we also obtain that and Let G m for m ≥ 1 denote the set of all distinct kmers each occurring with multiplicity m in the underlying genome. Recalling that g m denote the total number of positions in the genome each with multiplicity m, it follows that g m = m · |G m |. Consider a k-mer x belonging to G m . Let X 1 , X 2 , . . . , X m be m random variables each distributed as P oi(λ ′ ) and denoting number of times x was sampled from each of its m locations in the genome. Let X = X 1 +· · ·+X m denote the total number of occurrences of x in the final collection of true k-mers. By linearity of Poisson, it follows that X is Poisson distributed as P oi(mλ ′ ). That is, each true k-mer with multiplicity m in the genome is Poisson distributed as P oi(mλ ′ ) in the reads. We use the known fact that P oi(λ) has peak probability at value λ with corresponding probability value λ λ e −λ /λ! ≈ 1/ √ 2πλ. We consider the abundance histogram of all k-mers from G m present in the reads. It follows that this abundance histogram is expected to have peak at mλ ′ with peak value |G m |/ √ 2πmλ ′ = g m /(m √ 2πmλ). Consequently, corresponding to the k-mers in the genome with multiplicity m, for m = 1, 2, . . ., there would be a peak in the abundance histogram at mλ ′ with peak value g m /(m √ 2πmλ ′ ). In a diploid case, the first peak (at λ ′ ) would correspond to the heterozygous k-mers in the genome.
From this model, g m values can be easily computed using the corresponding peak positions and peak values in the histogram. The fractions g m /g can be computed using estimate for genome size g. Estimate for g can also be obtained from the k-mer abundance histogram, which we discuss in section II-C. By using Kmerlight for computing abundance histograms, estimates of g m for various k-mer sizes can be computed efficiently.
Erroneous k-mers
The generation of erroneous k-mers is modelled in [7] as following P oi(λ − λ ′ ). It is further assumed in [7] that erroneous k-mers result from single position errors. In other words, an erroneous k-mer at a position could be any one of the 3k possible candidates. Consequently, most erroneous k-mers occur only once in the reads and the cardinality of those with multiplicity two or more are significantly lesser, as typically observed in practice. Hence erroneous k-mers contribute to an initial sharp peak at position 1 in the abundance histogram, followed by peaks due to true k-mers with varying multiplicities. This is illustrated in Figure 1. Typically, the fraction of genome with a given k-mer multiplicity decreases with increasing multiplicity value and hence the abundance histogram will typically have peaks with decreasing peak values with increasing multiplicities. Since λ is related to c and l as in eq (1), increasing the coverage c results in increased value of λ and of λ ′ , resulting in a wider gap between the peak due to erroneous k-mers and remaining peaks.
Peak due to erroneous kmers kmers with multipicity 1 in the genome kmers with multiplicity 2 Typical k-mer abundance histogram of reads. x-axis corresponds to multiplicity values and y-axis corresponds to k-mer counts. Initial sharp peak is due to read errors and remaining peaks are due to true k-mers in the genome with different multiplicities.
C. k-mer Error Rate Estimation
k-mer error rate is the ratio of total number of erroneous kmers in the sequence reads to the genome size. Understanding k-mer error rate gives insights into sequencing errors. Fast estimation of k-mer error rate from reads was proposed in [7] by using estimates for F 0 and f 1 values of k-mers in the reads. For this purpose, a model for erroneous k-mer generation was proposed in [7] and k-mer error rate is inferred from this model by numerically solving a set of non linear equations involving estimates for F 0 and f 1 . Alternatively, kmer error rate can be estimated in a straightforward manner if the abundance histogram is available, which can be efficiently computed using Kmerlight. Let N ′ and N e denote the total number of true k-mers and total number of erroneous k-mers respectively in the sequence reads. Total number of k-mers N is thus given by N = N ′ + N e and the true k-mer rate λ ′ and k-mer error rate λ e are given by λ ′ = N ′ /g and λ e = N e /g respectively. It follows that λ e is given by We recall from the model for k-mer abundance histogram proposed in the previous section that λ ′ is easily obtained from the peak positions corresponding to true k-mers in the histogram. Value of N e can be estimated from the initial peak in the histogram due to erroneous k-mers as follows. EstimateN e for N e is given byN e = t i=1 i · f i . Typically the f i values for erroneous k-mers exhibit sharp decline as i increases. Hence good estimates for N e can be obtained by considering only first few initial histogram values.
We remark that this approach avoids detailed modeling of erroneous k-mer generation process and solving complex non linear equations as in [7]. From λ ′ and λ e , we obtain λ = λ ′ + λ e . Knowing λ, we can estimate coverage c using eq (1). Estimate for c can subsequently be used for estimation of g using eq (2).
A. Run time
We compared run time performance of Kmerlight with the KMC2 k-mer counting tool [11]. Kmerlight tool has multi-threaded C++ implementation. KMC2 was chosen for comparison because KMC2 was shown to outperform other state of the art k-mer counting tools [11] in terms of time and space requirements. We remark that KMC2 tool computes frequency counts for individual k-mers as against the k-mer abundance histogram. Abundance histogram can nevertheless be computed from individual frequency counts. Number of threads were kept seven for both tools. KMC2 we considered only the time taken for k-mer counting and excluded the additional time for computing the abundance histogram. The run times are given in Table I. KMC2 was run with strict memory mode. We give RAM and HDD usage for KMC2. For Kmerlight, only RAM usage is shown since it does not use any HDD space.
The first input contained 328 million reads with read length 90. The tools were run on a dual core desktop machine with 4 GB RAM and HDD where each core is a 3.10 GHz Intel i5 processor. The input contained 23 billion 21-mers and 9.2 billion 63-mers and the respective run times are given in the first two rows of Table I. KMC2 failed to process the 63-mers using 1GB RAM and aborted with error. There is only a minor variation in the Kmerlight run time for different RAM settings. Kmerlight run times for 63-mers with different memory settings are given in the Kmerlight was used to process reads from whole human genome (GRCh38). The reads were generated using ART tool [20] with read length 100 and coverage 50. The generated FASTQ file was 320 GB in size and contained 1.45 billion reads. Kmerlight processed 55 billion 63-mers present in this in 1hr and 20 minutes.
Next we compared the performance on metagenome scale data. Reads were generated using ART tool [20] from about 188039 reference genomes available from the NIH Human Microbiome Project [21]. Read length and coverage were 100 and 50 respectively. The resulting read file was 930 GB in size and contained about 3.8 billion reads. These experiments were conducted on a multi-core server with large memory due to larger memory requirement for KMC2 tool. The input contained 144 billion 63-mers and 61 billion 85-mers and the respective run times are given in the last two rows of Table I. KMC2 failed to process the 85-mers using 32GB RAM and aborted with error.
B. Accuracy
To measure the accuracy of Kmerlight, we compared Kmerlight output with the exact histogram. In particular, we compared output for different k-mer sizes and for different memory usages by Kmerlight. Reads were generated from human reference chromosome Y (GRCh38) using ART tool [20] with read length 100 and coverage 50 respectively. Figure 2 shows the accuracy of Kmerlight output for f 4 , . . . , f 60 , after processing the 15-mers in the input using 500 MB RAM. Histograms showing relative estimate of Kmerlight output for F 0 , f 1 , f 2 and f 3 respectively for 1000 trials by Kmerlight with 500 MB RAM setting.
C. Repeats in Chromosome Y
We used the probabilistic model and the approach discussed in Section II-B for de novo estimation of k-mer repeats in human reference chromosome Y (GRCh38) from reads. Validity of the model and the effectiveness of using histogram computed by Kmerlight in place of exact histogram were studied in these experiments. Reads from chromosome Y were generated using ART tool with read length 100 and coverage value 50. The built-in profile of Illumina HiSeq 2500 system was used by the ART tool. Table II gives the relative errors in estimation of g 1 , g 2 and g 3 values from reads using the model for k = 15.
The estimated values are compared against the true values computed directly from the chromosome Y sequence data. Relative errors are provided separately for estimation using the exact abundance histogram and using Kmerlight computed histograms with different memory settings. Kmerlight errors are averaged over 1000 trials and the standard deviations are also indicated in the table. These results indicate that our model is suitable for de novo estimation of g i values and Kmerlight can be used for the efficient computation of abundance histograms for this purpose. We refer to the supplementary material (Section B) for additional details including histogram plots, comparison of true peak values and peak values inferred using the model, true g i values etc.
D. k-mer Error Rate Estimation
We used Kmerlight to estimate k-mer error rate from reads. Reads were generated from human reference chromosome Y (GRCh38) using ART tool with coverage 50 and read length 100. The built-in profile of Illumina HiSeq 2500 system was used by the ART tool for read generation. The k-mer abundance histogram was then computed using Kmerlight with k = 15. Initial values of this histogram were then used to estimate N e , which denotes the total number of erroneous k-mers. In particular, initial 10 histogram values were used for N e estimation. The first true k-mer peak was observed at λ ′ = 39 in the abundance histogram. The k-mer error rate λ e was then estimated using eq (3). The average value of λ e over 1000 trials was obtained as 3.58. Using exact histogram was used in place of Kmerlight histogram, value of λ e obtained was 3.52, which is close to the Kmerlight estimate. To validate the k-mer error rate estimate, we estimated the genome length g using eq (1) and using the relation λ = λ ′ + λ e , with λ ′ = 39 and λ e = 3.58. Alternatively, we used eq (2) to estimate g by using the known values c = 50 and l = 100 used for read generation. The total number of valid nucleotides in the input chromosome Y data was also counted. All the three values for g were closeby and the difference between any two of them was within 1%. This indicates high accuracy of the estimated genome length and also the estimated k-mer error rate.
IV. DISCUSSIONS
We proposed the first streaming algorithm Kmerlight for efficient generation of k-mer abundance histogram. We provide analytical bounds for the error margins. We also show applications of Kmerlight in the de novo estimation of k-mer repeats in the genome using a model that we propose and also in the estimation of k-mer error rate and genome length. We discuss here few additional applications of Kmerlight.
Spectral alignment techniques for read error correction in de novo sequencing projects [1], [2], [3] usually depend on the set of 'trusted' k-mers G m k in the reads as an approximation to the spectrum G k which is the set of all k-mers in the underlying genome. The set G m k denotes the set of all k-mers whose frequency is above threshold m. The threshold m is usually determined from the region of erroneous k-mers in the abundance histogram. Kmerlight allows efficient generation of abundance histograms for various k values.
For choosing appropriate k-mer length in de Bruijn based assemblers, Chikhi et al. [4] propose that the most appropriate choice of k is the one that provides maximum number of distinct true k-mers to the assembler. This has been shown to yield excellent assembly on a diverse set of genomes. Recalling that F 0 denotes the total number of distinct k-mers in the sequence reads, F 0 can be written as F 0 = F e 0 + F ′ 0 , where F e 0 and F ′ 0 are the total number of distinct erroneous k-mers and true k-mers respectively. Thus, the objective is to choose k that maximizes F ′ 0 . In order to do this efficiently, they compute an approximate k-mer abundance histogram using down sampling. This is done to overcome the computational overhead of computing exact abundance histograms for different k values. This method has been incorporated into the KmerGenie tool. Alternatively, F ′ 0 can be estimated using estimates of F 0 and F e 0 as F ′ 0 = F 0 − F e 0 . Estimate for F e 0 can be obtained by summing values in the abundance histogram region due to erroneous k-mers. Kmerlight can be used here for efficient generation of histograms for multiple k values with high accuracy without requiring any down sampling. More details are provided in the supplementary material (Section C). We remark that estimating F e 0 say from k-mer error rate is infeasible because k-mer error rate estimates only the total number of erroneous k-mers as opposed to the total number of distinct erroneous k-mers.
Supplementary Material
Kmerlight: fast and accurate k-mer abundance estimation APPENDIX A ADDITIONAL ERROR PLOTS We provide additional error plots for the Kmerlight output. Input reads were generated from human reference chromosome Y (GRCh38) using ART tool [20] with read length 100 and coverage 50 respectively. Figure 4 shows Values f 1 , . . . , f 1000 were considered for this and Figures 6 and 7 illustrate the dependence of their estimation accuracy on λ. In these figures, the f 1 , . . . , f 1000 values computed by Kmerlight were segregated with respect to their associated λ = ⌈F 0 /f i ⌉ ratios. We note that the λ associated with an f i value is obtained by taking the ratio of F 0 to the true f i value. Range of λ was restricted to [1, . . . , 500].
In Figure 6, two plots are provided to show the dependence of estimation error on λ. The left side plot is for k = 15 and the right side plot is for k = 21. In each plot, the mean relative error as well as its standard deviation for all f i values associated with a given λ value are plotted. For any fixed λ, estimates from all 1000 trials for each f i value associated with λ were included in the mean and standard deviation calculations. All λ values need not have associated f i values and hence a piece wise linear plot of the values for λs with non empty set of f i values associated with them is given. Different Kmerlight memory settings, viz., 120MB (r = 16) and 500MB (r = 18), are considered in each of the two plots. As seen in Figure 6, relative errors increase with increasing λ and decrease with increasing memory size. Table III give frequency statistics of 15-mers, viz. g 1 , . . . , g 3 , of human reference chromosome Y (GRCh38) obtained using exact counting. There were in total 13493236 distinct 15-mers in the Y chromosome. Reads from chromosome Y were generated using ART tool with read length 100 and coverage value 50. The built-in profile of Illumina HiSeq 2500 system was used by the ART tool. Exact k-mer abundance histogram of the reads was computed using exact counting. Second and third columns of Table V gives the first three peak positions and corresponding values observed in the exact histogram, ignoring the initial peak due to erroneous k-mers. For these peak positions, the peak values were also inferred using the model, which are given by g i / √ 2πiλ ′ . The g i values from Table III were used. Value of λ ′ was obtained from peak positions since peak positions are given by iλ ′ according to the model. Estimated peak values and the relative errors are given in third and last columns respectively of Since computing exact histograms are resource intensive, we used histograms computed by Kmerlight in place of the exact histogram for the inferencing of g i values as given above. Figure 8 shows the abundance histogram computed by Kmerlight alongside the exact histogram. Kmerlight histograms computed with three different memory settings viz., 120 MB, 460 MB and 960 MB, are shown. Values for f 5 , . . . , f 200 are plotted in each histogram. Plot of f 1 , . . . , f 4 values, which are part of the initial sharp peak due to erroneous k-mers, are omitted from the plots because including them require a very large y-axis range which reduces the resolution of the remaining plot.
Table VI compares the relative errors in the g i estimation using exact histogram and using histograms computed by Kmerlight. Third and fourth columns of Table II give the relative errors in the estimation using histograms computed using Kmerlight with 500 MB and 940 MB memory respectively. Kmerlight errors are averaged over 1000 trials. The standard deviations are also given alongside these values. As seen in Table VI, relative errors in g i estimation are similar when histogram computed by Kmerlight is used in place of exact histogram.
APPENDIX C CHOOSING k-MER LENGTH IN GENOME ASSEMBLY
For choosing appropriate k-mer length in de Bruijn based assemblers, Chikhi et al. [4] propose that the most appropriate choice of k is the one that provides maximum number of distinct true k-mers to the assembler. This has been shown to yield excellent assembly on a diverse set of genomes. Recalling that F 0 denotes the total number of distinct k-mers in the sequence reads, F 0 can be written as F 0 = F e 0 + F ′ 0 , where F e 0 and F ′ 0 are the total number of distinct erroneous k-mers and true k-mers respectively. Thus, the objective is to choose k that maximizes F ′ 0 . Estimation of F ′ 0 can be done using estimates of F 0 and F e 0 as F ′ 0 = F 0 − F e 0 . Summing the f i values in the initial histogram peak due to erroneous k-mers provide an estimate of F e 0 . This along with F 0 estimate from Kmerlight can be used to estimate F ′ 0 . There could be overlap between the region of erroneous k-mers and true k-mers in the histogram, which for instance could happen in case of low coverage read generation. This can introduce error in F e 0 estimation obtained by summing the initial f i values. In this case, either by using regression techniques or by fitting models for erroneous k-mer generation, f i values corresponding to only erroneous k-mers in the overlap region can be inferred.
We make the simplifying assumption that all hash functions used are truly random. We refer the reader to [22] where it is shown that standard hash families such as 2-universal hash families 'well approximates' ideal hash functions when the data entropy is high. In this case, the probabilistic bounds obtained using ideal hash functions are 'close' to the bounds achievable using the universal hash families. We assume that parameters are suitably adjusted in such a way that the final failure probabilities accommodate for the additional difference arising due to use of universal hash families in practice.
Fix any f k for k ≥ 1 such that f k ≥ F 0 /λ. Consider the Kmerlight data structure and consider any fixed array T w corresponding to level w ≥ 1. We recall that counters of array T w can be either 'dirty' or 'non dirty'. Our algorithm considers only non dirty counters. The set of non dirty counters can contain both false positive counters and true positive counters. However, the algorithm cannot distinguish between these false positive and true positive counters. If a false positive counter has value k then it can erroneously contribute to f k calculation. Let X w denote the number of true positive counters in array T w each having value k after seeing the input. We will later bound the estimation error due to false positive counters.
Proof: Let x 1 , x 2 , . . . , x r denote r indicator random variables where x i = 0 if the counter i in array T w is true positive and holds value k.
The last inequality follows from facts r/2(r − 1) ≤ 1 for r ≥ 2 and 1 − 2x ≤ (1 − x) 2 . Observing that the second term of the last inequality is E 2 (X w ), we conclude that V ar(X w ) = E(X 2 w ) − E 2 (X w ) ≤ E(X w ). First we show lower and upper bounds on E(X w ). Let θ = r/(r − 1). We assume r ≥ 2. Let Using Claim 1, we obtain The last inequality follows from the inequality Similarly, we obtain where the last inequality follows from Claim (1), the definition of θ, and from the inequality It follows that Consider the interval [l, u], where l is the smallest level with F 0 /2 l ≤ 4r and u is the largest level with F 0 /2 u ≥ r/8. That is, From the above two inequalities, it follows that . Similarly, for w ≥ u + 1, H(w + 1) ≤ 2H(w)/3 and w≥u+1 H(w) ≤ 7 3 H(u + 1). Proof: For any w ≤ l − 2, using (7), we obtain Similarly, for any w ≥ u + 1, using (8), we obtain Hence, w≥u+1 H(w) ≤ H(u + 1) w≥0 (4/7) w ≤ 7H(u + 1)/3.
Proof:
First we show that for any given level w ∈ [l, u], |X w − E(X w )| ≥ ǫE(X w ) with probability at most 1/48. The result then follows from (9) by noting that there are at most 6 levels in [l, u].
Dealing with false positives
Consider any level w and a fixed array location T w [c] of array T w in level w. Let f p denote the probability that the counter value stored in T w [c] is false positive. Hence T w [c].v value is erroneously considered towards f k estimation for k ≥ 2. We recall that false positives do not arise in f 1 estimation.
Proof: As earlier, we assume that hash functions are truly random. Fix j ∈ {0, . . . , u − 1}. Consider the following two events: Event E 1 : Two or more elements are hashed to the pair (c, j) in level w Event E 2 : No elements are hashed to pairs (c, i) for any i = j in level w.
We observe that f p ≤ u · Pr(E 1 ∧ E 2 ). In the following, we show that Pr(E 1 ∧ E 2 ) ≤ 1/u 2 , which would then imply that f p ≤ 1/u. We can analyze Pr(E 1 ∧ E 2 ) by considering the following balls and bins experiment where F 0 = n balls are thrown into u bins in the following fashion. For each ball, a coin is tossed with success probability p = 1/(r · 2 w ). With probability 1 − p the ball is discarded. Upon success, the ball is thrown into any one of the u bins {0, . . . , u − 1} with equal probability. It is straightforward to verify that event E 1 ∧ E 2 is equivalent to the event that two or more balls are present in bin j and remaining u − 1 bins are empty. The probability of this event is given by: We use the facts that p ≤ 1 2r and r ≥ 2 to obtain that 1/(1 − p) 2 ≤ 2. Using this, we simplify the above expression as for u ≥ 8. The last inequality follows because x 2 ≤ exp(3x/4) for all x ≥ 0.
For the fixed k, let Y w denote the number of non dirty locations in the array T w each having value k at level w. Let Y w = X w + ∆ w , where X w as defined earlier is the number true positive locations and ∆ w is the number of false positive locations.
From Lemma 4, for any level w, we have, E(∆ w ) ≤ r/u.
F 0 computation
Estimate for F 0 can be computed by any of the existing approaches. Our algorithm uses the same approach of [7] to compute F 0 . It is straightforward to verify from the proof of Theorem 1 in [7] that for r = O(1/ǫ 2 ), the estimatep 0 of p 0 = (1 − 1/r) F0/2 w * computed in our F 0 estimation procedure, where w * is the level chosen by the algorithm, has the property that |p 0 − p 0 | > ǫ 3 p 0 with probability at most 1/2. Furthermore, we recall from the proof of Theorem 1 [7] that p 0 ≥ 1/3.
It follows that to estimateF 0 such thatF 0 ∈ (1 ± α)F 0 , α and ǫ are related as It is straightforward to verify that the above conditions are satisfied when ǫ/3 ≤ α ≤ ǫ. It follows that for r = O(1/α 2 ), estimateF 0 of F 0 can be computed such that |F 0 − F 0 | > αF 0 with probability at most 1/8. Using standard Chernoff bound, it follows that for s = O(log(1/δ)), more than half of s independent estimates of F 0 deviate by more than αF 0 from F 0 is at most δ/2. In other words, the median of s independent estimates deviates by more than αF 0 from F 0 with probability at most δ/2.
Probability amplification through median computation
Using again the median argument given above, it follows that by using median of O(log(λ/δ)) independent estimates as the final estimate, for any fixed f k for k ≥ 1, an estimatef k can be computed such that |f k − f k | ≥ ǫf k with probability at most δ/(2λ). Noting that there are at most λ such k with f k ≥ F 0 /λ, it follows by union bound that the probability of obtaining a bad estimate for either F 0 or for any f k where f k ≥ F 0 /λ, is at most δ.
The proof presented here does not attempt to obtain tight constants in the asymptotic space bounds. A more rigorous proof could possibly provide space bounds with tight constants that matches the experimental findings. | 2016-09-19T08:01:16.000Z | 2016-09-19T00:00:00.000 | {
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218955217 | pes2o/s2orc | v3-fos-license | Self-control Mediates the Relationship between Psychosocial Strengths and Perceived Severity of COVID-19 among Frontline Healthcare Professionals of Pakistan: A Single Center Experience
Objective: To examine the relationship between psychosocial strengths (resilience, self-efficacy beliefs and social support) and perceived severity of COVID-19 and also to gauge the mediating role of self-control among frontline health care professionals of Pakistan. Methods: A cross-sectional research design was utilized from March to April 2020 from one medical teaching hospital of South Punjab. As it was a single center experience so all the doctors were approached and asked to participate in this research. In total, 284 doctors (out of 300 approx.) completed online survey. The data were collected through online google forms consisting of self-report measures i.e. Brief Resilience Scale, Short General Self Efficacy Scale, Brief Scale for Social Support, Risk Behavior Diagnostic Scale and Brief Self-Control Scale. Results: The results were analyzed by using SmartPLS (3.0), direct effect of psychosocial strengths on perceived severity of COVID-19 and indirect effect of self-control were assessed through path coefficients, t-values and r-square values. The results confirmed that there was significant negative relationship between psychosocial strengths and perceived severity of COVID-19 (β = -0.854, t =14.279) with 72% variance in perceived severity due to psychosocial strengths. Further, the results also suggest that self-control proved significant mediator between psychosocial strengths and perceived severity (β = -0.604, t = 11.004, variance in perceived severity is 74%). Conclusion: In the time of pandemic, medical professionals are working as frontline force and can have several uncertainties regarding the risk associated with outbreak of COVID-19. This study concludes psychosocial strengths can play a significant role in subsiding the risk associated with severity of disease. Whereas, self-control can significantly contribute to buffer the negative influence of COVID-19 among frontline medical professionals. In line with findings of this study, there is a dire need to initiate psychotherapeutic studies for medical professionals to boost up their psychosocial strengths that would make them resilient against COVID-19.
INTRODUCTION
The outbreak of COVID-19 has created a sense of rapid emergency, everyone is victim of varied psychological responses. 1 It is not only an extraordinary public health concern but also a huge distressing factor for the healthcare providers and medical staff too. 2 Health professionals, especially those working in hospitals caring for people with confirmed or suspected COVID-19 cases, are more vulnerable to become victim of this deadly virus. That's why they are more prone to develop psychosocial health issues. In the same vein, a study conducted in Beijing, at the time of SARS outbreak exhibits that fear of contagion, depression, anxiety, and frustration is reported alarmingly high among frontline healthcare professionals; especially who are dealing with SARS-COV-2 positive patients. [3][4] Similarly, a recent study at Wuhan also reported that medical staff who had previous contact with COVID-19 patients exhibited high levels of psychosocial problems including fear of contagion. 5 It is reasonable to assume that outrageous growth is evident in the number of positive COVID-19 patients, why we should not quantify the role of protective shields (psychosocial strengths) that directly or indirectly safeguard psychological health of frontline medical professionals. These psychosocial strengths are key elements that boost well-being of individuals facing adversity. 6,7 Further, it has been reported that self-control is prime factor for the cultivation of resilience and other psychosocial strengths among individuals. 8 In general, self-efficacy is also an important factor that influences an individual's ability to exert self-control. 9,10 It is established that resilience, efficacy beliefs, accepting challenges and positive coping strategies reduce perceived severity of disease in individuals. 11 Prior studies on pandemics, found that good self-control is not directly link to psychosocial strengths but also intimidate negative effects, particularly, impact of perceived severity of disease. [12][13][14] In the light of above cited scenario, current study sought to examine the relationship between psychosocial strengths and perceived severity of COVID-19 through the lens of self-control among frontline healthcare professionals of Pakistan.
METHODS
The cross-sectional research was conducted in a teaching and medical hospital of South Punjab, where medical professionals are directly exposed to dealing with COVID-19 patients. It was a single center research so all frontline healthcare professionals (i.e. doctors) were approached to participate in this study. Study conducted from March to April, 2020. There were total 300 doctors deputed as frontline professionals and 284 willingly filled the online survey (through google forms) consisting of self-administrated questionnaires Psychosocial strengths were measured through three scales i.e. Brief Resilience Scale (BRS) 15 a six items short scale to measure resilience, Short General Self-Efficacy Scale (SGSE) 16 is also a six items inventory and measures self-efficacy beliefs within an individual and Brief Scale for Social Support (BSSC) consist of nine items which measure the emotional, interpersonal and material support. 17 Further, Risk Behavior Diagnostic (RBD) scale is three items measures which was used to measure perceived severity of COVID-19 18 and Brief Self-Control Scale (BSCS) was used to measure selfcontrol. 19 The ethical approval was obtained from ethical research committee of concerned department institution. The informed consent was also acquired from the participants and they were also ensured about the confidentiality of information they provided. Additionally, permission to employ measurement instruments was also obtained from the authors/institutions, wherever desired.
The data were analyzed through SmartPLS (3.0). To check psychometric estimates of measurement instruments, reliability and validity tests were employed. Further, the effect of psychosocial strengths on perceived severity (direct effects of three IVs) and mediating role of self-control (indirect effect of mediator) were observed through path coefficients (β), r-square and effect size through f 2 . Whereas, the t-values were considered for the significance of model through bootstrapping and Q 2 values depicted the relevance of model through blindfolding.
RESULTS
The baseline characteristics of respondents were obtained through the frequency distribution of gender; among 284 respondents, 176 were male (62%) and 108 were females (38%). Cronbach's alpha, composite reliability, average variance estimated and discriminant validity are shown in Table-II and Fig.1. Whereas, the mediation model shows significant mediating role of selfcontrol in Table-III and Fig.2. ates of constructs. The reliability and validity estimates of the constructs are given in Table-I.. For psychosocial strengths, three constructs for resilience, selfefficacy and social support measures were used. In perceived severity of COVID-19 on the frontline health care professionals with the significant negative correlation, (path coefficient), mean, SD, t value, p value, R square, effect size and cross validation redundancy estimates for model relevancy is shown in Table-II. The mediating role of self-control between psychosocial strengths and perceived severity of COVID-19 among the frontline health care professionals with the correlation value (path coefficient), mean, SD, t value, p value, R square, effect size and cross validated redundancy estimates Table-III. Here it can be clearly seen that self-control is a strong mediator between psychosocial strengths and perceived severity of disease.
DISCUSSION
This study proved significant negative correlation between psychosocial strengths and perceived severity of COVID-19 among health care professionals. It clearly depicts the importance of psychosocial strengths that safeguard medical professionals if these shields are well-nurtured. 21 Although, some of the psychosocial strengths are in-built, man cannot polish or boost them, but few are learned and can be refined with psychological training. At the time of crisis, we can select our medical professionals with high level of these strengths, so that other cohorts can be protected.
Plenty of studies proved that resilience and perceived social support can minimize the perceived severity of disease. 6,11,12 As Pakistani we are used to live in collectivistic settings either in homes or outside. This naturally provides social support all the time. At the time of adversity, this need is multiplied. It is good and prudent finding for those medical professionals whom facing this hardship directly, must maintain their social contact with others -family, friends, and significant others. Although it is advised to maintain a social distance to remain safe from SARS-CoV-2. In real terms, it is necessary to maintain physical distance rather social distance; here is a dire need to stay connected (either utilizing remote means) with others to get social support to maintain good mental health. Similarly, World Health Organization also suggesting the frontline medical professionals to take care their stress level and mental health along with physical health. 22 Self-control is also seen as mediating factor between psychosocial strengths and severity of COVID-19, that remined significant with 74% variance. It shows that we cannot minimize the role of self-control, when enhancing the psychosocial strengths of medical professionals. Self-control buffer in reducing the negative influence of perceived severity. 12,13 The current findings illustrate that prevalence of psychosocial strengths i.e. resilience, selfefficacy beliefs and social support in the health care providers, facilitate to manage the risk of pandemic related stigma and fears. Further, self-control is the strong psychological strategy for enhancing all the positive coping mechanisms within the frontline doctors. In Pakistan, this is the first study that encompasses psychosocial needs of frontline medical heroes that protect their mental health if well-cherished.
Limitations and future avenues of study:
Considering as a baseline research, this study collected data from doctors only, it is expected to include allied medical staff in upcoming researches. It was a single center experience, so we suggest the veterans to extend this study on other quarantine centers of Pakistan. There are several other psychosocial factors which need to be addressed and qualitative research should also be commissioned to understand the in-depth and lived experiences of affected.
CONCLUSIONS
Psychosocial strengths are very important indicators for minimizing the risk related to pandemic COVID-19. At this time of crisis, several stressors are faced by the health care professionals but those who have strong self-control, have resilient personality, self-efficacy beliefs and adequate social support can cope up well with the adversity. | 2020-05-07T09:08:43.637Z | 2020-05-01T00:00:00.000 | {
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73509916 | pes2o/s2orc | v3-fos-license | Interim 2018/19 influenza vaccine effectiveness: six European studies, October 2018 to January 2019
Influenza A(H1N1)pdm09 and A(H3N2) viruses both circulated in Europe in October 2018–January 2019. Interim results from six studies indicate that 2018/19 influenza vaccine effectiveness (VE) estimates among all ages in primary care was 32–43% against influenza A; higher against A(H1N1)pdm09 and lower against A(H3N2). Among hospitalised older adults, VE estimates were 34–38% against influenza A and slightly lower against A(H1N1)pdm09. Influenza vaccination is of continued benefit during the ongoing 2018/19 influenza season.
Influenza A(H1N1)pdm09 and A(H3N2) viruses both circulated in Europe in October 2018-January 2019.
Interim results from six studies indicate that 2018/19 influenza vaccine effectiveness (VE) estimates among all ages in primary care was 32-43% against influenza A; higher against A(H1N1)pdm09 and lower against A(H3N2). Among hospitalised older adults, VE estimates were 34-38% against influenza A and slightly lower against A(H1N1)pdm09. Influenza vaccination is of continued benefit during the ongoing 2018/19 influenza season.
Seasonal influenza vaccine is recommended in all European Union (EU) countries for older people and others at increased risk of severe influenza and its complications, including those with chronic diseases [1]. In the United Kingdom (UK), incremental introduction of a universal childhood influenza vaccination programme began in 2013/14 [2]. [3].
The early 2018/19 influenza season in Europe was characterised by both influenza A virus subtypes circulating widely. There was co-circulation in some countries, with others reporting dominance of either A(H1N1)pdm09 or A(H3N2) viruses. The season started late in most countries compared with previous seasons, with few influenza B viruses detected in the WHO European Region [4]. Since the 2008/09 season, the UK, Denmark, Spain, and several other EU countries conducting multicentre studies, have participated in I-MOVE (Influenza -Monitoring Vaccine Effectiveness in Europe), a network measuring influenza vaccine effectiveness each season.
We summarise interim 2018/19 season influenza vaccine effectiveness (VE) estimates from four singlecountry and two multi-country studies, including both outpatient and hospital settings, in order to help guide influenza prevention and control measures for the rest of the 2018/19 season.
Study setting
The primary care (PC) setting studies were conducted in Denmark (DK-PC), Spain (ES-PC), the UK (UK-PC) and via the European Union (EU) I-MOVE multi-country network (EU-PC). The hospital setting (H) studies were undertaken in Denmark (DK-H) and via the EU I-MOVE multi-country network (EU-H) ( Figure 1).
Study design and estimation of vaccine effectiveness
The methods of these six studies are described in detail elsewhere [5][6][7][8][9]. All six studies used a test-negative case control design, with differences between studies in how data were collected and how patients were selected (Table 1) [10]. Briefly, individuals presenting to participating healthcare settings with symptoms of influenza-like illness (ILI) (primary care settings) or severe acute respiratory infection (hospital settings) were swabbed. These samples were then tested by reverse transcription (RT)-PCR for influenza virus. Patients with positive results were classified as cases (by influenza virus (sub)type), and those with negative results as controls.
Patients were defined as vaccinated with the 2018/19 influenza vaccine if they were vaccinated at least 14 or 15 days (depending on the study) before symptom onset. Patients were excluded if they were vaccinated fewer than 14 or 15 days before symptom onset, or if the date of vaccination was unknown.
In eight EU-PC countries, DK-PC and DK-H, all or a random sample of influenza virus-positive specimens were selected for sequencing (haemagglutinin genome segment and/or whole genome). In ES-PC, in regions not included in EU-PC, an ad hoc sample of influenza viruses was sequenced. In UK-PC, all influenza viruses with sufficient genetic material (Ct value < 31) were sequenced, as well as all viruses derived from vaccinated cases. Sequencing results in Denmark were combined for both studies (DK-PC and DK-H).
We computed VE by comparing the odds of vaccination between cases and controls (VE = (1 -odds ratio (OR)) x 100%). All studies used logistic regression to adjust their VE for measured confounding variables (Table 1). Study-specific VE was estimated overall and where possible, by age group and target population (as defined locally in the various studies and study sites) against influenza A overall, A(H1N1)pdm09 and A(H3N2). If the number of cases (or controls if lower) per parameter was less than 10, a sensitivity analysis was performed using Firth's method of penalised logistic regression to assess small sample bias [11,12]. Where exposed case numbers were zero, exact logistic regression was used.
Virological results
All 265 A(H1N1)pdm09 viruses sequenced belonged to clade 6B.1 (A/Michigan/45/2015) ( Table 3). Among 240 viruses (91%) with information on substitutions in the haemagglutinin gene, all harboured additional substitutions of S74R (except one of the 83 sequenced in DK-H/DK-PC), S164T and I295V, and most of them also included the substitution S183P. The proportion of other substitutions identified (T120A, N129D, E235D and K302T) differed by study ( Table 3). None of these substitutions involve a change in potential glycosylation sites.
Sensitivity analyses
Sensitivity analyses for small sample size gave similar results (absolute difference range 1-9%). , not all specimens from the study period were processed. e All include additional substitutions S74R, S164T and I295V, and most also include S183P substitutions. f Representative strains for the clades.
Discussion
Interim results from six established influenza VE studies across Europe for the 2018/19 season indicate that VE against laboratory-confirmed influenza A ranged between 32% and 43% among all ages in primary care and hospital settings and was 59% in the target groups for vaccination.
Against influenza A(H1N1)pdm09, VE point estimates among all ages ranged from 40% to 71%, and were lower among older adults in DK-PC, DK-H and EU-H, ranging from 0% to 37%. Against influenza A(H3N2), the results of three of four primary care studies suggest that the vaccine was not effective among all ages combined. The VE point estimate against A(H3N2) was higher among older adults in EU-H and among 18-64-year-olds in DK-PC (47% and 48%, respectively). The low number of A(H3N2) cases in all studies resulted in less precise VE estimates against A(H3N2) than against A(H1N1)pdm09.
The influenza A(H1N1)pdm09 VE point estimates among all ages in EU-PC, among adults in DK-PC and EU-PC and among children in the UK-PC were similar to 2018/19 interim VE estimates in Canada [13]. For all ages combined, point estimates for this subtype for ES-PC and DK-H were similar to those recently reported from the United States (US) [14]. In UK-PC, the LAIV4 VE point estimate was high against influenza A(H1N1)pdm09, although sample size was very small. This suggests that the A(H1N1)pdm09 LAIV4 vaccine virus strain change from A/Bolivia/559/2013 to A/ Slovenia/2903/2015 that took place after the 2016/17 season may have improved vaccine performance against circulating strains in 2018/19. Compared with 2017/18 interim season estimates in studies where influenza A(H1N1)pdm09 VE results were available, the 2018/19 adjusted VE against influenza A(H1N1)pdm09 was similar in the 18-64 years age group in DK-PC (66% vs 60%, respectively, noting that in 2017/18 the setting in Denmark was primary care and hospital combined) and among all ages in EU-PC (71% vs 68%, respectively). VE was lower among those aged 65 years and older in DK-PC, but similar in the DK-H study.
The genetic diversity observed in the ongoing 2018/19 season did not seem to affect the VE against influenza A(H1N1)pdm09 in most groups and studies. To date, all A(H1N1)pdm09 viruses characterised in Europe were antigenically similar to the vaccine virus [15]. The lower VE among those aged 65 and older in DK-PC may be explained by small sample size, but needs further investigation.
As observed in the 2017/18 season, the 2018/19 interim primary care results suggest that VE against medically attended laboratory-confirmed influenza A(H3N2) was low or non-existent although, due to small sample size, these interim 2018/19 results need to be confirmed by the end-of-season results. End-of-season cladespecific VE results may help us understand whether regional differences in circulating clades of A(H3N2) viruses explain the difference in VE in DK-PC compared with all other primary care studies. Adaptation/alteration of the vaccine seed virus during propagation in eggs, impacting antigenicity, may have been an important explanation for low VE against influenza A(H3N2) in recent and current seasons [16].
The late start of the season resulted in small sample sizes and low precision of many VE estimates, which presents a limitation in this interim analysis. We thus conducted a sensitivity analysis to address potential small sample bias arising from this. Further limitations potentially present in all observational studies include residual confounding and bias.
Vaccination continues to be the most effective preventive measure against influenza and uptake of the 2018/19 influenza vaccines should still be promoted in countries with ongoing influenza virus circulation in line with national guidelines and recommendations. Our results further support the need for effective interventions against influenza A(H3N2) across all age groups. In the UK, the Joint Committee on Vaccination and Immunisation has recently advised the use of cellgrown influenza vaccine that will be licensed for the 2019/20 season for older children and adults in the UK [17]. In addition, given the observed non-effectiveness of the A(H3N2) component of the current vaccine in previous seasons, in settings with influenza A(H3N2) virus circulation, prophylactic and prompt therapeutic use of neuraminidase inhibitors is important to help prevent severe outcomes, irrespective of vaccination status [18]. End-of-season VE and antigenic studies will provide insight into age-and study-specific variation in VE estimates. In addition, monitoring effectiveness of the 2019 southern hemisphere influenza vaccine against influenza viruses and their genetic diversity will be important to prepare for the next influenza season in the northern hemisphere. | 2019-03-11T17:22:21.057Z | 2019-02-21T00:00:00.000 | {
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33779864 | pes2o/s2orc | v3-fos-license | Management of mandibular body fractures in pediatric patients: A case report with review of literature
Mandibular fractures are relatively less frequent in children when compared to adults, which may be due to the child's protected anatomic features and infrequent exposure of children to alcohol related traffic accidents. Treatment principles of mandibular fractures differ from that of adults due to concerns regarding mandibular growth and development of dentition. A case of a 4.5-year-old boy with fractured body of mandible managed by closed reduction using open occlusal acrylic splint and circum mandibular wiring is presented. This article also provides a review of literature regarding the management of mandibular body fracture in young children.
Introduction
The reported incidence of pediatric injuries accounts for 4-6% of the total. Below the age of 5 years, the incidence of pediatric facial fractures is even lower, ranging from 0.6 to 1.2%. [1] The principles of management of mandibular fractures differ in children when compared to adults. While in the adults, absolute reduction and fixation of fractures is indicated, in children minimal manipulation of facial skeleton is mandated. The goal of treatment of these fractures is to restore the underlying bony architecture to pre-injury position, in a stable fashion, as non-invasively as possible, with minimal residual esthetic and functional impairment.
Depending on the type of fracture and the stage of skeletal development the treatment modalities range from conservative non-invasive through closed reduction and immobilization methods to open reduction with internal fixation. Disruption of the periosteal envelope of the mandibular body may have an unpredictable effect on growth.
Thus, if reduction is required, closed reduction is favored.
Case Report
A 4.5-year-old boy reported to the dental clinic with bleeding from oral cavity following fall from bicycle. Clinical examination revealed bruise on the chin, open mouth appearance with profuse bleeding from the oral cavity and derangement of occlusion [ Figure 1].
Step deformity with tenderness and mobility was elicited along the lower border of the mandible on the left side canine region. Preoperative orthopantamogram (OPG) could not be taken due to the profuse bleeding needing immediate intervention.
Under sedation, upper and lower arch alginate impressions were taken and stone casts were poured. An open occlusal acrylic splint was fabricated [ Figure 2], and under general anesthesia, the mandibular body fracture was immobilized, fixed with the acrylic splint which was retained by circum mandibular wiring [Figures 3 and 4]. Patient was reviewed every week, and on the third postoperative week, the circum-mandibular wiring and splint was removed under local anesthesia. No mobility was present at the fracture site. Postoperative recovery was uneventful and occlusion achieved was satisfactory, although spacing was seen in incisor-canine region [ Figure 5]. Patient was reviewed monthly for 6 months. On 2 months follow-up, the spacing had closed and the child had good alignment of teeth [ Figure 6]. Patient had perfect occlusion and good masticatory efficiency.
Review of literature
Facial fractures in children account for the approximately 5% of all facial fractures. [2] A male predilection is seen in all age groups. The most common fracture in children requiring hospitalization and/or surgery generally involves the mandible and, in particular, the condyle. Fractures in the condylar region are the most common, followed by angle and body fractures. The etiologies of mandibular fractures in children are usually falls and sports injuries.
The protective anatomic feature of a child's face decreases the incidence of facial fractures. In young children (less than 5 years of age), the face is in a more retruded position relative to the "protective" skull, therefore, there is a lower incidence of midface and mandibular fractures and a higher incidence of cranial injuries. With increasing age and facial growth directed in a downward and forward direction, midface and mandible becomes more prominent; thus, the incidence of facial fractures increases, while that of cranial injuries decreases. [3] The high elasticity of young bones, a thick layer of the adipose tissue covering them, a high cancellous-tocortical bone ratio and flexible suture lines are some of the reasons contributing to the low incidence of facial fractures and minimal displacement of the fracture fragments. [4][5] The clinical features of a fractured mandible in a child are the same as in an adult, which includes pain, swelling, trismus, derangement of occlusion, sublingual ecchymosis, step deformity, midline deviation, loss of sensation due to nerve damage, bleeding, TMJ problems, tenderness, movement restriction, open bite and crepitus. Thorough clinical examination, however, may be impossible in uncooperative young trauma patients. Lacerations should be evaluated to reveal injuries to underlying structures. General palpation should be applied over all bony surfaces of the mandible. The mandibular range of motion must be examined as patients actively open and close their mouth.
For children, the imaging technique which is of value, especially following trauma, is a computed tomography (CT) scan, since plain radiographs in young children are less helpful than in adults due to unerupted tooth buds obscuring fractures, the increased incidence of greenstick fractures and the fact that the cortex is underdeveloped, leading to difficulty in visualizing fractures. [6] Treatment of mandibular fracture in children depends on the fracture type and the stage of skeletal and dental development. [7] Mandibular growth and development of dentition are the main concerns while managing pediatric mandibular fractures. In adults, absolute reduction and fixation of fracture is indicated, whereas in children minimal manipulation of the facial skeleton is mandated. The small size of the jaw, existing active bony growth centers and the crowded deciduous teeth with permanent tooth buds located in great proximity to the mandibular and mental nerves, all significantly increase the therapy related risks of pediatric mandibular fractures and their growth related abnormalities.
Intact active mandibular growth centers are important for preserving mandibular function, which have a significant influence on future facial development. Thus, restoration of the mandibular continuity after fracture is important not only for immediate function but also for future craniofacial development. Accordingly, the goal of treatment is to restore the underlying bony architecture to its pre-injury position in a stable fashion as non-invasively as possible with minimal residual esthetic and functional impairment.
Problems encountered in management of pediatric mandibular fractures • Loose anchorage system due to attrition of deciduous teeth and physiologic resorption of roots. [7] • Precarious dental stability in the mixed dental development period. • Difficulties in securing IMF using arch bars and eyelets as primary teeth are not sufficiently stable and may be avulsed due to the pressure exerted. In addition, the partially erupted secondary teeth are not sufficiently stable in the pediatric soft bone. [2,7] • Shape of the primary teeth: Conical shape with wide cervical margins and tapered occlusal surface makes placement of wires technically challenging. [4] • Restricted normal dietary intake in children on IMF was reported to result in significant weight and protein loss and reduced tidal volume. [7] • Children on IMF are at an increased risk of aspirating gastric contents should they vomit. [7] • The wires cause discomfort and damage periodontal tissues. [2,7] Despite the fact that the goal of treatment is to establish the bony architecture to pre-injury state as non-invasively as possible, the challenges with closed reduction and IMF are many. However, some authors have indicated that IMF using arch bars is safe in children, especially those older than 9 or 11 years.
Alternative devices for closed reduction
Several studies have recommended the use of pre-fabricated acrylic splints as a treatment for pediatric mandibular fractures. These splints are more reliable than open reduction or IMF techniques with regard to cost effectiveness, ease of application and removal, reduced operating time, maximum stability during healing period, minimal trauma for adjacent anatomical structures and comfort for young patients. [8] Laster et al, [9] described nickel titanium staples which are inserted in a relatively non-invasive and pain free manner and their eventual removal, if required, is done as quickly as their insertion, facilitated by the fact that the staples are not osseointegrated. Due to their superficial location, there is little risk for inhibiting and deforming facial bone development or having any proximal strategic structures such as nerves and developing dentition. Furthermore, the reduced compression rendered by the staples on the bony fragments result in primary healing with no callus produced.
• Modified orthodontic brackets have been used for maxillomandibular fixation (MMF). [10] • Orthodontic resin has been used for fixation of mandibular fractures in children. [2] • Orthodontic rubber elastics in combination with fixed orthodontic brackets were used to create compressive horizontal force marginally over the mandibular fracture site from one side to the other. [2] • A modified orthodontic splint appliance has been applied to fractures where two orthodontic bands are fit on the primary second molars with rounded stainless steel arch wires soldered to them on the buccal and lingual side. [11] The most common pediatric mandibular fracture: Greenstick fractures A greenstick fracture is a fracture in which one cortex of the bone is broken and the other cortex is bent. Pediatric patients are more likely than adults to sustain greenstick or incomplete fractures. The relatively high elasticity of the mandibular body's thin cortical bone and a thick surrounding layer of adipose tissue and the relatively larger amount of medullary bone held by a strong periosteal support results in a high incidence of greenstick fractures in children. [5] A greenstick fracture will ensure stability of the undisplaced segments in children less than 5 years. [5] Furthermore, the osteogenic potential of the periosteum in the developing craniofacial skeleton is very high and will lead to somewhat rapid and easier healing which occurs under the influence of masticatory stress, even when there is imperfect apposition of bone surfaces. [12] Thus, there is a greater degree of tolerance permissible in the alignment of fragments and restoration of occlusion, which will subsequently be corrected by alveolar bone growth at the time of eruption of permanent teeth. [5] Therefore, management of greenstick fractures without displacement and malocclusion would merely be close observation, a liquid-to-soft diet, and avoidance of physical activities (e.g. sports and analgesics). [4] There may be cases in which the fractures can be snapped back into a good reduced position and held by the periosteal sleeve, the fracture surfaces and even by the occlusion. [5] For greenstick/ minimally displaced fractures, conservative closed reduction is the most recommended treatment. [9] The closed reduction and immobilization approach can be achieved by means of acrylic splints, circumferential wiring, arch bar or gunning splints. [9] These techniques provide a good reduced position, continuity of periosteal sleeve and maintenance of the soft tissue, thus creating a positive environment for rapid osteogenesis and remodeling processes as well as prevention of any type of non-fibrous union. [12] Furthermore, in the splinted mandible, the fracture segments are tightly fixed and serve in reducing tenderness and pain reactions during a child's daily activity. [12] Open reduction management of mandibular body fractures in children Till the mid-seventies, closed reduction by means of IMF was used for all types of pediatric fractures. [12] Today, open reduction and rigid internal fixation (ORIF) has become the standard of care for management of displaced fractures. [4] ORIF includes micro or miniplates or biodegradable devices which significantly increase the therapy related risks previously mentioned. Nonetheless, this technique provides stable three-dimensional reconstruction, promotes primary bone healing and shortens treatment time. Posnick et al, [6] claimed that a decreased dependence in IMF improved postoperative respiratory care, nutritional intake and oral hygiene measures.
The treatment modality for displaced mandibular fractures is debatable between closed reduction and open reduction.
While different open reduction techniques have been the standard of care for adults for a long time, its suitability for children remains controversial. [5] The effect of implanted hardware in the mandible of a growing child is not completely understood. Damage to the periosteum and surrounding soft tissues and potential damage to primary teeth and permanent tooth gums is to be considered when choosing internal fixation. [6,7] Rigid internal fixation might create artifacts on CT scans or magnetic resonance imaging (MRI) and may be visible or palpated through the child's thin skin and cause pain and early or late infection. [4] Therefore, the decision to use ORIF in children should be taken with great caution and only if other means of reduction and fixation are not attainable. Miniplate and screw devices have revolutionized the modern management of facial fractures by enabling precise anatomical reduction and fixation under direct vision.
Problems with ORIF in pediatric trauma
• Presence of developing tooth germs, though the inferior border of the mandible can be plated either through an intraoral/extraoral approach. Additionally, the extraoral approach carries the risk of scarring and damage to the marginal mandibular nerve. [5,7] • Interference with growth due to placement of miniplates. [5] • General anesthesia and hospitalization is needed for removal of the hardware after complete healing. [2] • Allergic reactions to the metal resulting in inflammation that needs removal of plate have been reported. [2] • Stress shielding, especially after rigid plate fixation, has been reported and may cause weakening of the bone after removal of the implant. [2] • Corrosion and release of metal ions can lead to removal of the fixation device. [2] Recent advances in ORIF Currently, ORIF with resorbable osteosynthesis plates and screws is increasingly being used in children. These biodegradable materials do not interfere with radiodiagnostic techniques due to their radiolucency and they are sufficiently rigid and stable. They eventually degrade, resorb and are eliminated from the body. Although the secondary implant removal operations are avoided and there are no side effects on the growing skeleton, the risk of damaging tooth buds in the pediatric jaw is still present due to drilling for direct application of the resorbable plates and screws. [2] Eppley [2] claims that this risk is minimal since the drill hole and the tapping of the screw penetrate only the outer cortex of the bone. Even if the resorbable screw tip encroaches upon a tooth, its tip is blunt and non-penetrating. Subsequent resorption of the screw removes any potential obstruction to tooth eruption.
Despite the above reports, the literature advocates conservative management of mandibular body fractures at young ages with the added advantage of decreased immobilization time, decreased muscular atrophy and better oral hygiene. [2] Complications Complications per se are very rare in pediatric trauma due to the child's greater osteogenic potential, faster healing rate and less frequent requirement of ORIF. Furthermore, a greater number of fractures are minimal to non-displaced. Late complications such as damage to permanent teeth may occur in 50% of mandibular fractures. TMJ dysfunction (recurrent subluxation, noise and pain, limited condylar translation, deviation on opening, ankylosis) and growth disturbances (e.g. secondary mid face deformity, mandibular hypoplasia and asymmetry) usually occur in pediatric patients with severely comminuted fractures. [2,7] Malocclusion as a complication of pediatric facial fractures is rare. It has been attributed to short fixation time in alveolar fractures and may be caused by growth abnormalities following condylar fractures. [3] Spontaneous correction of malocclusion is seen as deciduous tooth shed and permanent teeth erupt. [7] Ellis et al, [2] did not find occlusal complication associated with the use of closed treatment and IMF.
Lois et al, [2] found no difference in the complication rates of fractures treated by MMF versus ORIF (4.3% and 5.45%, respectively). They concluded that in fractures with displacement in the range of 2-4 mm, there is no difference between MMF and ORIF.
A recent study of mandibular fracture complications in children [2] noted a lower complication rate (9.1%) with closed treatment of mandibular body, angle and parasymphyseal fractures, while open reduction using miniplate, mandibular plate and mandibular/miniplate fixation revealed a higher rate of complication (30%, 28.6% and 29.2%, respectively). Infection and wound dehiscence occur less frequently with closed versus open.
Nonunion occurs significantly less often in closed reduction than in open treatment. Nonunions of the mandible may develop due to a number of factors, including poor patient compliance with postoperative care, metabolic disturbances and generalized disease states, which can all lead to inadequate bone healing. [2] Other local causes for non union may be related to inadequate immobilization of fracture segments, infections at the fracture site, tissue or foreign body between the segments and the inadequate reduction the fracture segments. [2] However, Ellis et al, [2] found lower complication rates in patients with comminuted mandibular fracture, who underwent open reduction and fixation (10.3%) than in those who were treated with closed reduction with IMF (17.1%).
Yerit et al, [2] found uneventful healing and no complications when using resorbable osteosynthesis plates and screws for ORIF, while others described the same type of complications as mentioned for non-resorbable miniplates infections due to mucosal exposure of the plates, premature occlusal contact and TMJ disorders. [2] Long-term follow-up on facial and teeth development A long-term follow-up period is recommended postoperatively in cases of mandibular body fractures in children. [8] Facial growth pattern and mandibular movements should be recorded. Ranta and Ylipaavalniemi [2] pointed out that teeth in which root development has already begun at the time of fracture, appear to erupt normally; however, marked deformation of the crown and roots occur in teeth located on the fracture line when the calcification process is still in progress at the time of fracture.
Developmental disturbances occur in the lower tooth buds at the pre-calcification stages involved in the fracture and internal fixation site, which include damage to the pulp causing pulp obliteration and extensive root resorption as well as impaction. Koenig et al, [2] pointed out that the developing follicle is more elastic than the surrounding bone and better able to survive mechanical injury. Nevertheless, it is difficult to predict the facts of tooth buds and fracture and the implanted hardware fixation.
Suei et al, [2] mentioned that the presence of infection in the fracture site is a crucial factor affecting odontogenic cells in the dental follicle. Surgical procedures as well as fixation and reduction are also potential causes of impaction. Eleonora Schiller et al, [13] report that trauma occurring between 0 and 3 years of age is likely to disturb the formation and mineralization of the permanent teeth.
Nixon and Lowey [2] concluded that mandibular fractures which occur during mixed dentition can be associated with subsequent failed eruption of permanent teeth when the fracture line is reduced using an open surgical approach.
Yocheved Ben Bassat et al, [14] reported discoloration of the crown of permanent tooth in 16% of the children with the incisal one third being the most common site. Hypoplasia was evident in 9% of the permanent teeth.
In our case, the deciduous teeth seem to have aligned well over a period of 2 months, thereby closing the spacing initially seen between the teeth. We opted for the open occlusal acrylic splint because of its ease in lab fabrication, preservation of periodontal tissue integrity, patient compliance and ease in assessing the occlusion after reduction.
Conclusion
The anatomical complexity of the developing mandible and teeth and concerns regarding biocompatability of implanted hardware often mandate the use of surgical techniques that differ markedly from those used in adults. In cases of mandibular fractures of a young child, disruption of periosteal envelope may have unpredictable effects on growth. Thus, if intervention is required, closed reduction is favored. Due to the technical difficulties of IMF, acrylic splints with circumferential wiring are recommended. | 2018-04-03T06:07:16.384Z | 2010-10-01T00:00:00.000 | {
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14028595 | pes2o/s2orc | v3-fos-license | Scalar fields superdense gravitating systems
Solutions for scalar fields superdense gravitating systems of flat, open and closed type obtained in the frame of gauge theories of gravitation are discussed. Properties of these systems in dependence on parameter $\beta$ and initial conditions are analyzed.
Introduction
As it was shown at first in [1], generalized cosmological Friedmann equations (GCFE) deduced in the frame of gauge theories of gravity (GTG) [2] besides cosmological solutions lead to solutions for some hypothetical objects -so-called superdense gravitating systems (SGS). SGS have extremely high energy density and their dynamics is essentially noneinsteinian. In considered approximation of homogeneous isotropic space SGS oscillate between minimum and maximum values of the scale factor. Later solutions for such systems in the case of various equations of state for gravitating matter were discussed in [3][4][5]. Below SGS are investigated for systems including scalar fields.
2 Generalized cosmological Friedmann equations GCFE for systems including scalar field φ minimally coupled with gravitation and radiation have the following form [7] 1 .
where R is the scale factor, k = +1, 0, −1 for closed, flat and open models respectively, H is the Hubble parameter, ρ r is radiation energy density, V (φ) is a scalar field potential, parameter β with inverse dimension of energy density, the value of |β| −1 determines the scale of extremely high energy densities.
Besides Eqs. (1)-(2) gravitational equations of GTG lead to the following relation for torsion function S and nonmetricity function The conservation law in usual form follows from Eqs. (1)-(2), as result we obtain the equation for scalar field and the integral for radiation ρ r R 4 = const. If the condition is valid, solutions of GCFE coincide practically with that of general relativity (GR) and the torsion and nonmetricity functions are negligibly small.
As it was shown in [7], the GCFE allow to build regular inflationary cosmological models with dominating ultrarelativistic matter at a bounce, if the scale of extremely high energy densities is much less than the Planckian one |β| −1 ≪ 1M 4 p (β < 0). Numerical solutions obtained in [7,8] for closed and flat models show that after inflation (|φ| < 1M p ) during certain time intervals the dynamics of inflationary models has oscillating character, namely scalar field and the Hubble parameter oscillate near values zero. Such situation is typical for SGS. This means, if |φ| < 1M p and the relation (5) is not fulfilled, by choosing some initial conditions we can obtain numerical solutions of GCFE for SGS including scalar fields. We have to take into account that by given initial conditions for magnitudes (φ,φ,R, ρ r ) there are two different solutions corresponding to two values of the Hubble parameter, which according to (1) are Initial conditions will be chosen at the moment t = 0 corresponding to extremum of the scale factor R: Eq. (6) determines on the plane P with the axes (φ,φ) curves (H 0 -curves), in points of which the Hubble parameter vanishes for H +solutions or H − -solutions. From (2) the time derivative of the Hubble parameterḢ 0 at the moment t = 0 iṡ where Z 0 = 1 − β(4V 0 −φ 2 0 ). In order to analyze solutions properties near the origin of coordinates on the plane P we will examine H 0 -curves defined by (6) for some simple scalar field potentials.
3 Extremum H 0 -curves for simplest scalar field potentials According to (1) in the case of flat and closed models we have Z ≥ 0 (β < 0) and admissible values of scalar field φ and derivativeφ on the plane P are limited by two bounds L ± defined by equationφ H 0 -curves B 1 and B 2 for flat models (k = 0) are situated on the plane P between bounds L + and L − having with them common points (points K 1 and K 2 in Fig. 1) on the axiṡ φ (if V ′ (0) = 0). Each of H 0 -curves contains two parts -extremum curve for H +solutions (H 0+ -curves) and extremum curve for The family of H 0 -curves for closed models is situated on the plane P between curves B 1 and B 2 . The family of H 0 -curves for open models is situated on the plane P between bounds L ± and corresponding H 0 -curves for flat models. 2 For given scalar field potential V (φ) the behaviour of H 0 -curves depends on parameters of potential V , on parameter β and on the model type (k = 0, ±1). Below H 0 are analyzed for scalar field potential V = 1 2 m 2 φ 2 (m = 10 −6 M p ) in the case of ρ r = 0. For flat models H 0 -curves are given in Fig. 1 (β = −1.6 · 10 12 ). The derivativeḢ 0 is positive in points of H 0 -curves if the value of |β| < |β 1 | ∼ 1.8 · 10 12 . This means that H 0 -curves in considered case are bounce curves discussed in [8]. By increasing of the value of |β| the points K 1 and K 2 approach to the origin of coordinates and the derivativeḢ 0 becomes negative in the neighbourhood of points K 1 and K 2 (see Fig. 2). In points of H 0 -curves withḢ 0 < 0 the In the case of closed models by certain value of β there are three kinds of H 0 -curves (Fig. 3). For very large values of R 0 > R 01 H 0 -curves are closed curves with the center in origin of coordinates (curve (a) in Fig. 3), they correspond to H 0 -curves of GR. The value of R 01 depends on |β|, for example R 01 ∼ 7.14 · 10 7 M −1 p for |β| ∼ 1.0 · 10 14 M −4 p . In the case of R 0 < R 01 we have H 0 -curves (b) and (c) presented in Fig. 3. The Hubble parameter in points of curves (b) is positive (bounce-curves), and it is negative in points of curves (c). So, in points of H 0 -curves (φ 0 = 0,φ 0 = 0) the values of R 0 and V 0 are connected by usual relation of In points of H 0 -curves on the axisφ (φ 0 = 0,φ 0 = 0) we have The presence on H 0 -curves of regions with different signs of H + (H − ) leads to appearance of oscillating solutions. Note, that in points of H 0 -curves lying on coordinates axes we have The analysis shows that the behaviour of H 0 in the case of potential V 2 = 1 4 λφ 4 (λ = const) qualitatively is like to that of considered above potential V 1 = 1 2 m 2 φ 2 .
4 Numerical analysis of solutions for scalar fields SGS By using various scalar field potentials applying in inflationary cosmology (in particular, 2. SGS-solutions of closed type can have quasi-stationary as well as non-stationary regime in dependence on the value of β 4V 0 −φ 2 0 . The value of φ 2 0 has to be less than 4V 0 at several orders to conserve the stable character of discussed solutions. In connection with this the analysis of SGS-solutions will be given below in dependence on the value of x = 4|βV 0 |. [4] A. V. Minkevich, I. M. Nemenman, Class. Quantum Grav. 12, 1259 (1995). | 2014-10-01T00:00:00.000Z | 2003-10-10T00:00:00.000 | {
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248774836 | pes2o/s2orc | v3-fos-license | Novel Chemicals Derived from Tadalafil Exhibit PRMT5 Inhibition and Promising Activities against Breast Cancer
Breast cancer seriously endangers women’s health worldwide. Protein arginine methyltransferase 5 (PRMT5) is highly expressed in breast cancer and represents a potential druggable target for breast cancer treatment. However, because the currently available clinical PRMT5 inhibitors are relatively limited, there is an urgent need to develop new PRMT5 inhibitors. Our team previously found that the FDA-approved drug tadalafil can act as a PRMT5 inhibitor and enhance the sensitivity of breast cancer patients to doxorubicin treatment. To further improve the binding specificity of tadalafil to PRMT5, we chemically modified tadalafil, and designed three compounds, A, B, and C, based on the PRMT5 protein structure. These three compounds could bind to PRMT5 through different binding modes and inhibit histone arginine methylation. They arrested the proliferation and triggered the apoptosis of breast cancer cells in vitro and also promoted the antitumor effects of the chemotherapy drugs cisplatin, doxorubicin, and olaparib in combination regimens. Among them, compound A possessed the highest potency. Finally, the anti-breast cancer effects of PRMT5 inhibitor A and its ability to enhance chemosensitivity were further verified in a xenograft mouse model. These results indicate that the new PRMT5 inhibitors A, B, and C may be potential candidates for breast cancer treatment.
Introduction
Breast cancer accounts for approximately 30% of female cancers and exhibits a mortalityto-incidence ratio of 15% [1]. Approximately 10% of breast cancer patients have a family history or genetic predisposition. The most common germline mutations are located in the BRCA1 and BRCA2 genes, and these mutations indicate an average cumulative lifetime risk of approximately 70% [2][3][4]. At present, breast cancer is mainly treated by radiotherapy, chemotherapy, surgery, and targeted drugs according to the disease stage [4]. Chemotherapy plays a pivotal role in treatment. However, in the course of clinical treatment, an increasing number of breast cancer patients are less sensitive to anthracycline chemotherapeutics, such as doxorubicin, leading to drug resistance and reduced therapeutic effects [5][6][7].
Owing to the important role of PRMT5 in tumors, research into its inhibitors has generated intense interest. The inhibitors reported thus far, such as SAM-competitive inhibitors, ARG-competitive inhibitors, and allosteric inhibitors, suppress PRMT5 activities in different modes [28,29]. SAM, the cofactor that donates methyl group, and ARG, the cofactor that accepts methyl group, occupy distinct binding pockets [8]. Furthermore, allosteric inhibitors abrogate the canonical binding sites of PRMT5, such as compound 1a [8,29]. GSK3326595, which directly interacts with the departing methyl group of SAM and functions as an ARG-competitive inhibitor, is the first PRMT5 inhibitor tested in patients, and its phase I trial exhibited satisfactory clinical results. In addition, JNJ-64619178 and PF-06939999 were tested in phase I clinical trials [8,9,30]. However, to our knowledge, no approved PRMT5 inhibitors have been available for clinical use so far. This is a prominent challenge for targeting PRMT5 in breast cancer treatment. Therefore, finding suitable PRMT5 inhibitors to improve the sensitivity of chemotherapy drugs is of great significance for clinical breast cancer treatment.
Tadalafil was originally used to treat erectile dysfunction and pulmonary arterial hypertension [31][32][33]. Our group previously identified tadalafil as a new PRMT5 inhibitor that effectively improves the sensitivity of tumor cells to doxorubicin and other chemotherapeutic drugs in breast cancer, providing new insights into breast cancer treatment [27]. To further improve the binding specificity of tadalafil and PRMT5, we synthesized three chemicals based on the structural modification of tadalafil in this study and then tested their antitumor effects in vitro and in vivo when used alone or combined with chemotherapy drugs.
Drug Design of Chemicals A, B, and C
The C-terminus of PRMT5 has a Mtase domain, which binds with SAM to play a catalytic role [13,34]. Given its core structure and hydrophobicity, the mechanism of tadalafil may be similar to SAM binding domain inhibitors [12,[35][36][37].
First, Sybyl-X 2.0 software was used to analyze the molecular docking ability of some PRMT5 inhibitors that have been reported with PRMT5. The results showed that the binding affinity of tadalafil for PRMT5 was not as high as that of inhibitors, such as EPZ015666 ( Figure 1A) [9]. Since our research group has previously verified that the FDA-approved drug tadalafil can be used as a PRMT5 inhibitor [27], we aimed to further enhance the ability of tadalafil to bind to PRMT5. Therefore, we modified the structure of tadalafil as follows. First, groups containing nitrogen, oxygen, sulfur, and other heteroatoms were installed on the three relatively independent parts of tadalafil (the piperazine, indole, and benzene rings connected to the parent ring) to facilitate the formation of more hydrogen bonds between tadalafil and other proteins. Second, different alkyl substituents were introduced to enable tadalafil to interact with other molecules through hydrophobic interactions or aromatic groups. Third, the chirality of the chiral center was altered. Fourth, we introduced the above changes into tadalafil at the same time and explored the combined influence of these changes on the binding ability of tadalafil and other proteins.
Compounds A, B, and C Bind to the PRMT5 Protein through Different Binding Modes a Inhibit Histone Arginine Methylation
To predict the binding modes of the three inhibitors to the PRMT5 protein, chemi A, B, and C were used to dock to the ARG binding site, SAM binding site, and allost site of PRMT5 separately ( Figure 2A) [28,29]. The results showed that the skeleton st ture (the four rings) of compound A binds to the SAM binding site of PRMT5, whe the bromophenyl moiety of compound A extends to the ARG binding site ( Figure 2B Compound A binds with PRMT5 mainly through polar and hydrophobic interactions. interacting polar residues involved Thr323, Tyr324, Glu328, Lys333, Tyr334, Glu Glu444, and Ser578, and the interacting hydrophobic residues involved Pro314, Leu Phe327, Pro370, Leu436, and Trp579 of PRMT5 ( Figure 2D). Among these resid Phe327, Glu435, Glu444, Ser578, and Trp579 of PRMT5 are also involved in interac with the substrate ARG, which indicates that compound A might interfere with both S and ARG binding to human PRMT5 ( Figure 2D).
The molecular docking results suggest that compound B binds to an allosteric si PRMT5 ( Figure S1A,B). The binding interactions are mainly hydrophobic interacti and the involved residues include Leu315, Leu437, Pro447, Phe471, Leu472, Val
Compounds A, B, and C Bind to the PRMT5 Protein through Different Binding Modes and Inhibit Histone Arginine Methylation
To predict the binding modes of the three inhibitors to the PRMT5 protein, chemicals A, B, and C were used to dock to the ARG binding site, SAM binding site, and allosteric site of PRMT5 separately ( Figure 2A) [28,29]. The results showed that the skeleton structure (the four rings) of compound A binds to the SAM binding site of PRMT5, whereas the bromophenyl moiety of compound A extends to the ARG binding site ( Figure 2B,C). Compound A binds with PRMT5 mainly through polar and hydrophobic interactions. The interacting polar residues involved Thr323, Tyr324, Glu328, Lys333, Tyr334, Glu435, Glu444, and Ser578, and the interacting hydrophobic residues involved Pro314, Leu319, Phe327, Pro370, Leu436, and Trp579 of PRMT5 ( Figure 2D). Among these residues, Phe327, Glu435, Glu444, Ser578, and Trp579 of PRMT5 are also involved in interacting with the substrate ARG, which indicates that compound A might interfere with both SAM and ARG binding to human PRMT5 ( Figure 2D).
To gain further insight into the binding specificity of the three above-mentioned compounds, we used Surface Plasmon Resonance (SPR) to investigate the binding affinity of chemicals A, B, and C to the PRMT5/MEP50 complex. The binding of chemicals A, B, and C to the PRMT5/MEP50 complex was dose dependent, as indicated by the fast associationdissociation process, and the response units at equilibrium against the PRMT5/MEP50 complex were plotted. These results verify that the three compounds could bind to PRMT5 ( Figure 3A). pounds, we used Surface Plasmon Resonance (SPR) to investigate the binding affini chemicals A, B, and C to the PRMT5/MEP50 complex. The binding of chemicals A, B, C to the PRMT5/MEP50 complex was dose dependent, as indicated by the fast associa dissociation process, and the response units at equilibrium against the PRMT5/ME complex were plotted. These results verify that the three compounds could bin PRMT5 ( Figure 3A). PRMT5 could catalyze the symmetric dimethylation of histones H2AR3, H3R2, H3R8, and H4R3 [16,38,39], so we added compounds A, B, and C and tadalafil at 50, 100, and 150 µM to MDA-MB-231 triple-negative breast cancer cells [40] and performed western blotting to detect the total protein levels of H4R3me2s and H3R8me2s 48 h later. Compared with the control, compounds A, B, and C and tadalafil reduced the total levels of H4R3me2s and H3R8me2s in the cells in a dose-dependent manner. Among them, compound A exerted the strongest effect ( Figure 3B). The above results indicate that these three compounds could bind to PRMT5 through different binding modes and inhibit histone arginine methylation, thus representing new PRMT5 inhibitors.
Compounds A, B, and C Inhibit Breast Cancer Cell Proliferation
Since compounds A, B, and C have inhibitory effects on PRMT5, we analyzed the effects of the three compounds on the proliferation of breast cancer cells in comparison with that of tadalafil. First, we used CCK-8 cell proliferation and toxicity testing experiments to observe the effects of compounds A, B, and C on MDA-MB-231 cell viability. At a concentration of 75 µM or more, compound A was better than the other two compounds, and the tendency to inhibit cell viability was stronger. Under 150 µM culture conditions, the gap between compound A and the other two compounds was the most obvious. Therefore, we performed a statistical analysis of cell viability at this concentration ( Figure 4A). The CCK-8 results for ER-positive MCF-7 breast cancer cells ( Figure 4B) and BRCA1-mutant HCC1937 breast cancer cells ( Figure 4C) were similar to those for MDA-MB-231 cells [41,42]. pared with the control, compounds A, B, and C and tadalafil reduced the total levels of H4R3me2s and H3R8me2s in the cells in a dose-dependent manner. Among them, compound A exerted the strongest effect ( Figure 3B). The above results indicate that these three compounds could bind to PRMT5 through different binding modes and inhibit histone arginine methylation, thus representing new PRMT5 inhibitors.
Compounds A, B, and C Inhibit Breast Cancer Cell Proliferation
Since compounds A, B, and C have inhibitory effects on PRMT5, we analyzed the effects of the three compounds on the proliferation of breast cancer cells in comparison with that of tadalafil. First, we used CCK-8 cell proliferation and toxicity testing experiments to observe the effects of compounds A, B, and C on MDA-MB-231 cell viability. At a concentration of 75 μM or more, compound A was better than the other two compounds, and the tendency to inhibit cell viability was stronger. Under 150 μM culture conditions, the gap between compound A and the other two compounds was the most obvious. Therefore, we performed a statistical analysis of cell viability at this concentration ( Figure 4A). The CCK-8 results for ER-positive MCF-7 breast cancer cells ( Figure 4B) and BRCA1mutant HCC1937 breast cancer cells ( Figure 4C) were similar to those for MDA-MB-231 cells [41,42]. Next, we used EdU incorporation experiments to further clarify the effects of the three compounds and tadalafil on the proliferation of MDA-MB-231 cells. In the CCK-8 cell proliferation and toxicity test, the difference in efficacy between the compounds at a concentration of 150 µM was the most significant. Therefore, this concentration was selected for detection in the EdU incorporation experiment. Similar to the results of the CCK-8 experiment, the three compounds showed varying degrees of inhibition of nucleotide incorporation during DNA replication, but compound A had the strongest inhibitory effect on nucleotide incorporation during DNA replication ( Figure 5).
In summary, compounds A, B, and C could inhibit the proliferation of breast cancer cell lines with different degrees of malignancy when used alone, and compound A had a more pronounced effect than tadalafil.
Next, we used EdU incorporation experiments to further clarify the effects of the three compounds and tadalafil on the proliferation of MDA-MB-231 cells. In the CCK-8 cell proliferation and toxicity test, the difference in efficacy between the compounds at a concentration of 150 μM was the most significant. Therefore, this concentration was selected for detection in the EdU incorporation experiment. Similar to the results of the CCK-8 experiment, the three compounds showed varying degrees of inhibition of nucleotide incorporation during DNA replication, but compound A had the strongest inhibitory effect on nucleotide incorporation during DNA replication ( Figure 5). In summary, compounds A, B, and C could inhibit the proliferation of breast cancer cell lines with different degrees of malignancy when used alone, and compound A had a more pronounced effect than tadalafil.
Compounds A, B, and C Promote Breast Cancer Cell Apoptosis
Since knocking down PRMT5 in tumor cells can not only inhibit tumor cell proliferation but can also increase cell apoptosis [26], we further analyzed the effects of compounds A, B, and C on MDA-MB-231 cell apoptosis. The MDA-MB-231 cell line was treated with 150 μM compounds A, B, C, and tadalafil, and the changes in cell survival were analyzed by annexin Ⅴ/propidium iodide (PI) double staining. After 48 h of drug treatment, the three compounds and tadalafil all promoted cell apoptosis. Among them, compound A had a stronger ability to promote apoptosis of cells than other compounds, as the compound A group had the lowest proportion of living cells and the highest proportion of annexin Ⅴ + PIand annexin Ⅴ + PI + cells ( Figure 6). The apoptosis effect in MCF7 cells was similar to that in MDA-MB-231 cells ( Figure 7). Overall, the addition of compound A significantly inhibited cell survival and promoted cell apoptosis, and its efficacy was stronger than that of tadalafil and the other two compounds.
Compounds A, B, and C Promote Breast Cancer Cell Apoptosis
Since knocking down PRMT5 in tumor cells can not only inhibit tumor cell proliferation but can also increase cell apoptosis [26], we further analyzed the effects of compounds A, B, and C on MDA-MB-231 cell apoptosis. The MDA-MB-231 cell line was treated with 150 µM compounds A, B, C, and tadalafil, and the changes in cell survival were analyzed by annexin V/propidium iodide (PI) double staining. After 48 h of drug treatment, the three compounds and tadalafil all promoted cell apoptosis. Among them, compound A had a stronger ability to promote apoptosis of cells than other compounds, as the compound A group had the lowest proportion of living cells and the highest proportion of annexin V + PI − and annexin V + PI + cells ( Figure 6). The apoptosis effect in MCF7 cells was similar to that in MDA-MB-231 cells (Figure 7). Overall, the addition of compound A significantly inhibited cell survival and promoted cell apoptosis, and its efficacy was stronger than that of tadalafil and the other two compounds.
Compounds A, B, and C Promote the Antitumor Effect of Chemotherapeutics In Vitro
According to our previous work, tadalafil can enhance the sensitivity of doxorubicin to breast cancer treatment [27]. First, we analyzed the effects of compounds A, B, C, and tadalafil in combination with doxorubicin on MDA-MB-231 cell proliferation. In the CCK-8 experiment, MDA-MB-231 cells were treated with compounds A, B, C, and tadalafil at a concentration of 150 µM, and a concentration gradient of doxorubicin treatment was established to observe the effect of the compound and doxorubicin on cell viability. The results showed that when the concentration of doxorubicin treatment was 0.2 µg/mL, compounds A, B, C, and tadalafil could further enhance the inhibition of doxorubicin on cell viability, but compound A combined with doxorubicin had a more obvious effect on the inhibition of cell viability ( Figure 8A). Moreover, we found that treatment with A, B, C, and tadalafil at higher concentrations of doxorubicin no longer enhanced the effect of doxorubicin, indicating that compounds A, B, C, and tadalafil can enhance the inhibitory effect of doxorubicin on cell proliferation within a certain concentration range ( Figure 8A). Similar results were observed in MCF-7 cells ( Figure 8B).
The clinical efficacy of cisplatin is dose-dependent, and high-dose cisplatin has greater toxicity and side effects for patients than low-dose cisplatin [43,44]. Therefore, we attempted to explore whether compounds A, B, C, and tadalafil can promote the antitumor effect of cisplatin, thereby reducing the dosage of cisplatin needed for clinical application. In MDA-MB-231 cells and MCF-7 cells, with 16 µM cisplatin treatment, compounds A, B, C, and tadalafil further promoted the inhibitory effect of cisplatin on cell proliferation, and the combined use of compound A and cisplatin had the most obvious inhibitory effect on cell viability (Figure 9). tablished to observe the effect of the compound and doxorubicin on cell viability. The results showed that when the concentration of doxorubicin treatment was 0.2 μg/mL, compounds A, B, C, and tadalafil could further enhance the inhibition of doxorubicin on cell viability, but compound A combined with doxorubicin had a more obvious effect on the inhibition of cell viability ( Figure 8A). Moreover, we found that treatment with A, B, C, and tadalafil at higher concentrations of doxorubicin no longer enhanced the effect of doxorubicin, indicating that compounds A, B, C, and tadalafil can enhance the inhibitory effect of doxorubicin on cell proliferation within a certain concentration range ( Figure 8A). Similar results were observed in MCF-7 cells ( Figure 8B). The clinical efficacy of cisplatin is dose-dependent, and high-dose cisplatin has greater toxicity and side effects for patients than low-dose cisplatin [43,44]. Therefore, we attempted to explore whether compounds A, B, C, and tadalafil can promote the antitumor effect of cisplatin, thereby reducing the dosage of cisplatin needed for clinical application. In MDA-MB-231 cells and MCF-7 cells, with 16 μM cisplatin treatment, compounds A, B, C, and tadalafil further promoted the inhibitory effect of cisplatin on cell proliferation, and the combined use of compound A and cisplatin had the most obvious inhibitory effect on cell viability (Figure 9). The PARP inhibitor olaparib has a more obvious effect on patients with BRCA1 deficiency in the treatment of breast cancer, but patients with BRCA1 deficiency account for only a part of the population [45][46][47]. This is one of the constraints in the clinical application of PARP inhibitors. Previous results from our group showed that PRMT5 inhibition in breast cancer cells can promote the m6A modification of BRCA1 mRNA and reduce its stability [27]. Therefore, we speculated that the combination of compounds A, B, C, and tadalafil could promote the efficacy of the PARP inhibitor olaparib against breast cancer cells. In MDA-MB-231 and MCF-7 cells, the CCK-8 proliferation and cytotoxicity test showed that when the concentration of compounds A, B, C, and tadalafil was set to 150 µM, all the compounds could enhance the inhibitory effect of olaparib on cell viability, and compound A had the most pronounced effect (Figure 10).
In addition, we analyzed the effects of compound A and tadalafil on MDA-MB-231 cell apoptosis when combined with doxorubicin by FACS. Combined treatment with tadalafil and compound A, compared with doxorubicin alone, significantly reduced the proportion of living cells, and the proportion of cells in early apoptosis and late apoptosis was also significantly increased. Instead, the PI + necrotic cell population did not change after compound A treatment. Overall, the combined use of compound A and doxorubicin significantly inhibited cell survival and promoted cell apoptosis, and the effect was stronger than that of doxorubicin alone and that of tadalafil and doxorubicin combined ( Figure 11). The PARP inhibitor olaparib has a more obvious effect on patients with BRCA ciency in the treatment of breast cancer, but patients with BRCA1 deficiency acco only a part of the population [45][46][47]. This is one of the constraints in the clinical a tion of PARP inhibitors. Previous results from our group showed that PRMT5 inh in breast cancer cells can promote the m6A modification of BRCA1 mRNA and red stability [27]. Therefore, we speculated that the combination of compounds A, B, tadalafil could promote the efficacy of the PARP inhibitor olaparib against breast cells. In MDA-MB-231 and MCF-7 cells, the CCK-8 proliferation and cytotoxic showed that when the concentration of compounds A, B, C, and tadalafil was se μM, all the compounds could enhance the inhibitory effect of olaparib on cell vi and compound A had the most pronounced effect (Figure 10). The PARP inhibitor olaparib has a more obvious effect on patients with ciency in the treatment of breast cancer, but patients with BRCA1 deficiency only a part of the population [45][46][47]. This is one of the constraints in the clin tion of PARP inhibitors. Previous results from our group showed that PRM in breast cancer cells can promote the m6A modification of BRCA1 mRNA a stability [27]. Therefore, we speculated that the combination of compounds tadalafil could promote the efficacy of the PARP inhibitor olaparib against b cells. In MDA-MB-231 and MCF-7 cells, the CCK-8 proliferation and cyto showed that when the concentration of compounds A, B, C, and tadalafil w μM, all the compounds could enhance the inhibitory effect of olaparib on and compound A had the most pronounced effect (Figure 10). portion of living cells, and the proportion of cells in early apoptosis and late apoptosis was also significantly increased. Instead, the PI + necrotic cell population did not change after compound A treatment. Overall, the combined use of compound A and doxorubicin significantly inhibited cell survival and promoted cell apoptosis, and the effect was stronger than that of doxorubicin alone and that of tadalafil and doxorubicin combined ( Figure 11).
Compounds A, B, and C Promote the Antitumor Effects of Chemotherapeutics in Xenograft Tumor Models
To further confirm the antitumor effect of the novel PRMT5 inhibitor in vivo, we subcutaneously inoculated nude mice with MDA-MB-231 cells, and the mice were treated with chemical A in combination with doxorubicin, cisplatin, and olaparib. Consistent with the results from the in vitro experiments, the use of compound A alone slowed the growth of tumors ( Figure 12). This finding differs from that obtained in our previous work, in which mice treated with tadalafil alone did not exhibit tumor growth suppression in the MDA-MB-231 mouse xenograft model [27]. The use of compound A in combination with chemotherapeutics further enhanced the efficacy of the chemotherapeutics (Figure 12). We also confirmed that the protein level of H3R8me2s was downregulated in tumors
Compounds A, B, and C Promote the Antitumor Effects of Chemotherapeutics in Xenograft Tumor Models
To further confirm the antitumor effect of the novel PRMT5 inhibitor in vivo, we subcutaneously inoculated nude mice with MDA-MB-231 cells, and the mice were treated with chemical A in combination with doxorubicin, cisplatin, and olaparib. Consistent with the results from the in vitro experiments, the use of compound A alone slowed the growth of tumors ( Figure 12). This finding differs from that obtained in our previous work, in which mice treated with tadalafil alone did not exhibit tumor growth suppression in the MDA-MB-231 mouse xenograft model [27]. The use of compound A in combination with chemotherapeutics further enhanced the efficacy of the chemotherapeutics (Figure 12). We also confirmed that the protein level of H3R8me2s was downregulated in tumors treated with compound A and that cleaved PARP was upregulated in the olaparib-treated group ( Figure S3).
Furthermore, the results of Ki67 immunohistochemistry showed that when compound A was used in combination with chemotherapy drugs, the proliferation ability of tumor cells was further inhibited (Figure 13A,C) [48]. Additionally, the expression of γH2A.X in tumor tissues increased, suggesting increased levels of DNA damage ( Figure 13B,C) [49]. In summary, compound A can enhance the antitumor effects of doxorubicin, cisplatin, and olaparib in vivo. treated with compound A and that cleaved PARP was upregulated in the olaparib-treated group ( Figure S3). Furthermore, the results of Ki67 immunohistochemistry showed that when compound A was used in combination with chemotherapy drugs, the proliferation ability of tumor cells was further inhibited ( Figure 13A,C) [48]. Additionally, the expression of γH2A.X in tumor tissues increased, suggesting increased levels of DNA damage ( Figure 13B,C) [49]. In summary, compound A can enhance the antitumor effects of doxorubicin, cisplatin, and olaparib in vivo.
Discussion
Breast cancer is an important disease endangering women's health [1,4]. In the treat-
Discussion
Breast cancer is an important disease endangering women's health [1,4]. In the treatment of breast cancer, resistance to chemotherapeutic drugs, such as doxorubicin, has become a key factor affecting the treatment response [7,50,51]. PRMT5 is involved in the methylation modification of proteins, which can regulate DNA damage and repair, and plays a critical role in cell survival and growth [8,9,14]. Additionally, it can directly affect the therapeutic effect of doxorubicin [26]. However, research on PRMT5 inhibitors is in the preliminary stage, and new inhibitors require further development [8,12,36]. Our previous study showed that tadalafil can act as a PRMT5 inhibitor [27], but its binding ability to PRMT5 still needs to be improved. In this study, three new PRMT5 inhibitors were designed and synthesized based on tadalafil, and the antitumor effects of the new PRMT5 inhibitors alone and combined with chemotherapy drugs were evaluated in vitro and in vivo.
First, through molecular docking experiments, we modified the structure of tadalafil and obtained chemicals A, B, and C, which have a stronger binding ability to PRMT5. The SPR experiments confirmed that compounds A, B, and C can bind to PRMT5, and the molecular docking results suggested that the three compounds could bind to PRMT5 through different binding modes. Moreover, the new compounds decreased the histone modification levels of H4R3me2s and H3R8me2s in breast cancer cells. Then, we verified the proliferation-inhibiting and apoptosis-inducing effects of chemicals A, B, and C in breast cancer cells. Among them, compound A had the strongest antitumor effect when used alone. These results confirm that the newly synthesized compounds can act as novel PRMT5 inhibitors.
This study has its own strengths demonstrated in several aspects. First, based on the restructured tadalafil, we screened out more potent drug compound A, which showed a promising antitumor effect. Second, compound A could facilitate chemotherapeutic efficacy. Chemotherapy is currently one of the main clinical treatments for breast cancer, but the dose dependence and side effects of various chemotherapeutic drugs restrict them from wider application [7,50,52,53]. Compounds A, B, and C can promote the antitumor effect of chemotherapeutics, such as doxorubicin, cisplatin, and olaparib. Among these, compound A was the most effective in this context. Finally, it is worth noting that the PARP inhibitor olaparib has a more favorable effect on BRCA1-mutated patients in the clinical treatment of breast cancer, but the BRCA1-mutated patients account for only a portion of the population. Encouragingly, compounds A, B, and C could not only enhance the antitumor effect of olaparib in BRCA1-mutated HCC1937 cells but also function in BRCA1-wildtype cells, such as MDA-MB-231 and MCF-7 cells. This is of great significance for the expanded clinical application of PARP inhibitors [54,55].
However, our current study has several limitations to be acknowledged. The three new PRMT5 inhibitors have higher working concentrations than the commercially available PRMT5 inhibitors. Nevertheless, PRMT5 −/− mice exhibited embryonic death at E6.5 due to the abrogation of pluripotent cells in blastocysts, suggesting that PRMT5 plays a critical role in development [56]. For this reason, although commercially available PRMT5 inhibitors may possess a higher affinity to PRMT5, their corresponding toxicity and side effects cannot be ignored [12]. Taken together, inhibitors A, B, and C have potential advantages in controlling drug doses and toxicity, but their safety and pharmacokinetics still need to be further investigated.
Molecular Docking
Molecular docking was performed using Sybyl-X 2.0 software and MOE software. In the analysis using Sybyl-X 2.0 software, the energy of the compounds was minimized under the Tripos force field. The distance was used as the dielectric function, and the Gasteiger-Hockel atomic charge was applied. The Powell energy gradient method was applied to optimize the lowest energy conformation, and the maximum number of energy optimizations was set to 10,000. The energy convergence criterion was set to 0.001 kcal·mol −1 , which mimicked the stable conformation of molecules in natural systems. The structure of the PRMT5:MEP50 complex was downloaded from the PDB database and imported into Sybyl-X 2.0 for molecular docking. The semiflexible docking method was used for molecular docking, and the optimized compound and processed protein were docked with the Surflex-Dock module. The total score in Surflex-Dock can reflect the binding affinity between the ligand and the receptor, considering factors such as polarity, hydrophobicity, entropy, and solvation.
MOE software was used to predict the binding modes of the compounds when binding to PRMT5. The crystal structure of human PRMT5 (PDB ID: 4X61) was retrieved from the protein data bank for ARG and SAM binding site docking [28], and the crystal structure with PDB ID 6UXX was retrieved for allosteric site docking [29]. Hydrogen atoms were added, and the proteins were protonated using Protonate3D. Missing residue sidechains and loops were built. The structures of the compounds were built in MOE 2014 and optimized using the MMFF94x forcefield. The binding sites of SAM and ARG in the structure of 4X61 and the allosteric site in the structure of 6UXX were defined as active sites for molecular docking. The placement method was set to Triangle Matcher and rescored with London dG. The docking conformations were refined with the forcefield and rescored with GBVI/WSA dG. Finally, 30 conformations of each compound were retained, and the conformation with the best S-score was used for structural analyses. In the two methods, a larger absolute value of the docking score indicated a stronger binding ability.
SPR Analysis
A Biacore T200 instrument (GE Healthcare, Little Chalfont, UK) was used for the SPR analyses. The recombinant PRMT5/MEP50 complex (31521, Active Motif, Carlsbad, CA, USA) was immobilized on carboxymethylated dextran CM7 sensor chips (GE Healthcare) using an amine-coupling strategy. A solution of N-hydroxysuccinimide and 3-(N,Ndimethylamino)-propyl-N-ethylcarbodiimide (1:1) was used to activate the sensor chip surface. Then, the PRMT5/MEP50 complex was solubilized in acetate buffer and injected in PBS running buffer (10 µL/min) to reach an immobilization level of 20,000 relative units on the CM7 sensor chips. Then, an ethanolamine solution was used to block the surfaces. The binding kinetics of chemicals A, B, and C to the PRMT5/MEP50 complex sensor chip were evaluated in PBS buffer with concentrations ranging from 31.75 to 500 µM. All experiments were performed at 25 • C (30 µL/min). The surfaces of the CM7 sensor chips were then regenerated with two injections of a glycine-HCl solution. Binding sensograms were acquired by subtracting the reference flow cell, and BIA Evaluation Software was used for data analysis.
Cell Culture
The breast cancer cell lines MDA-MB-231, MCF-7, and HCC1937 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MDA-MB-231 and HCC1937 cells were maintained in DMEM containing 10% fetal bovine serum and 1% antibiotics, and MCF-7 cells were maintained in DMEM containing 0.2 IU/mL insulin, 10% fetal bovine serum, and 1% antibiotics. Cells were cultured at 37 • C in a 5% CO 2 incubator. For the Cell Counting Kit-8 (CCK-8) cell proliferation and cytotoxicity experiment, cells were seeded into 96-well plates at 1 × 10 4 cells/well. For western blotting, cell cycle investigation, and apoptosis detection, cells were seeded into 6-well plates at 1.5 × 10 5 cells/well. For an EdU incorporation experiment, 2 × 10 4 cells/well were seeded in a 48-well plate. In the above experiments, the drug treatments were performed on the second day after cell inoculation, and detection was performed 48 h later. Treatments with cisplatin (Selleck, S1166, Houston, TX, USA), doxorubicin (Selleck, S1208), olaparib (Selleck, S1060), and tadalafil (MCE, HY-90009A, Monmouth Junction, NJ, USA) were performed as described in the main text.
Cell Proliferation Assays
CCK-8 (MCE) experiments were performed to assess cell proliferation according to the manufacturer's instructions. The CCK-8 solution was added to cells in 96-well plates, and incubation was continued for another 1 h at 37 • C. The plates were read at 450 nm with a microplate reader.
EdU assays were performed with a Cell-Light EdU DNA Cell Proliferation Kit (Ribo-Bio, Guangzhou, China). Cells were incubated with a 50 µM EdU solution in the medium for 2 h, fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature and stained with an Apollo staining solution. Images were acquired under a fluorescence microscope (NI-U, Nikon, Tokyo, Japan).
Apoptosis Analysis
For apoptosis analysis, the cells were harvested and washed twice with cold PBS. Afterward, 400 µL of Binding Buffer was added to the cells, followed by the addition of 5 µL of an annexin V staining solution and incubation in the dark at room temperature for 15 min. Thereafter, 10 µL of a PI staining solution was added, and incubation was continued on ice for 5 min. The analysis was performed with a FACSCalibur TM flow cytometer.
Mouse Xenograft Model
All animal experiments were approved by the Animal Experiment Administration Committee of Fourth Military Medical University (Approval Code: 20203285-1, Approval Date: 8 April 2020). MDA-MB-231 cells (5 × 10 6 ) were subcutaneously injected into the right flanks of 6-week-old female Balb/c nude mice. Ten days post-inoculation (dpi), the tumorbearing mice were randomly sorted into different groups. Chemical A (2 mg/kg) were administered by gastric gavage every day from 10 dpi. Olaparib (50 mg/kg) was injected intraperitoneally (i.p.) every day from 10 dpi. Doxorubicin (2 mg/kg) was delivered intravenously once per week from 10 dpi. Cisplatin (2.5 mg/kg) was injected i.p. every three days beginning from 10 dpi. The compounds were formulated according to the product instructions. Tumor size was monitored once every 3 days using a caliper and calculated as π × [d 2 × D]/6 (d, short diameter; D, long diameter). The tumors were dissected at 31 dpi, fixed with 4% PFA at 4 • C overnight, and then embedded in paraffin using a routine procedure. Paraffin-embedded tissue was stained with anti-Ki67 (1:200, ab15580, Abcam, Cambridge, UK) and anti-γH2A.X (1:400, 9718, CST) antibodies.
Statistical Analysis
Quantitative analysis was performed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA), Fiji v2.0.0 (National Institutes of Health, Bethesda, MD, USA), and FlowJo V7.6.5 (BD Biosciences, Franklin Lakes, NJ, USA) software. Statistical analysis was performed using GraphPad Prism 8.0 software. All quantitative data are presented as the mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison test. A p < 0.05 was considered to indicate statistical significance.
Conclusions
In summary, we designed and synthesized three new PRMT5 inhibitors through the chemical modification of tadalafil. These three inhibitors have favorable antitumor effects when used alone or in combination with chemotherapeutics. Therefore, these new PRMT5 inhibitors have the potential to become novel molecular targeted drugs that clinically enhance the patients' tumor cell chemosensitivity, but further study is still needed. | 2022-05-15T05:21:46.202Z | 2022-04-27T00:00:00.000 | {
"year": 2022,
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98053988 | pes2o/s2orc | v3-fos-license | Xanthones and Other Constituents of Vismia parviflora
Vismia parviflora, planta da família Guttiferae, é uma espécie da tribo Vismeae encontrada na região de Ouro Preto MG. Dos extratos benzênico e etanólico dos galhos e etanólico dos frutos de um especimen desta planta, cromatografados em sílica gel, foram isolados sitosterol, ácido betulínico, lupeol, friedelina, β-friedelinol, ácido chiquímico, ácido 3,4-diidroxibenzóico, quercetina e as antraquinonas madagascina, ácido crisofânico, vismiaquinona-A e vismiaquinona-C, além das xantonas 1,7-diidroxixantona e 1,5-diidroxi-8-metoxixantona. Estas substâncias tiveram suas estruturas elucidadas com base nos seus dados espectrométricos de IV, UV, EM e RMN de H e C uni-(1D) e bidimensional-(2D).
Introduction
According to Engler 1 , Vismia parviflora belongs to the Guttiferae family, subfamily Hyperiocoideae, tribe Vismieae.The Vismia genus consists of small trees inhabiting the tropical and subtropical regions of South and Central America, where they are used in folk medicine as strong purgative, whereas their barks are considered to be tonic and febrifugal [2][3] .
Although the chemistry of the Guttiferae family has been widely studied 4 , no more than fifteen species of Vismia have been investigated among the fifty known species.Several species of this genus have been shown to contain xanthones and anthranoides [5][6][7][8][9][10] .In the present study we have identified the chemical constituents from stems and fruits of Vismia parviflora, which have not been reported previously.
Results and Discussion
Fractionation of the ethanol extracts of stems and fruits on silica gel, followed by gel filtration and recrystalization, afforded the isolation the compounds 1-6.The UV spectra of 1 and 2 exhibited absorption bands characteristic of xanthones 11 .The bathochromic shift observed upon addition of AlCl3 indicated the presence of a chelated hydroxyl group at C-1 or C-8 for the both compounds 1 and 2. The 1 H-NMR spectra of both 1 and 2 showed , absorptions at: δ 7.68 (t, J = 8.1 Hz H-3), 6.75 and 6.97(dd, J = 8.1 and 1,0 Hz H-2 and H-4) for 1 and δ 7.63(t, J = 7.30 Hz H-3), 6.72 and 6.94 (dd, J = 7.3 and 1.3 Hz H-2 and H-4) for 2, indicative for both compounds the same three hydrogens vicinal pattern for one of the aromatic ring.In addition, the 1 H-NMR of compound 1 showed three orthoand metacoupled hydrogens at δ 7.50 (d, J = 8.8 Hz H-5), 7.40 (dd, J = 8.8 and 2.7 Hz H-6), 7.57 (d, J = 2.7 Hz H-8).The OH on C-7 was proposed by the absence of the bathochromic shift of the UV maxima in the presence of sodium acetate, and confirmed by methylation of 1 with an ether solution of diazomethane.The 13 C-NMR spectra was compatible with 1,7-dihydroxyxanthone 1.The mass spectrum showed peak at m/z 228 ([M] +• ), which is in accordance with the molecular formula C13H8O4.For the compound 2 the 1 H-NMR showed two ortho coupled hydrogens at δ 6.85 and 7.32 (d, J = 7.3 Hz, H-7 and H-6 respectivelly), besides the singlet at δ 3.89 (3H, s, OCH3).The 13 C-NMR was compatible with 1,5-dihydroxy-8-methoxyxanthone.The mass spectrum showed peak at m/z 258 ([M] +• , 96%) in accordance with molecular formula C14H10O5, and the base peak at m/z 240 ([M -18] +• , 100%).The loss of water from the [M] +• is due to the operation of an ortho effect caused by the methoxy substituent at C-8 12 , what is in agreement with the proposed structure.(Table 1).
Compounds 3-6, appeared to be 1,8-dihydroxyanthraquinone derivatives based on their UV-Vis and the IR absorptions bands characteristic of anthraquinones 13 .The 1 H-NMR spectra of these compounds showed the same pattern of substitution for one of the aromatic ring, all of them showing two meta-coupling hydrogens, between δ 7.23 -7.61 (H-4) and 6.59-7.06(H-2), besides the methyl group at C-3 in δ 2.4 and a chelated hydroxy group at C-1 for all of them.The 1 H-NMR spectra of compounds 4 and 5 showed signals compatible with a γ, γ'-dimethylallyl side chain, an O-dimethylallyl at C-6 for 4, whereas 5 has a dimethylallyl at C-7.In addition compound 5 showed a methoxy group at 4.01 and an aromatic hydrogen at δ 7.34 (s, H-5).For compound 6 the 1 H-NMR spectra showed signals in accordance with the presence of a ∆ 1 -isopentenyl side chain at C-7, a methoxy group at δ 3.93 and an aromatic hydrogen at δ 7.46 (s, H-5).(Table 2).
Experimental
Plant material, A specimen of Vismia was collected in Três Moinhos district, Ouro Preto city, State of Minas Gerais, Brazil, in March 1986.The specimen was identified by Prof. José Badini, botanic garden of the Universidade Federal de Ouro Preto -Brazil. | 2017-09-16T00:19:21.014Z | 1997-01-01T00:00:00.000 | {
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54028609 | pes2o/s2orc | v3-fos-license | Improving territorial accessibility of mental health services : The case of Spain
Background and Objectives: Citizens choose their health care services not only depending on their needs, but also on where they are located. The location of the services is especially important in the case of mental health due to the specific features of mental disorders. This article provides an analysis of temporal access by road to outpatient mental health centres in Andalusia (Spain) with a view to improving accessibility for the greatest volume of population possible. Methods: Firstly, accessibility by road to the outpatient mental health centres was calculated in terms of time by establishing journey times using the ArcGIS Geographical Information System’s (GIS) Network Analyst module. These journey times by road enabled travel times to be established for these sections, temporal accessibility areas to be plotted from each of the outpatient mental health centres and the number of people included in each accessibility area to be calculated. Results: The accessibility analysis enabled the sitting of the centres to be evaluated for 2006, a comparison to be made with 2011 (with six new facilities having been set up since 2006) and new locations for the siting of these six new facilities to be proposed. Conclusions: This study has enabled the optimum territorial locations to be proposed for the six mental health centres created between 2006 and 2011 that would allow travel times to be reduced for the greatest numbers of people possible. It can be stated on the basis of this study that, if territorial criteria had been taken into account, 97,720 inhabitants would have seen their travel times to their nearest mental health centres reduced using the same resources. Received: 4 May 2011 Revised: 18 April 2012 Accepted: 1 June 2012 228 ENRIQUE LÓPEZ-LARA, MARCO GARRIDO-CUMBRERA AND M. PILAR DÍAZ-CUEVAS
Introduction
Countries such as Spain, where there is a National Health Service (NHS) with universal coverage, provide a wide range of free health care services.However, inequalities continue to be an issue, even in western countries with an NHS model 1,2 .
Territorial accessibility is one of the most important elements that define equity in health care.For this reason, one of the provisions of the 1986 Spanish Health Act lays down that the national health policy should aim to iron out any territorial and social imbalances.This Act also states the criteria for defining health areas in accordance with geographical, socio-demographic, epidemiological and communication features.However, there is no consensus regarding the standard methodology for systematic calculations to be made to enable the territorial siting of new health care services.
It is important to distinguish between "effective" and "territorial" accessibility."Effective" accessibility refers to a service's opening times, its economic and social availability and people's own funds.However, "territorial" accessibility refers to a service's physical proximity and can be measured using Geographical Information Systems (GIS) 3 .Access to health care services is determined by geographical features, such as distance overland, journey times, the region's geographical features and service availability, as well as socio-demographic factors, such as rurality and ethnicity, income and the state of health care coverage 4 .A service's territorial accessibility refers to the distance between a service and its users' places of residence, and is an essential factor for consideration when siting any planned service.It has thus been demonstrated that the territorial location of health services determines their access and use, two parameters that are inversely related.People who live nearer services are more likely to use them and obtain better outcomes from the care received 5 .Determining where health care facilities should be located is a controversial issue since they are perceived as symbols of local power 6 , especially in the field of mental health, where services are scarce compared to the burden of disease in comparison with services for other health care specialities 7 .
Studies on territorial accessibility to health care facilities have been implemented in a great variety of areas, including hospitals 6,8 , palliative care 5,9 , cancer care 4 and mental health services 10 .
The territorial location of services is a crucial topic for the health care sector in general terms, but it is especially important for the field of mental health due to the specific features of mental disorders.In the past, mental health patients have been stigmatised, discriminated against and even confined in isolated, remote psychiatric hospitals, which prevented them being included in society.A large number of studies have demonstrated that community care has better effects than hospitalisation, improving the development of the illness and reducing the number of flareups in people who suffer from mental disorders 7,11 .People with mental disorders need to live in the community, near their family and friends.Mental health services are therefore more efficient when they are located in the community 12 .People with mental disorders benefit from their improved quality of life when they are not excluded from society but, instead, take part as citizens in their own right.
Improved spatial accessibility has the potential to reduce journey times for mental health patients (enabling them to live nearer the community), for mental health professionals (especially for home visits) and for family members (when they visit their relatives).
When the responsibility for health care was transferred to the 17 Autonomous Communities or regions, and two autonomous cities, 19 different and independent health care systems were created 13 .This is why an evaluation of the Spanish National Health care system as a whole is impossible, and each of the Autonomous Communities must be assessed individually.
This article presents an analysis of the temporal accessibility by road of the outpatient mental health facilities in Andalusia using the ArcGIS Network Analyst module.Andalusia is the southernmost Autonomous Community in mainland Spain with a population of eight million spread over an area of 87,268 km 2 .The territory extends almost completely along the southern strip of Spain and borders with Portugal to the west and Morocco to the south.Andalusia is split into eight provinces: Almeria, Cadiz, Cordoba, Granada, Huelva, Jaen, Malaga and Seville.The provincial capitals are among the most highly populated cities in the region along with Jerez and Algeciras (see Figure 1).
Method
Polygons were created to represent the temporal accessibility by road of each of the outpatient mental health centres and the size of the population within their boundaries was calculated.A number of variables were taken into account to calculate access by road to the outpatient mental health centres: the location of the facilities, the road network (routes, distances, road classification, state of the road surface, sinuosity, etc) and the population (location, age range, etc).
The first step was to locate the mental health centres using the city centroid in which they were located.On a regional scale, as is the case in Andalusia, the number of facilities in a single city does not affect spatial accessibility.This is why cities like Seville and Malaga, where there is more than one facility, were represented by a single icon.
With regard to the road network, the Andalusian Cartographic Institute's 1:100,000 Digital Map of Andalusia spatial road network coverage (2009) was used and a relative score was awarded to restraints on traffic speed for each section of road.These scores were established on the basis of the official speed limit for each category of road, the specific features of the traffic and the sinuosity of each segment of the road.A sinuosity index was established to calculate speed restraints as follows: where W is the distance in a straight line between the start point and the end point of a given section (s&e) divided by the actual length of said section (l), with a straight section given a sinuosity score of 1.This score falls the more the shape of a circumference is approached and the distance between the start point and the end point diminishes, to a point where W equals 0. With respect to the population, a number of inhabitants was assigned to each municipality for 2006 and 2011 respectively, taking the central point of each population centre as a reference point.
Once these centre-points, the population distribution and the travel speed for each section of the road network had all been established, the road network was analysed using the ArcGIS Network Analyst module.This analysis of the road network enabled mini-mum journey times to be established from each of the outpatient mental health centres, and areas of temporal accessibility to be plotted (>30 minutes, from 30 to 60 minutes and >60 minutes).Once these accessibility areas had been established the numbers of people in each area were calculated for each of the centres that existed in 2006 and in 2011 (including the six new facilities set up by the Andalusian Health Department between 2006 and 2011), and new optimum locations were proposed for these six new facilities based on geographical criteria using the GIS.What is understood by a centre's "optimum" territorial location is one that enables access by the greatest number of people in the shortest journey time, thus improving temporal accessibility.The suitability of the potential municipalities for relocating these six facilities was assessed; they had to be geographically distant from each other and with no other outpatient mental health facility in their surrounding areas.The suitability of future locations for new mental health services was thus assessed.Each map plotted included three temporal boundaries (<30, from 30 to 60 and >60 minutes) which were set as specifically appropriate journey distances for evaluating accessibility to outpatient mental health centres.
The hypothesis that supports the research is based on the idea that the greater the distance to a health centre, the less the pull of the services provided there.Shorter distances to health centres and lower temporal costs for travelling there result in a greater ability to attract people and increased numbers of people tended to.In this way, greater accessibility (lesser physical-geographical difficulty of access) increases the pulling-power of health centres and, consequently, the degree to which users' needs are met, as they can make more frequent and efficient use of them.
Results
The temporal accessibility by road of the mental health centres that existed in 2006 was analysed in comparison with accessibility in 2011 by adding the six new facilities at their official locations established by the Andalusian Health Department, and a new location was proposed for each of these six new facilities in keeping with geographical criteria that improve the temporal accessibility of part of the population.Temporal road accessibility areas (<30, from 30 to 60 and >60 minutes) were plotted round the outpatient mental health centres with a distinction being made between the situations in 2006 (when there were 72 facilities, Figure 2.A) and 2011, when six new facilities had been opened (78 facilities, Figure 2.B) at their current official locations and, finally, a proposal was made for the relocation of the six new facilities based on territorial criteria for their optimum locations, whereby a greater percentage of the population would see their journey times reduced (78 facilities, Figure 2.C).Table 1 presents the population distribution in the three areas of temporal accessibility (<30, from 30 to 60 and >60 minutes) by road of the outpatient mental health centres that existed in 2006, in 2011 with the six new fa-cilities in locations according to the official distribution, and with these six facilities in locations according to the territorial criteria proposed for improving temporal accessibility.
Despite the mental health centres being spread across the whole region and providing medical care to the majority of the population, a shortfall can be seen in 2006 in services to the northern mountain areas, which are characterised by low population density and a large proportion of elderly people.Accessibility in these areas had not improved in 2011 despite six new centres being set up.In fact, the setting up of six new centres between 2006 and 2011 did not result in any major changes in temporal accessibility compared to six years previously (2006).However, our geographical criteria-based proposal for the location of these six new facilities results in a major improvement in temporal accessibility compared with the official situation.Indeed, if territorial criteria had been used when the locations of these six new facilities were determined, accessibility would have improved for a large percentage of the population.Accessibility would have improved for 1.16% of the population (approximately 97,720 inhabitants), who would have moved up from the 30-60 minutes band to the 30 minutes band from the nearest outpatient mental health facility.
Discussion
Geographical factors, such as distance, affect the use of mental health services.As a result, an increase in territorial and temporal distance between a person's place of residence and these services means the usage rates decrease 14 .Territorial analysis techniques are useful tools for analysing the distribution of a variable and for explaining factors relating to the availability and appropriate access to health care services 15 .
This study has mainly examined geographical access to outpatient mental health centres.However, apart from territorial aspects, there are other factors, such as the type of medical insurance, patient preferences, socio-economic factors, level of education, waiting lists and the availability of medical care that are barriers to access to medical care that should be analysed along with territorial factors to assess service use.
Increased journey times to medical care services have been linked with a greater risk of mental disorders.Access to specialised services that are not widespread across the territory can be limited, mainly by proximity, which can be measured as travel time 4 .Individuals with serious mental disorders often have no alternative but to move to another town or city to ensure that they are a reasonable distance from mental health services, which are mainly located in the larger towns and cities.However, patients are often obliged to live far from their own environment, which has a negative impact on the way that their illnesses develop.
In the 2008-2012 Andalusian Integrated Mental Health Plan we find a reference to the need to undertake specific studies on the territorial accessibility of mental health centres as a way of detecting and reducing territori-al inequalities in isolated rural areas.In this way, the need to take into account territorial factors in decision making processes that affect the locations of new mental health resources is mentioned 16 .
A variety of tools have been implemented to calculate the territorial accessibility of health care facilities.It is possible to define accessibility areas according to the places of residence of patients tended to 17 and establish the accessibility areas of the specific medical care facilities 10 .Temporal distances appear to be more interesting than physical distances as they include information regarding journey features and the condition of the transport infrastructure.The changes and technical improvements in transport infrastructure are capable of modifying the ease and speed with which people are able to access medical care services.However, not all regions are organised in the same way in territorial terms, which leads to disparities and inequalities which can be reduced when an integrated management and planning system is implemented.Geographical Information Systems (GIS) are suitable tools for detecting the areas with the greatest shortfalls in certain services 18 .
As has been shown in this study, the greater part of the population lives near one or other of the outpatient mental health centres, although a number of inhabitants still live a certain distance away and only have limited access to mental health care.The analysis of the territorial accessibility of mental health centres has been seen to be useful for detecting peripheral and neglected areas.These findings show that the locations of the new services created between 2006 and 2011 were chosen without taking into account any criteria for territorial equity.However, it has been demonstrated that it is possible and viable to reduce temporal distances and territorial inequalities, using the same resources, if geographical analysis methods are used for their location.GIS tools and methods could be included among mental health system management and planning tools.
Conclusion
The information produced has enabled us to detect and propose optimum locations for the new mental health centres established during the 2006-2011 period.On the basis of this study it can be stated that if territorial criteria had been taken into consideration, 97,720 inhabitants would have seen their journey times to the nearest mental health centre reduced using the same resources.The proposed methodology could support the work done by managers and planners when deciding on where future mental health care centres are to be located.
The initial hypothesis has been demonstrated; reduced distances and temporal costs for travelling to health centres result in these having a greater pulling-power and in an increase in the number of people tended to, and GIS is an effective method for determining their optimum locations.
Figure 1 .
Figure 1.Andalusian territorial population distribution and road network.
Figure 2 .
Figure 2. Temporal road accessibility of outpatient mental health centres (<30, from 30 to 60 and >60 minutes) in Andalusia (Spain).A) Situation in 2006.B) Situation in 2011 with six new facilities located according to the official distribution.C) Situation in 2011 with these six new facilities located according to territorial accessibility criteria.
Table 1
Population distribution in areas of temporal accessibility (<30, from 30 to 60 and >60 minutes) by road of the outpatient mental health centres that existed in 2006, in 2011 according to the official distribution, and according to the territorial criteria proposed in Andalusia (Spain) | 2018-11-30T01:20:40.305Z | 2012-12-01T00:00:00.000 | {
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124472607 | pes2o/s2orc | v3-fos-license | The LHCb Trigger System: Present and Future
The current LHCb trigger system consists of a hardware level, which reduces the LHC inelastic collision rate of 30 MHz to 1MHz, at which the entire detector is read out. In a second level, implemented in a CPU farm, the event rate is reduced to about 5 kHz. The major bottleneck in LHCb's trigger efficiencies for hadronic heavy flavour decays is the hardware trigger. The LHCb experiment plans a major upgrade of the detector and DAQ system in the LHC shutdown of 2018. In this upgrade, a purely software based trigger system is being developed, which will have to process the full 30 MHz of inelastic collisions delivered by the LHC. Both the current trigger system and its planned upgrade are discussed in these proceedings.
Introduction
The LHCb detector at the LHC is a precision experiment dedicated to beauty and charm physics, covering a rapidity range of 2 < η < 5 [1]. Ref. [2] summarizes the performance of the LHCb detector in Run 1 (2010 -2012). The trigger system during Run 1 [3,4] consisted of a fixed latency hardware level which reduced the visible bunch crossing rate to 1 MHz. At this rate, the whole detector was read out and a flexible software High-Level Trigger (HLT) was run to further reduce the rate to about 5 kHz, which was written to offline storage. This configuration allowed LHCb to record the largest beauty and charm hadron samples, at a very high signal purity.
For the second data taking period (Run 2, 2015 -2018), the detector will be mostly unchanged with respect to Run 1, while the trigger software will undergo an incremental update. The software trigger will be split in two separate instances, the first one running synchronous with the LHC collisions while the second stage will be run asynchronously. This split allows to to perform a real-time calibration and alignment of the detector. The software trigger is thus able to select events based on a reconstruction with a quality almost identical to the offline processing.
The LHCb experiment plans major upgrades in preparation for the third LHC data taking period (Run 3, 2019++). As part of the detector upgrade, all of the front-end devices will be replaced in order to read the detector out at the full bunch crossing rate of 40 MHz [5]. Removing the readout bottleneck allows the complete event reconstruction at the full collision rate and hence an unprecedented trigger performance, especially for hadronic modes.
This note discusses the LHCb trigger system and its performance in Run 1 as well as the prospects and preparations for Run 2 and 3.
Run 1: Trigger Performance in 2009 -2012
The trigger system of the LHCb experiment in Run 1 consists of two levels: the first level, implemented in hardware (L0), and the High Level Trigger (HLT), implemented in a CPU farm of about 29 000 logical cores. The LHCb trigger system and its performance during 2011 data taking has been described in detail in Ref. [4]. Here, the adjustments of the system for 2012 and its performance are discused. The LHCb detector was originally designed for an instantaneous luminosity of L = 2 · 10 32 cm −2 s −1 . During the running period of 2012, this design luminosity was doubled to L = 4 · 10 32 cm −2 s −1 , which was possible thanks to the excellent performance of both the detector hardware and the trigger system. In these conditions, the average number of visible interactions per bunch crossing is µ = 1.6. One novel feature of the Run 1 trigger was that of event deferral: 20% of all L0 accepted events were stored on disk to be processed by the HLT during the LHC inter-fill time, which made an effective 25% of extra CPU available.
The trigger efficiency is evaluated using events that are reconstructed using the full offline software and selected with the final analysis selection for the respective channel. Thus, the trigger efficiency contains only the additional inefficiency due to simplifications used in the trigger, possible alignment inaccuracies, worse resolution than the offline reconstruction or harder cuts imposed by rate and processing time limitations. The method to evaluate the trigger performance and its implementation are discussed in Refs. [4,6].
The rate of visible collisions in LHCb is about 13 MHz, which then is reduced by the L0 trigger to 1 MHz, at which the full detector is read out. The L0 trigger is implemented in custom hardware and has a latency of 4 µs. It triggers on high transverse momentum (p T ) muons and on large transverse energy (E T ) deposition in the calorimeter.
The High Level Trigger consists of two stages, HLT1 and HLT2. The first stage, HLT1, performs a partial event reconstruction and an inclusive selection of signal candidates. At the reduced rate of 80 kHz, HLT2 performs a full event reconstruction with only minor adjustments as compared to the offline reconstruction sequence. After this reconstruction, a set of inclusive and exclusive selections reduces the trigger rate to 5 kHz, which are saved for later offline analysis. The rates discussed above are average rates from the 2012 run of the LHC, in 2011 the HLT1 output rate was approximately 40 kHz and the HLT2 output rate was 3 kHz.
Hadron trigger performance
The L0 hardware trigger performance for hadronic modes is shown in Fig. 1. It is most efficient on two prong beauty decays (∼40%), and least for four prong charm decays (∼20%). The other modes lie in between.
Hadronic signals are generally selected in the HLT most efficiently by the inclusive beauty and charm trigger lines for both levels of the HLT. The HLT1 line selects good quality track candidates based on their p T and displacement from the primary vertex. This trigger line gets the dominant part of the HLT1 bandwidth allocated. It is the dominant trigger line for most physics channels that do not contain leptons in the final state. The performance of HLT1 for hadronic signatures is shown in Fig. 2 as a function the p T of the decaying charm or beauty hadron.
A multivariate selection is used to trigger B decays into charged hadrons in an inclusive selection based on two-three-and four-prong vertices. These trigger lines are based on a BDT classifier that uses discretized input variables [7] which ensures a fast and robust implementation. A crucial input to the BDT is the corrected mass, which takes the missing momentum transverse to the direction of flight into account. This allows the trigger to select heavy flavour decays even when some final state particles are not reconstructed. Fig. 3 shows the efficiency for the inclusive HLT2 trigger lines on a set of hadronic beauty decays.
Lepton trigger performance
The L0 hardware trigger selects muons based on their track segment reconstructed in the Muon chambers. The corresponding efficiency is shown in Fig. 4 for the decay B − → J/ψK − with J/ψ (µ + µ − ). The single muon trigger contributes the dominant part to the efficiency. The largest inefficiency originates in the tight muon identification requirements inside the L0 reconstruction algorithm. The L0 dimuon trigger selects a small fraction of additional candidates at lower transverse momenta. The combined efficiency for both L0 muon triggers, integrated over p T , is 89%.
In the first part of the HLT, muons are selected both by an inclusive single track line (analogous to the hadron line discussed above) and by dimuon triggers. The dominant inefficiency for these lines originates in the online muon identification algorithms. The performance of HLT1 at selecting muonic signatures is shown in Fig. 5 as a function of p T of the B + candidate.
In HLt2, several trigger lines select events with one or two identified muons. The muon identification procedure is identical to the one used in offline analysis. Single muon candidate events are selected with very tight p T and vertex separation requirements. Dimuon candidate events are selected with either mass or vertex separation requirements, or with a combination of both. The efficiency of the HLT2 muon selections is shown in Fig. 6.
Run 2: Trigger preparation for 2015 -2018
In 2015, the collision energy will be increased compared to 2012 (from 8 TeV to 13 TeV), and the bunch spacing will reach the design value of 25 ns. In these conditions the 2012 instantaneous luminosity of 4 × 10 32 cm −2 s −1 can be achieved at a lower pile-up (1 interaction/crossing instead of 1.6). The yield will further benefit from increased cross-sections for b-and c-hadron production. The requirements on the High Level Trigger will be partly relaxed by increasing the output rate for storing data to tape (12.5 kHz). A part of this data will be saved in a reduced data format for analysis without offline processing. On the other hand, the maximum read-out rate will be still limited to 1 MHz, and the new conditions will be challenging for the L0 trigger.
The computing resources will be adjusted to the 2015 conditions by doubling the Event Filter Farm CPU power and increasing the disk space for deferral. Changes will also be introduced in the architecture: both HLT levels will be split in two separate physical processes, allowing the second stage to run asynchronously. This allows a more efficient use of disk space and of the deferral mechanism, ultimately leading to an increase in the resources available for the High Level Trigger processing. A real time alignement and calibration can be performed on the output of HLT1 and used to improve the quality of the reconstruction in HLT2.
The track reconstruction sequence will be improved such that it allows to adopt multi-track trigger selections already in the first stage of the High Level Trigger.
Run 3: Trigger strategy from 2018 onwards
The LHC will provide LHCb with an instantaneous luminosity of 2 × 10 33 cm −2 s −1 . This implies signal rates of about 300 kHz of beauty hadrons and 800 kHz for charm hadrons that are reconstructible inside the detector [8]. This represents a fundamental change in trigger requirements of LHCb. It is no longer the case that the trigger must reject background from signal, it must now categorise signal according to physics requirements. Such a strategy requires significantly more information in the trigger than can be provided by low-latency, hardware based solutions.
With the removal of the L0 hardware trigger, LHCb will be the first hadron collider experiment to deploy a trigger exclusively in software, using off-the-shelf hardware. The upgraded LHCb trigger will select and categorise events at the full collision rate. The advantages of such a system are clear. Software is easily modified, allowing an unprecedented flexibility as and when the LHCb physics programme changes. It also has the advantage that computing power is readily upgradeable and benefits from the ability to purchase more CPU power for the same price at a later stage. The deferral technique used in Run 1 will be leveraged in Run 2 and Run 3. Subdetector alignment and calibration will be performed while events are buffered to
The inclusive beauty trigger
After the tracking sequence, the remaining timing budget is available to apply offline-quality trigger disk, permitting the use of offline-quality reconstruction, and reducing the need for reprocessing of data at a later stage. In the Run 3 trigger, all tracks will be made available at the earliest stages of the trigger and the full tracking sequence will be performed upfront [9]. In the trigger tracking sequence the same algorithms as in the offline sequence are used, but the sequencing and configuration are modified to reconstruct the most valuable tracks first and perform some basic candidate selection using these. Slower more specialised reconstruction algorithms only need to be executed later in the HLT algorithm flow.
After the tracking sequence, the remaining timing budget is available to apply offline-quality trigger selections. An inclusive, multivariate beauty signal selection is then performed directly on the 30 MHz of inelastic collision data. The performance of the inclusive trigger selections is summarised in Fig. 7. This inclusive trigger selection is complemented by a set of exclusive trigger selections, that are designed to maximize the efficiencies for specific core modes or to minimize systematic uncertainties. One example are trigger selections that do not bias the decay time of the selected mesons.
Summary
The LHCb trigger system has performed extraordinarily well in the first running phase of the LHC. It is designed to select charm and beauty hadrons in a large range of decay modes. The flexible design of the HLT, fully deployed in software, allows to quickly adjust to changes in running conditions and physics goals. Inclusive selections in the full trigger chain allow an efficient trigger for basically any beauty decay to charged tracks. Several innovative concepts have enabled this performance: the deferred triggering allows to optimise the trigger usage for mean instead of peak usage of the available computing resources. Multivariate selections allow the inclusive selection of beauty decays into charged tracks with high efficiency.
For the upcoming Run 2, several improvements of the trigger system are planned: the two software trigger levels will be decoupled which allows calibrations between them and thus a performance much closer to the one achieved offline. The use of event buffering for alignment and calibration permits selections that would otherwise only be available in the offline analysis environment.
The upgraded LHCb trigger represents a turning point in the design of hadron collider trigger systems. The approach to trigger purely in software running on commercially available hardware allows an inexpensive, flexible and scalable design. The upgraded trigger will perform offlinequality tracking at the full collision rate, which allows a number of very efficient inclusive and exclusive trigger selections. Subject to output bandwidth requirements, gains of up to a factor of four in efficiency are possible for several LHCb benchmark modes. | 2019-04-21T13:05:28.399Z | 2015-06-11T00:00:00.000 | {
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270223373 | pes2o/s2orc | v3-fos-license | Primary pericardial synovial sarcoma requiring emergency salvage right atrial debulking: a case report
Abstract A 52-year-old gentleman presented with symptoms of breathlessness and type 1 respiratory failure. His CT pulmonary angiogram showed a heterogenous, oval-shaped lesion between the heart and diaphragm with a right atrial (RA) filling defect, pericardial thickening and pulmonary metastasis. An RA debulking salvage operation confirmed this to be a pericardial tumour and further cytology and immunohistochemistry testing confirmed a primary synovial sarcoma. After 12 days in intensive care for ventilation, the patient was successfully discharged on warfarin and underwent oncology follow-up for chemotherapy. Following a 15-month follow-up, no mortality was observed despite the aggressive nature of the tumour.
Introduction
Primary pericardial synovial sarcoma is a rare pathology with an often, uncertain prognosis.This report presents a case of a gentleman with acute respiratory failure as a result of a pericardial synovial sarcoma requiring emergency salvage debulking.
Case summary
A 52-year-old gentleman was admitted to a district general hospital for shortness of breath causing type 1 respiratory failure with oxygen saturations of <90% despite high f low oxygen.His past medical history was unremarkable, and he was usually fit and well.A CT Pulmonary Angiogram (CTPA) was undertaken, which showed a heterogeneous oval-shaped lesion measuring 15 × 9 × 14 cm 3 between the heart and the diaphragm, with a median attenuation of 35 HU and no enhancement during the arterial phase.Further solid lesions of 14 mm in the right lower lobe and 5 mm in the right upper lobe raised the suspicion of a primary cardiac sarcoma with pulmonary metastasis.This is shown in Fig. 1.Subsequent urgent CT staging (Fig. 2) was undertaken, which confirmed an indeterminate space occupying the inferior pericardial space, right atrial (RA) filling defect and anterior nodular pericardial thickening.No intra-abdominal or bone lesions were identified.An echocardiogram also confirmed these finding with the addition of no f low through the tricuspid valve (TV), as shown in Fig. 3.
Due to the critical condition of the patient, he was transferred to a tertiary centre for an emergency salvage debulking of the RA tumour via sternotomy.The goals were to improve haemodynamics, save his life and to obtain tissue histology samples.The chest was divided with a median sternotomy and multiple pericardial adhesions were encountered on approach.Following adhesiolysis, the tumour was found to be invading the inferior surface of the diaphragm and right ventricle.This was determined to be non-resectable.Hence, the cannulation strategy was through superior vena cava, right femoral venous percutaneous cannulation and the ascending aorta.The right atrium was incised, and the tumour was debulked.Once the inferior vena cava and TV were free, the atrium was closed with 4/0 sutures.The cardiopulmonary bypass time was 43 minutes and the crossclamp time was 15 minutes.Tissue samples were sent for histology.Macroscopically, three pieces of grey and tan tissue measuring 100 × 80 × 15 mm 3 were found to be heterogeneous.Microscopic findings showed a malignant hypercellular neoplasm with vague fascicular to haemoangiopericytic architecture with scant intervening stroma and amphophilic cytoplasm.There some cytoplasmic dot staining with pancytokeratin, and patchy staining with CD56 and CD99.This confirmed a synovial sarcoma that was Grade 3 (FNCLCC) with the tumour extending to the margins.
Post-operatively, the patient required stay in intensive care for 12 days for ventilatory and inotropic support.During this period, the patient required a short course of antibiotics for a hospital-acquired pneumonia.However, he was subsequently stepped down to the ward and discharged on warfarin with a target INR of 2.5.Following the diagnosis of synovial sarcoma, the patient was referred to oncology for follow-up and chemotherapy.On a 15 months' follow-up, the patient was still alive and undergoing adjuvant therapy, although the prognosis is still uncertain.
Follow-up CT scans were undertaken during chemotherapy that showed continued reduction in the size of the RA tumour, as demonstrated in Figs 4 and 5.The pericardial mass following chemotherapy is visualized in Fig. 6.
Discussion
The incidence of a pericardial synovial sarcoma is rare with only a few cases being reported in literature [1,2].A series of 12 485 with an incidence of 80 and 20%, respectively [ 4].Synovial sarcomas have been described to belong to three subtypes: biphasic, monophasic and poorly differentiated [2].The relationship between immunohistochemistry markers and the variants of synovial sarcoma is not fully established; however, Bcl-2, CD99 and cytokeratin have been described as characteristic markers of synovial sarcomas [5,6].
Factors for a good prognosis and increased survival from the limited data available were noted to be location on the left side of the heart, limited necrosis, low mitotic count, no metastasis on diagnosis.Their case series had 75 patients of which the mean and median survival were 11 and 6 months, respectively [4].However, this patient had none of these features and was undergoing chemotherapy under oncology 15 months post-operatively.Prognosis can also be directly linked to debulking, and the quantity of the tumour resected.In fact, less than a quarter of published cases have been identified to have been completely resectable due to the anatomical location and typically aggressive spread [2].
Symptoms of pericardial synovial tumours were mostly attributed to chest pain and dyspnoea, which have been noted to be attributed to an increased pericardial effusion.However, despite the large tumour, no effusion was demonstrated on echocardiogram.This case report demonstrates the patient was that of an acute scenario and an atypical presentation.Typically, careful pre-operative planning and staging is required with the use of echocardiogram, CT imaging, MRI and cytology of any pericardial effusion.However, this case has demonstrated that a successful salvage operation is feasible with adequate debulking and prompt oncology referral.A swift response is paramount to maximize prognosis.
Figure 1 .
Figure 1.CTPA showing oval-shaped heterogenous lesion as well as RA filling defect.
Figure 2 .
Figure 2. CT thorax-abdomen pelvis, depicting the RA filling defect as well as pericardial bulk.
Figure 3 .
Figure 3. Pre-operative apical four-chamber view echocardiogram showing RA bulk blocking TV f low.
Figure 4 .
Figure 4. Post-operative CT scan during chemotherapy showing reduction of size RA bulk.
Figure 5 . 3 Figure 6 .
Figure 5. Post-operative CT scan showing further reduction of RA bulk following chemotherapy. | 2024-06-05T05:10:16.189Z | 2024-06-01T00:00:00.000 | {
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211554652 | pes2o/s2orc | v3-fos-license | Anxiety and hemodynamic reactivity during cardiac stress testing: The role of gender and age in myocardial ischemia
Background The prevalence of myocardial ischemia is associated with anxiety. State and trait anxiety are more common in younger women compared to men, and high anxiety levels could affect hemodynamic reactivity during cardiac stress testing. The aim is to examine whether anxiety plays a role in gender differences in patients ≤ 65 and > 65 years in hemodynamic reactivity and ischemia during cardiac stress testing. Methods and results Included were 291 patients (66.8 ± 8.7 years, 45% women) with suspect ischemia undergoing myocardial perfusion single-photon emission computed tomography (MPI-SPECT). Primary outcomes were semi-quantitative summed difference score (SDS) and summed stress score (SSS), as continuous indicators of myocardial ischemia. Analyses were stratified by age. Trait anxiety was measured using a validated questionnaire (GAD-7) and state anxiety using facial expression analyses software. Overall, trait and state anxiety were not associated with the prevalence of ischemia (N = 107, 36%). A significant interaction was found between gender and trait anxiety in women ≤ 65 years for SDS (F(1,4) = 5.73, P = .019) and SSS (F(1,10) = 6.50, P = .012). This was not found for state anxiety. Conclusion SDS and SSS were significantly higher in women younger than 65 years with high trait anxiety. This interaction was not found in men and women over 65 years. Electronic supplementary material The online version of this article (10.1007/s12350-020-02079-3) contains supplementary material, which is available to authorized users.
CAD
Coronary artery disease HRR Heart rate reserve LVEF Left ventricular ejection fraction IHD Ischemic heart disease MPI Myocardial perfusion imaging SDS Summed difference score SRS Summed rest score SSS Summed stress score TPD Total perfusion deficit
INTRODUCTION
Traditional cardiovascular risk factors (e.g., smoking, hypertension, and diabetes mellitus) are clinically important but do not fully account for the gender differences in the increasing rates of cardiovascular risk and hospitalizations in younger women. 1,2 Large epidemiological studies 3 and meta-analyses 4 have shown that high levels of psychosocial risk factors are associated with an increased risk of IHD. Anxiety 5 and depression 6 are among the most prominent psychological risk factors for incident ACS and adverse outcomes in patients with IHD. 7 However, depression has been extensively studied 8 and has an official recommendation in clinical cardiology guidelines. 9,10 Anxiety has received less attention, but multiple studies show its importance in predicting higher rates of cardiac risk factors and events. 11,12 Findings on anxiety and patients with suspected myocardial ischemia have been mixed, 13,14 warranting further investigation. The population attributable risk of psychological factors for incident CAD events is found to be higher for women than men because women report higher levels of distress-related psychological factors than men. 15,16 However, the relative risk of incident CAD events related to psychological factors does not markedly differ between women and men. 4,17 Specifically, the prevalence of anxiety is higher among women compared to men. 18 In addition, especially younger women in the cardiac patient population (compared to the general population) have significantly more anxiety-related problems than older women and men. 19 Although evidence indicates that younger age may be associated with different pathophysiological processes in women with IHD, 20,21 it is unclear whether the association of anxiety with inducibility of ischemia is age dependent.
Epidemiological data indicate that women younger than 65 years of age display increasing rates of acute coronary syndromes (ACS) relative to men in the same age range. 22,23 Hospitalization and 30-day mortality rates are also increasing in women younger than 55 years of age compared to men. 24 Elevated hemodynamic reactivity in younger women may partially explain the relatively high prevalence of myocardial ischemia in response to adenosine administration during cardiac stress testing. 21 For example, baseline heart rate and baseline systolic blood pressure (SBP) were significantly higher in women compared with men during adenosine cardiac stress testing, and SBP significantly increased during stress testing for women but not for men. 21 The gender differences in IHD incidence and progression as well as inducibility of ischemia indicate that further investigations are needed on the interplay between gender, age, and cardiovascular risk factors.
High levels of trait anxiety are associated with higher rates of ischemia in women without a history of CAD compared to women with lower levels of trait anxiety. 25 Another study showed that, among patients with non-obstructive CAD, the risk of developing ischemia during cardiac stress testing was higher among women with high levels of trait anxiety, but not state anxiety. 26 It has also been documented that trait anxiety is associated with more frequent and severe cardiac symptoms. 27 The association between anxiety and ischemia may be accounted for in part by higher hemodynamic responses during cardiac stress testing. Anxiety is generally related to increased sympathetic nervous system activity. 28 In addition, elevated levels of anxiety have been associated with higher sympathetic nervous system activity in postmenopausal women. 29 These data indicate that there may be an interplay between gender, age, anxiety, and hemodynamic responsiveness as related to inducible myocardial ischemia during cardiac stress testing.
The present study examines whether anxiety plays a role in the gender differences in hemodynamic reactivity and inducible ischemia during cardiac stress testing. Given that evidence suggests that these associations seem to be stronger in young women, we will also explore age-stratified analyses. Specifically, it was hypothesized that: (1) women will report higher levels of anxiety than men; (2) high levels of anxiety in patients undergoing cardiac stress testing will be associated with elevated hemodynamic reactivity in women, and less so in men; (3) high levels of anxiety will be associated with the presence and severity of inducible ischemia during cardiac stress testing in women, but less in men. Furthermore, we expect that these genderspecific associations primarily occur among female patients of younger age (B 65 years) versus older age ([ 65 years).
Patients
The study sample consisted of 291 patients who underwent a pharmacological or exercise stress/rest myocardial perfusion imaging single-photon emission computed tomography (MPI-SPECT, henceforth referred to as MPI) protocol with adenosine (n = 203) (140 mcg -1 kg -1 min -1 for 5 minutes) or exercise (n = 88) (i.e., bicycling to maximum exertion using the modified Bruce protocol) between January 2017 and December 2018 at Institute Verbeeten Tilburg, the Netherlands. Inclusion criteria were: (1) referral for the MPI protocol with adenosine or exercise; (2) ability to fill out questionnaires; and (3) sufficient knowledge of the Dutch language. There were no exclusion criteria. Reason for referral were categorized into 'examination of new or worsening symptoms (no previously known CAD)' (N = 174), 'examination of known CAD (returning symptoms)' (N = 114), or 'preoperative cardiovascular risk assessment' (N = 3). All patients underwent the protocol as described below with MPI images obtained at rest and following cardiac stress testing.
In order to obtain state anxiety measures, patients' facial expressions were video-recorded using a webcam (Logitech C920-HD Pro) attached to the stationary exercise bicycle, for both adenosine and exercise stress testing protocols (GE Healthcare, Ergometer ebike comfort 162202, Freiburg, Germany), as further described below. During cardiac stress testing (both protocols), patients were asked whether they experienced anginal chest pain symptoms (anginal chest pain present/absent) similar to their ''typical'' cardiac symptoms.
Sociodemographic and psychosocial data were collected before and after cardiac stress testing. Information on cardiovascular risk factors (hypertension, hypercholesterolemia, familial risk, and smoking), medical history, and medication was obtained from electronic patient records. In the present study, we refer to differences between women and men as gender rather than sex differences, since in human study participants the contributions of social, environmental, cultural, and behavioral factors and choices cannot be excluded as factors influencing the research findings. 30 The study was approved by the regional Medical Ethics Committee (METC Brabant, Protocol number: NL56707.028. 16), and all patients provided written informed consent.
Myocardial Perfusion Imaging (Rest-Stress Protocol)
All patients underwent a two-day protocol with the first day allocated to the at-rest imaging and the second day to the stress (adenosine or exercise) imaging. Patients were instructed to refrain from consuming caffeine-containing beverages for 24 hours before each protocol day. The first day consisted of 99m Tc-tetrofosmin injection (dosage: 370 MBq), and a rest period of 45 minutes followed by myocardial perfusion imaging. The second day, patients performed cardiac stress testing, either pharmacologically by intravenous adenosine injection (140 mcg -1 kg -1 min -1 for 5 minutes) or by exercise (i.e., bicycling to maximum exertion using the modified Bruce protocol). As per standard clinical protocol, the pharmacological adenosine cardiac stress testing also involved mildintensity cycling to limit adenosine side-effects and reduce extracardiac activity. 31 For the adenosine protocol, 99m Tctetrofosmin injection was at 2 minutes after adenosine administration. During the exercise protocol, the injection was at peak exercise, at 85% of the maximum heart rate (0.85*(220patient age)).
Image acquisition was performed using a hybrid dualheaded gated IQ SPECT/CT system (Symbia T, Siemens Medical Solutions AG) equipped with multifocal collimators (SmartzoomTM) of 128 9 128 matrix size and zoom factor of 1. Acquired data were then reconstructed using an iterative reconstruction. Attenuation correction was applied using a patient-dedicated low-dose CT-derived mu map. A symmetric 15% window was centered at 140 keV, with a three-lead electrocardiographic monitoring. Perfusion images were inspected by qualified staff before interpretation by experienced observers.
Image Analysis and Hemodynamic Reactivity Parameters
Bull's eye generation, and visual analysis using a 17segmental model, 32 was performed by two experienced observers. Semi-quantitative analyses of perfusion were performed with QPS software from Cedars-Sinai Medical Center. The interpretation of the scan was assessed both semiquantitatively and by visual analysis, as is recommended by the American Society of Nuclear Cardiology (ASNC). 33 Both AC and no-AC images were reviewed during interpretation. Perfusion was graded on a 0-4 scale: 0 = normal, 1 = equivocal, 2 = moderate, 3 = severe perfusion defect, and 4 = very severe perfusion defect. Quantitative summed rest scores (SRS), summed stress scores (SSS), and summed difference scores (SDS) were tabulated. Myocardial ischemia was semiquantitatively defined as SDS C 2. 34 Pixel-wise total perfusion deficit (TPD) was used as an automated perfusion deficit parameter and TPD C 3% was considered abnormal and indicative of a perfusion defect. 35 Hemodynamic Reactivity Baseline and peak heart rate (HR) and blood pressure (BP) were recorded in beats per minute (bpm) and mmHg, respectively. Peak HR was defined as the HR at 2 minutes after adenosine injection and maximum heart rate during the exercise protocol. Peak BP was recorded at 2 minutes after adenosine injection and at peak exertion during the exercise protocol. HR response for both protocols was defined as the % heart rate reserve (HRR, (peak HR -baseline HR)/baseline HR)*100). Left ventricular ejection fraction (LVEF) was determined, and also end diastolic volume (EDV) and end systolic volume (ESV) were calculated.
Trait Anxiety Measure
The generalized anxiety disorder (GAD)-7 scale is a clinical screening measure for assessing generalized anxiety disorder. The GAD-7 consists of seven items that are answered on a four-point scale from 0 (''not at all'') to 3 (''nearly every day''). The GAD-7 has been validated in the general population, 36 yielding a high internal consistency (Cronbach's alpha = .89). For analyses, the continuous score of the GAD-7 was used, with a higher score indicating more anxiety symptoms. A cut-off score of 10 is also used indicating patients with 'low anxiety' (GAD-7 B 10) and 'high anxiety' (GAD-7 [ 10).
State Anxiety Measure
Video-Recording State anxiety measures were based on analyzed video-recordings during cardiac stress testing for both the adenosine and exercise protocol. For the analyses of anxiety expressions, the software package FaceReader 7.0 37 was used to analyze the video-recordings made during cardiac stress testing. Although this is a relatively novel method, it has been validated and used in multiple research settings. [38][39][40] After taking a seat on the exercise bicycle (both adenosine and exercise), the video-recordings started with 'baseline', approximately 1 minute before cardiac stress testing actually began, and served as the baseline measure of facial expression of anxiety. The three other time blocks during cardiac stress testing were 'start cardiac stress testing' when patients started exertion, 'maximal cardiac stress testing', when heart rate was at peak level (or at 2 minutes after adenosine injection), and 'recovery', when patients stopped exertion and slowly recovered.
FaceReader Analysis of Video-Recordings FaceReader first synthesizes an artificial face model, which describes the location of over 500 keypoints in the face and the facial texture of the area entangled by these points. Then, based on this, classification of the facial expressions is done by an artificial neural network. 41 Based on over 10.000 images, the network was trained to classify the six universal emotions: anxiety, sadness, anger, surprise, happiness, and disgust. 42 Finally, scores of intensity of facial expressions are computed on a continuous scale from 0 to 1, yielding the percentage (0%-100%) of facial expression of anxiety. In addition to the percentage of facial expression of anxiety, a dichotomous measure (median split) of facial expression of anxiety is used in statistical analyses.
Statistical Analysis
Data are presented as means ± standard deviation (SD) for continuous variables and frequencies and percentages for categorical variables, stratified by gender. Group comparisons for categorical variables were examined using v 2 tests and continuous variables using independent t-tests, for the adenosine and exercise protocols separately. Associations of ischemia with trait and state anxiety and the interaction with gender and age were examined using a two-way ANOVA with a gender by (trait and state) anxiety interaction term, stratified for two age groups: B 65 and C 65 years of age. Residual analysis was performed to test for the assumptions of the twoway ANOVA. Outliers were assessed by inspection of a boxplot, and homogeneity of variances was assessed by Levene's test. SRS, SSS, and SDS were square root transformed to account for outliers. The primary outcome measure is ischemia, semi-quantitative operationalized as SDS, and SSS, inferring diagnostic value. 43 Hemodynamic measures included HR, HRR, SBP, DBP, and LVEF. Statistical analyses were performed using SPSS version 24.0 (SPSS Inc., Chicago, Illinois), two-sided P values are reported and statistical significance was set at P \ 0.05. Table 1 displays the patient characteristics. The mean age of the sample was 66.8 ± 8.7 years. The mean age for women was slightly lower (65.8 ± 8.5) than for men (67.7 ± 8.8; P = .071). Men more often had a previous MI (16% vs 5%, P = .006), PCI (34% vs 18%, P = .002), and CABG (22% vs 5%, P \ .001) than women. Women more often had no history of previously known CAD compared to men (72% vs 50%, P = .001).
Gender Differences in Myocardial Ischemia
Myocardial ischemia was present in 107/291 (37%) of the patients. Overall, women less often displayed ischemia than men; 28% vs 39% (X 2 = 6.98, P = .008), and women had lower mean SRS (1.16 ± 2.83 vs 2.59 ± 5.0, P = .004), SSS (2.52 ± 4.74 vs 4.12 ± 6.16, P = .016), and TPD (2.09 ± 3.27 vs 3.18 ± 4.17, P = .016) values than men, and a similar but non-significant difference was found for SDS (1.45 ± 2.46 vs 1.84 ± 3.13, P = .253). We also explored the interaction between gender and age as related to ischemia, but no significant interactions between gender and age were found with regard to SRS, SSS, SDS and TPD values (all P [ .753). Table 2 shows the analyses per cardiac stress testing protocol (adenosine and bicycle exercise). Ischemia occurred in 40% of the 203 patients undergoing the adenosine protocol and 28% in the 88 patients undergoing the bicycle exercise protocol. During the adenosine protocol, men displayed slightly more ischemia then women (46% vs 34%) but this difference was not significant (P = .077) ( Table 2). During the exercise protocol, ischemia was more prevalent among men than women (38% vs 16%, P = .022).
During the adenosine protocol, SSS was higher for men compared to women (4.9 ± 6.9 vs 3.1 ± 5.2, P = .046). TPD was higher for men only during the exercise protocol (0.8 ± 1.6 vs 2.3 ± 3.1, P = .003). No differences were found for typical anginal chest pain between men and women for both protocols.
Gender Differences in Trait and State Anxiety
No significant gender differences were found for the presence of trait anxiety (18% vs 18%, P = .883) with similar total scores (women 5.48 ± 5.2 vs men 5.49 ± 5.6; P = .982). When comparing the sample by age, no significant differences were found for the presence of trait anxiety (23% (younger patients) vs 14% (older patients), P = .058). The interaction between gender and age as related to trait anxiety was also not significant (F(1, 270) = .633, P = .427, partial g 2 = .002) Measures of state anxiety based on facial expression revealed that no gender differences were found for presence of state anxiety for all time blocks (baseline: 44% vs 38%, P = .270; start cardiac stress testing: 38% vs 35%, P = .660; max cardiac stress testing: 47% vs 40%, P = .260; recovery: 45% vs 38%, P = .189). Mean scores for each time block were also similar for men and women (baseline: 2.94 vs 2.74, P = .552; start cardiac stress testing: 2.52 vs 2.60, P = .796; max cardiac stress testing: 3.26 vs 2.90, P = .449; recovery: 3.77 vs 3.33, P = .267). The interaction between gender and age as related to state anxiety was not significant for all time blocks (all P [ .252).
Gender and Age Differences in Hemodynamic Reactivity During Cardiac Stress Testing
Women had a higher heart rate after 2 minutes of adenosine injection (100.4 ± 16.8 bpm vs 90.8 ± 18.4 bpm, P \ .001) and HRR (40.9 ± 23.3 vs 30.7 ± 22.7, P = .002) than men. Baseline heart rate was higher for women prior to the exercise protocol (77.9 ± 12.7 bpm vs 71.7 ± 14.5 bpm, P = .039) and no gender differences for peak heart rate or HRR during exercise were found. No significant differences in blood pressure during rest or peak were found between men and women for both protocols ( Table 2).
State Anxiety and Hemodynamic Reactivity
During start cardiac stress testing, patients with high state anxiety during the adenosine protocol (n = 76) had a lower systolic blood pressure than patients with low state anxiety during cardiac stress testing (n = 127) (125.5 ± 20.5 vs 132.8 ± 26.7 mmHg, P = .032). No other differences in hemodynamic reactivity were found for patients undergoing the adenosine protocol. In the exercise protocol, patients with high state anxiety during recovery (n = 43) had a higher peak LVEF than patients with low state anxiety (n = 39) (64.7% ± 11% vs 59.2% ± 11.9%, P = .030). No other differences were found in hemodynamic reactivity for patients in the exercise protocol.
Trait Anxiety and Ischemia
There was no significant overall association between trait anxiety with inducibility of ischemia, SSS, SDS, or TPD values (all P [ .221 and partial g 2 \ .006). There were also no gender-by-trait anxiety interactions for these measures (P values [ .217; partial g 2 \ .003).
For patients [ 65 years, a significant interaction effect was found for gender with trait anxiety for TPD only (F(1, 80) = 4.05, P = .046, partial g 2 = .025). For patients with high trait anxiety, the mean TPD was 4.71 (95% CI 0.96-8.46) higher for men than for women, whereas other subgroup comparisons were not significant.
State Anxiety and Ischemia
A two-way ANOVA showed that the interaction effect between state anxiety and gender for SSS, SDS, and TPD was not statistically significant for either of the four time blocks (baseline, start cardiac stress testing, max cardiac stress testing, and recovery) during cardiac stress testing (all P values C .071 and partial g 2 \ .023) for both patients under and over 65 years.
DISCUSSION
The present study examines whether anxiety plays a role in the gender differences in hemodynamic reactivity and inducible ischemia during cardiac stress testing. The first hypothesis was not confirmed, as men and women did not differ on overall trait and state anxiety. The second hypothesis was confirmed, since women had higher heart rate responses to cardiac stress testing when undergoing the adenosine protocol and, overall, a higher heart rate response was associated with high trait anxiety. For the third hypotheses, we found that measures of ischemia (SDS and SSS), albeit low in each subgroup, were significantly higher in women with high trait anxiety, but only in patients younger than 65 years of age.
Gender differences during cardiac stress testing were examined for both the adenosine and exercise protocol. For the adenosine protocol, the peak heart rate and HRR were significantly higher in women compared to men. This difference was not observed during the exercise protocol, in which peak heart rate was higher for men and no gender differences in HRR were found. Consistent with these results, previous studies determined gender and age as independent factors associated with HR responses. 44,45 Mechanisms for this gender difference are not yet fully clear, but research suggests that women exhibit higher parasympathetic stimulation of the heart, which is ultimately protective during periods of increased cardiac stress. 46 However, no marked gender differences were found for SBP and DBP during baseline and peak for both protocols. Consistent with earlier studies, SBP was slightly blunted during adenosine in women, but not in men. 47 A significantly higher LVEF was observed in women compared to men for both protocols which is consistent with previous findings. 48 Reduced LVEF has been associated with inducibility of myocardial ischemia, 49 which may partially explain the overall higher prevalence of ischemia in men than women.
For patients younger than 65 years, a significant interaction effect was found between trait anxiety, but not state anxiety, and gender. Being female, younger than 65 years, and having high trait anxiety was associated with a significantly higher SSS, SDS, and TPD compared to men. This finding was not observed in patients of 65 years and older. The observed state versus trait anxiety contrast in association with ischemia is consistent with earlier findings. 26,50 These two studies, however, were conducted with a healthy elderly (male) patient sample and a cardiac syndrome X patient sample, respectively, suggesting that trait anxiety is a recurring significant factor in both patients with and without cardiac history and studies with different cardiac outcomes. In the study by Paine and colleagues, 25 trait anxiety in women with no CAD history was significantly related to increased ischemia extent compared to women without anxiety, which is comparable to the findings by Vermeltfoort and colleagues, 26 but in the study of Paine, state anxiety was not examined. In the present study, both trait and state anxiety were examined in relation to ischemia and groups were stratified for age, as is done in the study by Gebhard and colleagues, 21 which concludes that younger women with ischemia have a stronger hemodynamic response to adenosine stress testing than men, a finding that was partly confirmed in our study when comparing women to men.
Consequently, when examining previous studies, our finding that trait anxiety is related to myocardial ischemia in younger women could possibly be attributed to increased sympathetic nervous system activity inherent to anxiety. 51 Increased sympathetic nervous system activity could result in an increased presence of ischemia through increases in heart rate and vasoconstriction. 52 However, increased sympathetic nervous system activity is beyond the scope of the present study and requires additional research. In addition, it is unclear whether this rationale would be more applicable to trait anxiety or state anxiety.
The present study had some strengths and limitations that should be taken into consideration. Limitations include the relatively small patient sample and the crosssectional design, making discussions on causality only speculative. The incorporation of a validated questionnaire to measure trait anxiety can be considered a strength of this study. A unique aspect is the measurement of state anxiety with facial expression recognition software during cardiac stress testing. Because of the transient nature of cardiac stress testing, it should be linked with a transient measure of facial expressions of emotions to optimally reflect the dynamic association between emotions and ischemia. 53 The low intensity of detected state anxiety during cardiac stress testing for men and women (under 10%), could account for the non-significant reported findings for state anxiety in this study.
When evaluating the gender differences in patient characteristics, it was observed that men more often had an elaborate cardiac history compared to women, which is consistent with previous studies. 54 Reasons accounting for this difference are discussed with increasing attention for psychological factors. 55,56 Furthermore, no significant differences in typical anginal chest pain during cardiac stress testing were found for men and women in both protocols. This study also confirms the trait and state anxiety contrast reported in previous studies and suggests that high trait anxiety is an important factor to take into account in women with myocardial ischemia. Larger studies with more sociodemographic variation in the patient groups are needed to further clarify the risk of anxiety in patients with myocardial ischemia.
To conclude, this study shows that female patients younger than 65 years of age with high trait anxiety have increased SDS, SSS, and TPD following cardiac stress testing compared with men and patients older than 65 years. Thus, the implications of psychological constructs on cardiac health are intertwined with gender and age. This interplay is worthy of further investigation and larger observational and intervention studies in patients who are at high risk of myocardial ischemia and adverse long-term cardiovascular prognosis.
NEW KNOWLEDGE GAINED
Our data indicate that SDS and SSS were significantly higher in women younger than 65 years with high trait anxiety. This interaction was not found in men and women over 65 years. State anxiety is not significantly associated with myocardial ischemia. | 2020-02-29T14:55:29.845Z | 2020-02-28T00:00:00.000 | {
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236755399 | pes2o/s2orc | v3-fos-license | A Novel Framework Design for Test Case Authoring and Auto Test Scripts Generation
Every product has defects and identifying defects in the process of development and rectifying them before the launch of the product is very important. Embedded software testing process find the bugs in the software and report to the developer to fix issues. Sometimes to meet the product release deadlines, test engineers will not get much time to cover all test cases. That is why most software testing depends on test automation. In this paper, we focused on the area of automotive and home appliances embedded software test automation. Test automation is the only solution to improve the test phase and meet the timeline of the product launch. There are many test Automation tools like LabVIEW, test stand, and automation desk to automate testing embedded software. However, there is still manual efforts are required to use these tools. This paper deals automate those manual efforts. This Works shows how to generate test scripts from test cases to reduce the manual efforts, time, and cost.
Introduction
Embedded Software Development Life Cycle aims to deliver high-quality product software that satisfies all the requirements. Its starts with Planning and requirements analysis where senior people design product, based on market requirements and technical experts' suggestions. Once the product design has been done then the next stage is to document called SRS (Software Requirement Specification) the product requirements and get the approval from the client. The SRS document decides the product architecture and gets reviewed by the top management for final changes. Based on the SRS document, software developers develop the product software and develop test cases that cover all software functionalities. The software quality assurance team used the test cases and performed either manual Regression or do automation testing. SWQA team used test management software to automate the manual test scenarios. If there is any part of the software updated, the testing team must perform entire software testing to find any other aspects affected with part of the software update, and this is called the regression test.
Software developers follow one of the common software life cycle development models which are mainly waterfall model, Iterative model, Spiral model, V-model and Agile model. The waterfall model divides software development into different phases, and each phase depends on the previous phase. Moreover, it is not suitable for complex projects as it has high risk and uncertainty. The iterative model repeatedly improves the developing versions until the full system is ready as this suite for large projects, but more resources are required. In the spiral model, the development covers three phases like identification, design and build. Requirement changes can easily manage in the spiral model, but the process is complicated. In the Verification and Validation model, implementation of processes happens sequentially like a V-shape. Here each phase of development is directly linked with testing. The V -model is best suited for a small project where requirements are well defined, but it is not suitable for significant and ongoing projects. The agile model divides the product into minor incremental builds and a very realistic approach to software development. These builds follow the iteration method. Each iteration naturally takes from about one to two weeks. Every iteration contains sub functional teams working simultaneously on various areas.
Manual regression is essential for software validation and its time-consuming task. The solution is test automation where one needs to create script manual test cases using any test management software. Later, scripts can be executed as many as times to get results reports and can reduce manual effort and time. In embedded software industries, mostly using test management software are NI Test Stand and dSPACE Automation Desk. The Test Stand is test management software and allows test engineers to develop automated test scripts using code modules written in any programming language. One of the popular languages is LabVIEW, and it is a graphical programming language. At the same time, the automation desk is using mostly in the automotive industry for Hardware in loop test automation. Software testing is a crucial stage in the software development and maintenance cycle. It has been estimated that software testing involves between 30-50 percent of software development. With the wrong choice of testing, the software development process may become costly, inefficient and causes a high frequency of product release delay. We can script manual test cases using test automation tools, but this is costly, and maintenance required highly skilled test engineers. Our framework gives the solution to this problem.
Related Work
Software developers seek test automation methods owing to the costs involved in manual testing. Reusable functionality is widely useful and can reduce engineering costs. Researchers worked on IEC 61131-3 programming languages and classified script generation methods. The study provides a fast method for generation for automation in different industries. The full validation of system functions by simulation at the sub-system and component level can significantly reduce system simulation time. This paper implemented a new framework for automation in the wood and the chemical industry. The framework talks about the requirements of key intelligent automation features and data characteristics, also presented the development of support decision systems into automation modules. A Middleware for Intelligent Automation has been proposed in the experiment using Big data and cloud infrastructure.
A robot operation and testing simulation framework have been proposed to detect logical and physical design flaws. The study uses High Level Architecture (HLA) as a middleware for real-time testing for any hardware device. Test automation approach reduces the software cost and improves the quality. An attempt has been made to measure the effect of automation of testing. A well-trained model can generate test scripts from the design document in the web application testing. For the experiment, a testing tool, TesMa was developed at the NTT Labs. Test automation method has been explored for various appliances employing embedded software. It introduced a novel method to select required test cases from large test suites to reduce time and cost.
Methodology
The proposed script generation framework is implemented in three phases. Phase-1 is the process of writing test cases as per the required format using the software requirements document. Phase -2 is the key part that generates test scripts using pre-defined library set based on intelligent logic. Phase -3 is about to execute scripts and to get test reports. Test script execution depends on the test management software. The three phases have been described in figure 1.
Test Case Authoring
Phase-1 aims to generate standard test cases and deals with Requirements documents, Natural Language Processing-based Test case Generation (NLP TCG) and Test case editor. Test case editor is an interface where we can write test cases manually using software requirement documents. Test case authoring is built on the structure of a pre-defined key with sections. The test case editor user interface is shown in figure 2.
Test steps are divided into three groups: setup, Main steps and Clean-up. Setup group covers pre-condition steps, Main steps group covers the actual test steps of the test scenario, and clean group covers the postconditions of the test scenario.
Five sections are provided to write the test step to cover most of the functionalities.
Action:
Action has the following key terms to define the action of the test step. We used four key terms write, read, set and check to cover the significant test actions. We can add new key terms into action depending on the requirement as shown in figure 3. The keys belong to this section to determine the service type of test step. For example, if we are using protocols like LIN, CAN or Ethernet in software development, writing into these protocol bus messages and reading from these protocol bus steps can be determined here as shown in figure 4.
Figure 4
Sample key set at Function/Service type section
Parameter:
Test parameterization is an efficient method and utilizing parameters rather than fixed values. So, one can execute the same test with different input values of test steps used as inputs or expected outputs that are stored separately from the test steps. Test Parameterization is a powerful practice to enhance the testing scenarios and reuse the same. During the execution of test steps, the parameters are filled in with their actual value, so a parameterized variation of the original test case is produced. The main benefits of parameterization are as follows: • By externalizing the changing parts of a test case(s) as parameters, it is easier to manage complex test cases • Users can automatically execute multiple variations of each test. Multiple Parameter values provided that will cause several executions of each test for each variation of the parameters.
Test Parameterization allows sharing information between multiple test cases.
Value:
In this section, we can define the value of the parameter and can define the expected output value. The remarks section can use to explain any additional notes, which is useful to consider complicated test steps.
Oven, a home appliance used for baking food items and given below is sample oven software requirement. The oven light should turn ON when the oven door is open, or the oven light switch is turned on. Also, the count of oven light ON and OFF, count of the door open and close should be stored. Test case writing using the proposed framework test case editor is simple and easy to understand as shown in figure 5.
Figure 5 Sample Testcase
Embedded software development uses standard documents like JSON or XML files to maintain microcontroller variable data. Here we do parse these files and save them into test label data. Test label data useful write test cases easily. OvenDoorOpenCloseCount, Oven Door and oven light are the test label which parsed from the oven software JSON file.
Phase -2: Test Script Generation
Test steps library based on test management software required to prepare before script generation. Test steps library is part test automation process. Test steps library need to fill with all basic functionality with respective test management software's code modules. In NI test stand mostly, LabVIEW code module used, and for Dspsce automation desk python code used. The test configuration is part of the proposed framework which configure test management software. Script Generation Engine is the business logic which generates test script taking test cases as input and identify the functions based on key terms then replace parameters and values. For example, Step1 action key is Set, the Service type is Function, Parameter is Test Unit and value is Power ON. Based on this combination, it identifies the test unit function and set the write value as Power ON. Research Article Vol.12 No.6 (2021), 1479-1487
Figure 6 Test Script generation block diagram
The general process takes a minimum of 6 hours to write test cases and for scripting 8 hours. So total time approximately 14 hours to automate this requirement testing. Our proposed framework takes 4 hours to write test cases and script generation and validation take a maximum of 15 minutes.
The Script generation Engine has almost had all combinations of logic to generate script generation. Moreover, it is practically not possible to write all combinations of logic. Artificial intelligence and machine learning-based algorithm to predict the unknown combination have been introduced.
Machine learning is part of the Artificial Intelligence technique which will minimize the code to cover unknown cases. Supervised Machine learning algorithms are used to train the model using labelled data, such as an input where the desired output unknown. We trained the script generation model using the k-nearest neighbours (KNN) algorithm with pre-defined key section combinations to predict the test function from the test library for the unknown combination of input keys. However, we have not tested much prediction accuracy and precision. We will discuss the ML algorithm related information in future work.
Phase -3: Test Script execution
The Scripts generated in phase-II is loaded into test management software which is used to execute the test scripts and will get the automation report. We can see the sample automation report in figure 9.
As per the above
Figure 9
Test Automation Report generated by Test Stand Table 1 shows the time required for traditional agile test automation and new approached test automation.
Conclusion
The proposed method is very useful in the field of test automation to reduce the time and cost. This paper need improvements in the selection of right library for key combination. In future, this library selection can replace with trained model with machine learning algorithms. | 2021-08-03T00:04:07.558Z | 2021-04-05T00:00:00.000 | {
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225380840 | pes2o/s2orc | v3-fos-license | Impact of an 11-Week Strength and Conditioning Program on Firefighter Trainee Fitness
: Physical fitness is an important aspect of physical health and wellbeing. The aim of this study wasto investigatethe e ff ectsof 11-weeksof formalstrengthand conditioningtrainingconducted during fire academy training on the physical fitness characteristics of firefighter trainees. Archived physical fitness data for 23 male fire academy trainees (age: 27.6 ± 4.3 y; height: 178.5 ± 6.9 cm; body mass [BM]: 83.9 ± 1.8 kg; BM index [BMI]: 26.8 ± 2.8 kg / m 2 ) were analyzed for this study. These data included vertical jump height, maximum pull-up repetitions, hand grip strength, lower-body strength (3RM Hexbar) and aerobic fitness. Trainees performed three sessions per week (two resistance training sessions and one aerobic conditioning session) in addition to fire academy training. A repeated measures ANOVA revealed significant ( p < 0.05) pre-post decreases in BM and BMI and significant increases in upper- and lower-body strength, and aerobic fitness. Strength and conditioning programs are beneficial for improving firefighter trainees’ physical fitness even when run concurrently with fire academy training and with limited space and equipment. This increased fitness may aid in mitigating known occupational injury risks to this population and improve task performance. data curation, J.J.D., B.L.S. and J.G.; writing—original draft preparation, B.L.S., J.J.D. and Q.R.J.; writing—review and editing, B.A.A., Q.R.J., R.M.O. and R.G.L.; supervision, J.J.D. and R.M.O. All authors
Introduction
As part of their normal occupational duties, firefighters perform physically and cognitively demanding work while enduring extremely high temperatures (i.e., 155 • C or greater) [1]. These professionals must respond effectively and efficiently in these high-stress and austere working environments to preserve personal safety and protect life [2]. Many tasks performed by firefighters, such as carrying heavy tools, victim rescues, pulling objects upwards, and working with heavy objects that may exceed 60 kg, require adequate levels of strength and muscle endurance if they are to be performed safely and successfully [2][3][4]. While performing these tasks, firefighters must wear personal protective equipment (PPE) that can weigh upwards of 22 kg to maintain personal safety [1,2]. This load significantly increases their physiological burden, and as such aerobic fitness requirements, when performing these tasks [5]. Due to the complex demands and the dynamic physiological requirements of this occupation, attaining and maintaining an adequate level of physical fitness is of importance in firefighter populations [2,4,6,7]. This requirement is further made evident given that musculoskeletal injuries in the firefighter's workplace are most often caused by 'muscle bending, lifting and squatting, or muscle stressing' [8].
Relationships between physical fitness and firefighting performance have been reported in the literature. Rhea et al. [6] reported strong correlations between performance on firefighting specific tasks and upper body strength (r = −0.66), upper body muscular endurance (r = 0.61-0.71), lower-body strength endurance (r = 0.47) and anaerobic fitness (r = 0.79) [6]. In addition, Michaelides et al. [7] found that successful performance in simulated firefighting tasks was significantly related to vertical jump (VJ) height (r = −0.44, p < 0.01), isometric abdominal strength (r = −0.53, p < 0.01), upper-body and trunk muscular endurance (push-ups, r = −0.27, p < 0.05, sit-ups, r = −0.41, p < 0.01), and upper-body strength (1 repetition maximum (RM) bench press, r = −0.41, p < 0.01) [7]. These researchers also discovered that both fat mass and body mass index (BMI) were positively correlated to completion times on simulated firefighting tasks. In other words, as fat mass and BMI increased, firefighters required a longer duration of time to complete the measured tasks. In a profession where time literally can mean the difference between life and death, these findings are of significant importance to fire agency instructors responsible for developing training programs aimed at improving the health, fitness, and performance of firefighters.
While muscular (e.g., strength and endurance) and anaerobic fitness (i.e., short bouts of high-intensity activity) are critical components of performance, the need for aerobic fitness in firefighting cannot be overlooked. Previous research conducted by Gledhill and Jamnick [4] reported that, to successfully complete strenuous firefighting tasks, a mean oxygen uptake (VO 2max ) value of 41.5 mL/kg/min was necessary [4]. This value equated to a cardiorespiratory demand of approximately 85% of maximal oxygen uptake (VO 2max ) for the firefighters participating in their study, with the majority of work being performed at 23 mL/kg/min (approximately 50% VO 2max ) [4]. Research by Johnson et al. [5] also provides further evidence on the need for aerobic fitness as they reported average maximum heart rates (MHR) ranging from 85 to 102% of the firefighter's age predicted MHR when performing training evolutions [5]. These values for cardiovascular fitness continue to serve as baseline fitness values by most fire agencies and support the need for aerobic fitness within firefighting populations.
Based on the aforementioned literature, it is evident that firefighting requires a unique combination of muscular, anaerobic, and aerobic fitness in order to have a sufficient work capacity and resistance to fatigue when performing essential job tasks. For example, a seminal study by Davis et al. [9] investigated the potential relationship between standard neuromuscular performance measures, physiological measures, and performance on firefighting tasks. The standard neuromuscular performance measures included grip strength, sit-up repetitions in 2-min, chin-ups and push-ups to exhaustion, standing long jump, and the Wells and Dillon hamstring flexibility test. The physiological measures included systolic and diastolic blood pressure, pulse pressure, fractional heart rate, 5-min step test, and the Balke treadmill test protocol. Lastly, the firefighting tasks included a ladder extension, standpipe carry, hose pull, simulated rescue, and simulated forcible entry. These authors reported that the average within-task heart rates indicated that near maximal aerobic efforts were required to perform these tasks. It was discovered that the main predictors of physical work capacity in the simulated firefighting tasks were MHR, sit-ups, grip strength, and submaximal oxygen pulse (r 2 = 63). Furthermore, the tests that best predicted (r 2 = 39) resistance to fatigue were lean body mass, MHR, final treadmill grade, and body fat. For these reasons, developing muscular strength, endurance, and aerobic fitness, as well as maintaining healthy body composition levels, are critical for improving work capacity within these populations.
There is a paucity of research that examines the ability of a structured strength and conditioning program to bring about changes in the aforementioned components of fitness among firefighter trainees. Cvorovic et al. (10) investigated the effects of a 4-week physical training program, emphasizing high-intensity intermittent training and high-intensity functional training, on the physical fitness and body composition of professional firefighters. Significant improvements in upper-body strength, as measured by chin-ups to failure, (p = 0.021) and estimated VO 2 max (p = 0.012) were reported. However, no significant changes were observed in body mass, BMI, standing long jump, 50 m speed performance, grip strength, lower-back and hamstring flexibility via the sit and reach test, or a 1 min push-up and sit-up test. While significant improvements were not observed in all aspects of fitness, the hort-term improvements observed in upper body strength and aerobic performance are encouraging when minimal blocks of training time are available, such as during fire academy.
Fire academy training provides an ideal environment to educate and train incumbent firefighters on proper exercise regimes as well as exploring their individual needs regarding their personal levels of fitness. This reinforces the purpose of fire training academy, which is to prepare trainees for the rigors of performing fire suppression tasks across the duration of their careers. While prior evidence suggests the importance of physical training programs and their effectiveness in improving trainee physical fitness in first responders [10,11], little research has investigated the effects of specific physical training programs among firefighter trainees [12]. Furthermore, often the limitations during academy training (e.g., time and access to strength and conditioning equipment) present challenges for both the trainer and trainee. Therefore, the primary aim of the present study was to determine baseline physical fitness in firefighter trainees and assess potential physiological changes throughout an 11-week firefighter academy training period. This information may be of value to agencies with limited resources (i.e., budgets, exercise equipment and time) in the development of physical training programs to improve health, fitness, and performance among firefighter trainees, as well as part-time and full-time firefighters.
Materials and Methods
A retrospective cohort analysis of fire academy trainees from a firefighting agency located in the USA was utilized for this analysis. In order to compare physical changes after 11-weeks of training in a 16 week fire training academy, various anthropometric and performance measures were assessed at weeks 4 and 15. Based on the structure of this academy, trainees performed only classwork for the first five weeks of training, and began physical training sessions at week 5. Academy training consisted of a 4-day per week schedule (three, twelve-hour days and one four-hour day) with trainees participating in formal physical conditioning twice per week under the supervision of at least one Certified Strength and Conditioning Specialist (CSCS) and one day per week focused on aerobic conditioning (i.e., long, slow distance running up to approximately 3 miles) with members of the cadre.
Subjects
Archived data for all 23 of the male fire academy trainees (age: 27.6 ± 4.3 y; height: 178.5 ± 6.9 cm; body mass: 83.9 ± 1.8 kg; BMI: 26.8 ± 2.8 kg/m 2 ) who completed training at the academy were analyzed for this study. Prior to the release of this data, all subjects signed an informed consent allowing release of this data to the senior investigator. Furthermore, prior to the commencement of this study ethics approval was granted by a university Institutional Research Board (IRB 17-227).
Procedures
For both pre-and post-testing, trainees reported to the firefighting training academy at 0730. All assessments were conducted at the same firefighter training academy location and were completed at the same time of day by the same testers. Anthropometric and performance measures were taken in the following order: height, body mass, BMI, vertical jump height and lower-body power with a VJ, upper-body muscular strength (pull-up), grip strength with a handgrip dynomometer, lower-back and leg strength with a leg/back dynomometer, and aerobic fitness with a 20 m multi-stage fitness test (20MSFT).
Height, Body Mass, and BMI-Height and body mass (BM) were measured using a portable stadiometer (Seca, Chino, CA, USA) and portable digital scale (Health-O-Meter, McCook, IL, USA).
All height measurements were rounded to the nearest centimeter, whereas all BM measures were rounded to the nearest tenth of a kg. Using this information, BMI was calculated using the following equation: BMI = body mass (kg)/[height (m)] 2 . BMI classifications provided by the National Institutes of Health [13] were used to categorize trainees' potential health risk.
VJ-The VJ was conducted using a Just Jump electric contact system (ProBiotics Inc., Huntsville, AL, USA), which has been previously utilized in tactical populations [14]. The Just Jump Mat system (Probotics, Huntsville, AL, USA) calculates vertical displacement based on flight time. Trainees were instructed to step onto the center of the mat with their feet placed hip to shoulder width apart. Trainees then jumped as high as possible using a counter movement jump with arm swing. Trainees performed two jumps with approximately 10 s rest between each attempt. The best of the two jumps was recorded in inches then converted to metric units (i.e., centimeters) for analysis. During analysis, VJ height was converted to power using the following equation by Sayers et al. (PAPw = (60.7 × VJ Height (cm)) + (45.3 × Body Mass (kg)-2055) [15].
Maximal Pull-up Repetitions-A maximal pull-up repetition test was utilized to measure upper-body muscular strength among the trainees. Trainees began this test by hanging from the top bar of a quad pull-up station, with the arms extended, and the hands wrapped around the bar in an overhand position approximately shoulder width apart. Trainees were then instructed to flex their arms and pull their body upward until their chin was above the bar. After each pull-up was completed, the trainee was required to return to the starting position before performing the next repetition. The trainees' final score was the maximum number of pull-ups that could be completed with good technique and form (no swinging, kicking, or bending of the knees was allowed for the repetition to count). Trainees were allowed to rest at the beginning of this exercise movement, with the arms fully extended. The test was terminated when they were unable to maintain proper technique or reached volitional fatigue. The total number of repetitions performed with correct technique was recorded as the final score for this test.
Handgrip Dynomometer-Grip strength was measured for each trainee using a handgrip dynamometer (Takei Scientific Instruments, Niigata, Japan). All assessments were conducted in accordance with previously established guidelines [16]. Prior to the test, the dynanometer was adjusted for each trainee so that the second joint of the fingers of the hand being assessed fit under the handle. Trainees were then instructed to hold the instrument down to the side of, and in line with, their body without contacting their hip or thigh. The trainee then squeezed the dynamometer as hard as possible for up to three seconds. Two attempts were performed with each hand in alternating fashion, with approximately 60 s of rest between trials with the same hand. The best scores for both right and left hands were recorded to the nearest kilogram and used for analysis.
Lower-body strength-Lower-body strength was measured using a three-repetition maximum (3RM) high-handle hex bar deadlift using the multiple repetition max protocol as outlined by the National Strength and Conditioning Association (NSCA) [17]. Trainees were instructed to perform three warm-up sets of 3-5 repetitions, progressively increasing the load until they worked up to their first 3RM attempt. Additionally, trainees had a maximum of five attempts (or load increases) to reach their 3RM [17]. When ready to test, the trainees were instructed to lift the bar from the ground to a standing position, lowering the bar to lightly touch the ground between repetitions. The testing was observed by a CSCS who assisted with weight adjustments after each attempt. If any technique issues were observed during an attempt, such as rounding of the back, the test was terminated for safety reasons. During analysis, 1 RM estimations were calculated using the Brzycki equation (Weight ÷ (1.0278 − (0.0278 × Number of repetitions)) [18]. Relative strength was also calculated by dividing the trainees' estimated 1RM by their BM [13].
20MSFT-Aerobic capacity was measured using the 20MSFT. This test has been used to measure aerobic capacity in other tactical populations [19,20]. Trainees were instructed to run a 20-m shuttle keeping pace with a recorded audio track of beeps. The test started at 8.5 km/h and increased in speed by 0.5 km/h for every stage, with the beeps getting concomitantly quicker. If the trainee did not make it to the line two subsequent times before a beep, or they reached volitional fatigue, the test was terminated. The final shuttle stage and shuttle level (number of shuttles per stage) were converted to total number of shuttles completed for the purpose of data analysis. These scores were also used to estimate aerobic fitness (i.e., VO 2max ) using the equation developed by Ramsbottom et al. [21].
Training Program-Two CSCSs and the firefighter training cadre collaborated to design and implement the training program. The training schedule for the academy consisted of three 12-h days and one 4-h day with 75 min of formal physical training on two of the days ( Table 1). The formal training consisted of a dynamic warm-up (~10-12 min), agility training (~7-8 min), speed and power training (~3-4 min), hypertrophy/strength training (~30-35 min), trunk, mobility and conditioning (~5-10 min), and a cooldown (~5 min). The trainees also performed an aerobic fitness session, interspersed with callisthenic exercises, as a group, once per week for approximately one hour. It is important to note that the resources available for formal physical training were limited. This is a common challenge when working with tactical populations. For this study, only dumbbells and sandbags (weight range 4.5-29.5 kg), two TRX suspension training systems, and a quad pull-up station were available to provide external loading. Manual resistance (MR) provided by an exercise partner was also used to overload specific bodyweight exercises, such as the push-up. A metal picnic table with three benches and two weight benches were also used by the trainees to perform many of the single-leg) exercises (e.g., front and lateral, step-ups, Bulgarian squats, etc.) featured in this program (Table 1). An alternating push-pull, upper-lower body exercise rotation was used to ensure undue fatigue between stations was not a factor based on the design of the circuit.
Analysis
Following descriptive analysis, a repeated measures ANOVA was used to determine if significant changes in anthropometric and performance factors occurred over the 11-week academy period. Results were analyzed using the Statistics Package for Social Sciences (SPSS) (Version 26.0; IBM Corporation, New York, NY, USA). Effect sizes (d) were also calculated to determine the smallest worthwhile difference between the means. In accordance with Hopkins [22], a d less than 0.2 was considered a trivial effect; 0.2 to 0.6 a small effect; 0.6 to 1.2 a moderate effect; 1.2 to 2.0 a large effect; 2.0 to 4.0 a very large effect; 4.0 and above an extremely large effect. All statistical analyses for pre-and post-test measures were set at a priori p ≤ 0.05.
Results
The results of the descriptive analysis (Mean ± SD) are presented in Table 2. The results of the repeated measures ANOVA identified significant changes from pre-to-post academy for BM, BMI, pull-up, lower-body absolute strength, lower-body relative strength, and 20MSFT. All other measures were not found to be significant. For significantly different changes, effect sizes ranged from small (BM, Pull-up and HexBar 1RM) through moderate (HexBar relative) to large (BMI and 20MSFT).
Discussion
The aim of the present study was to investigate the impact of an 11-week formal strength and conditioning program, completed during fire academy training, on the anthropometric and physical performance characteristics of firefighter trainees. Significant improvements (e.g., reductions) in both BM and BMI were observed. Moreover, improvements in upper-body strength and endurance as well as lower-body maximal and relative strength were also found in the study. Furthermore, significant improvements were seen in aerobic fitness as measured by the 20MSFT. However, no significant changes were found for grip strength, VJ height, or lower-body power. These results provide evidence that an 11-week strength and conditioning program with minimal resistance training equipment, in conjunction with standard fire academy training, can elicit significant changes in physical fitness among firefighter trainees, and thus in turn prepare them for their careers as fire fighters.
While BMI is considered a crude method of determining health status, it may be useful in identifying trainees that require further exercise and nutrition interventions [20]. It was discovered that after only 11 weeks of participation, both BM and BMI significantly changed and large effect sizes were observed. In fact, the average BMI for trainees in this class dropped their overall classification from overweight (BMI = 25-29.9 kg/m 2 ) to healthy (BMI = 18.5-24.9 kg/m 2 ) [23]. These findings may be of significance when seeking to improve firefighter health and wellness, as prior evidence has suggested that the largest mortality event for firefighters was cardiac in nature and increased body mass may contribute to the physiological strain of the cardiovascular system [4,24]. Participation in a training program similar to the one presented in this investigation may help improve health status, thereby reducing a firefighter's risk of developing cardiovascular disease.
VJ height has been shown to correlate to job task performance within firefighting populations [6,7,22]. For example, Sell [25] found that VJ height had the strongest relationship (r = −0.58) with simulated firefighting task scenario (SFTS) completion times. In Sell's [25] research it was determined that firefighters able to achieve a VJ height of greater than 17 inches (43.18 cm) were more likely to achieve a passing rate (95.6%) on the SFTS than those that jumped less than 17 inches (46.2%).VJ height has also been associated with increased simulation performance of a rescue mannequin drag (r = −0.31, p < 0.05) and charged hose advance (r = −0.28, p < 0.05) [7]. While the present investigation found no significant changes in VJ height or power output, this may have been due to a relatively high initial VJ height score by this trainee class (61.2 cm) when compared to that reported by Sell [25]. This suggests that there may be ceiling effect in this measure and increasing VJ capability beyond a certain point may be more difficult to achieve as an individual approaches their genetic potential. Furthermore, it is also possible that neuromuscular fatigue accrued over the 11-week training program may have impacted acute performance in this test [26]. Future studies should seek to measure VJ height in the weeks following training academy to see if increased restitution from training may yield greater improvements in these measures post training.
Grip strength has been shown to be critical for performing numerous firefighting tasks including hydrant opening, carrying equipment, and hose deployment [27]. In this investigation, no changes in grip strength were observed over the 11-week training period. These findings support those of Roberts et al. [12], who likewise did not find significant changes in grip strength pre-to post-training program in firefighters (p > 0.05). Given that the results of this study (right hand grip strength = 55.8 kg) were notably higher than those of Roberts et al. [12] (best hand grip strength = 46.8 kg) who likewise did not find improvements, a ceiling effect may again have been present, whereby trainees, in general, presented with a level of grip strength above that required. Conversely, it is also possible that the training load utilized in this study may not have been sufficient to elicit a significant increase in grip strength given the high initial standards. Future studies should investigate the impact on grip strength when performing firefighter job tasks and attempt to set baseline recommendations for this measure.
Based on the physically demanding and uncertain nature of firefighting (e.g., victim rescue, hydrant removal, fire hose deployment, room breach), muscular strength is of importance within this population. Similar to the findings of Cvorovic et al. [10], significant improvements from pre-to post-test in upper-body strength (as measured by the pull-up to fatigue test) was observed among trainees. These findings are important, as performing heavy lifting and pulling tasks are inherent to this profession [9,21]. Additionally, significant improvements in both absolute and relative lower-body muscular strength in response to the program also occurred. With the limitations in strength and conditioning equipment, these findings are of value to fire departments and agencies with limited resources. These findings also suggest that the use of single-leg and unilateral lower-body strength training can significantly increase bilateral force production when performing lifting tasks. While the back and lower extremities are a leading site of non-fire-related injuries in firefighters and with the mechanism of these injuries often being reported as muscle bending, lifting and squatting or muscle stressing, improvements in absolute lower-body strength may reduce injury risk in this population [8]. This information may be used by firefighters, agencies, and administrators when determining the type of fitness equipment necessary in firehouses or when working with limited budgets.
Aerobic capacity is essential for successful job performance, as well as for reducing the risk of a firefighter experiencing a cardiac incident while on the job [2,4,6,7,12,24,25,28,29]. The present findings are in alignment with the recent meta-analyses conducted by Andrews et al. [24], who reported significant improvements in aerobic capacity (∆ = 1.20, [0.52-1.87] z = 3.48, p < 0.001) amongst firefighters with exercise training. Based on the average age and number of shuttles completed on the 20MSFT by trainees in this study, the estimated V0 2 for this sample would be 42.2 mL/kg/min, respectively [21]. This level of aerobic fitness would meet the minimum recommendations of Gledhill and Jamnick (i.e., 41.5 mL/kg/min) [4]. The traditional focus of aerobic training in tactical occupations has primarily focused on longer, paced-based runs, which are aimed at enhancing aerobic capacity [27]. Prior evidence suggests that a focus on intensity-based interval training may elicit greater enhancements in both aerobic and anaerobic fitness [30] and supports the findings of this study. Trainees in this study only spent~1 h per week performing long, slow distance training. Thus, a majority of the adaptations observed in this study may have been due to the circuit-based style of the strength and conditioning program that was implemented, as well as those imparted by the firefighting specific skills training performed as part of the academy curriculum [6]. Future research should explore the impact of participation in skills training alone on aerobic fitness among firefighter trainees.
This study was not without limitations. The nature of this study did not allow for a control group. However, based on the need to ensure that trainees are adequately prepared for the job of a firefighter, not training these individuals may be considered unethical. Additionally, the population size, while incorporating the entire completing cohort, is relatively small. However, this is the typical cohort size and as such the findings bear relevance. Further limitations also include the order of tests. While the order of tests was conducted based on best practice to reduce the impact of fatigue, performing all tests in a single session may have a negative effect on performance. Specifically, grip strength is an underlying component of both the pull-up and the hexbar deadlift assessment. It is possible that grip strength may have been impacted by performing the pull-up prior to the grip strength test, which in turn may have impacted on the hexbar deadlift. Additionally, performing the hexbar deadlift and fatiguing the legs prior to the 20MSFT may have impacted on shuttle run performance. Additionally, nutritional and dietary status was not controlled or tracked over the course of the academy, which could influence the rate of recovery for trainees and their training adaptations. However, the results reflect the practical nature of this intervention, as it is not practical to control these elements during fire academy training.
Conclusions
The results of this study highlight that fitness can be made in firefighter trainees attending fire academy, even with training academy specific requirements. Furthermore, these changes can be achieved with minimal equipment. Finally, it should be noted that not only was fitness increased but BMI, a measure associated with cardiovascular risk, was improved, thereby aiding in the mitigation of the leading cause of mortality in these future firefighters. These findings can be used by fire agency administration and staff to develop an evidence-based physical conditioning program to enhance fitness and occupational performance of firefighter trainees. | 2020-08-20T10:09:31.393Z | 2020-08-13T00:00:00.000 | {
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5400534 | pes2o/s2orc | v3-fos-license | Examining Effects of Anticipated Stigma, Centrality, Salience, Internalization, and Outness on Psychological Distress for People with Concealable Stigmatized Identities
Understanding how stigmatized identities contribute to increased rates of depression and anxiety is critical to stigma reduction and mental health treatment. There has been little research testing multiple aspects of stigmatized identities simultaneously. In the current study, we collected data from a diverse, urban, adult community sample of people with a concealed stigmatized identity (CSI). We targeted 5 specific CSIs – mental illness, substance abuse, experience of domestic violence, experience of sexual assault, and experience of childhood abuse – that have been shown to put people at risk for increased psychological distress. We collected measures of the anticipation of being devalued by others if the identity became known (anticipated stigma), the level of defining oneself by the stigmatized identity (centrality), the frequency of thinking about the identity (salience), the extent of agreement with negative stereotypes about the identity (internalized stigma), and extent to which other people currently know about the identity (outness). Results showed that greater anticipated stigma, greater identity salience, and lower levels of outness each uniquely and significantly predicted variance in increased psychological distress (a composite of depression and anxiety). In examining communalities and differences across the five identities, we found that mean levels of the stigma variables differed across the identities, with people with substance abuse and mental illness reporting greater anticipated and internalized stigma. However, the prediction pattern of the variables for psychological distress was similar across the substance abuse, mental illness, domestic violence, and childhood abuse identities (but not sexual assault). Understanding which components of stigmatized identities predict distress can lead to more effective treatment for people experiencing psychological distress.
Introduction
People with concealable stigmatized identities -socially devalued identities that can be hidden from others -show great variability in their experience of psychological distress. Some people are quite resilient while others suffer greatly. The goal of the current work is to examine the extent to which beliefs and experiences related to the stigmatized identity can predict variance in psychological distress. We attempt to replicate and then extend previous research on concealable stigmatized identities. Specifically, we will address the following: (a) Utilizing a low-SES, diverse, community sample of adults, we will test replication of research conducted with college students showing that anticipated stigma, centrality, and salience predict psychological distress [1]; (b) We include two additional stigma variables in our prediction model: stigma internalization and level of outness of the identity. Testing these 5 stigma constructs simultaneously will help give clarity to a research field that often uses the constructs interchangeably; (c) Unlike previous research where many different concealed stigmatized identities were lumped together or only one identity was examined, our sample includes 5 distinct concealed stigmatized identities-mental illness, substance abuse, sexual assault, domestic violence, and childhood abuse-that we can compare. We will examine both between group differences in mean level of stigma experience as well differences in ability of the stigma variables to predict distress across the groups.
Research on a diverse range of concealable stigmatized identities such as mental illness, sexual assault, childhood sexual abuse, HIV-status, and minority sexual orientation has found increased levels of psychological distress among these groups compared to their non-stigmatized peers [2][3][4][5]. Although measuring between group differences in level of distress is important, it is crucial to not lose sight of the immense within group variability that exists. Drawing on research and theory on specific concealed identities such as minority sexual orientation (minority stress model [2]), mental illness [6], and HIV+ status [7], we have been developing and testing a more general model of how concealable stigmatized identities affect psychological distress [1], health outcomes, and behaviors [8][9][10].
In the current work, we focus on predicting level of psychological distress, as measured by a composite of depression and anxiety. Psychological distress is important for several reasons. Anxiety and depression are the two most common psychological disorders, affecting nearly a quarter of the adult U.S. population, and yet among those who are diagnosed only about a third receive adequate treatment [11][12]. The consequences of untreated anxiety and depression can have profound implications at individual, family [13][14][15][16][17], community [18], and societal levels. Indeed, the National Institute of Mental Health [19] estimated that the costs of mental health care service totaled $57.5 billion in 2006, making it the third most costly medical condition (tied with cancer).
Concealable Stigmatized Identity (CSI) and Predicting Distress
In the current work we examine the ability of 5 stigma identity constructs to predict psychological distress for people with concealable stigmatized identities (CSIs). Although many variables (i.e., environmental, genetic, interpersonal, dispositional, structural) account for variance in distress, in the current work we focus on cognitions and experiences related to the stigmatized identity by the person with the CSI. Based on our theoretical model, we divide our variables into two types (see [20] for full model description). First, constructs with emotional valence are those that are related to specific experiences or beliefs about the identity and contain affective and evaluative components, including anticipation of future stigma, internalization of negative stereotypes about the identity, and level of ''outness'' or disclosure to others. Second, constructs that capture the magnitude of the identity within the self include identity centrality and identity salience. These two constructs measure the size and meaningfulness of the identity to the self but not the emotional valence. This set of predictors allows us to examine a full range of identity experiences, beliefs, and meaningfulness. Below, we detail each construct and how we hypothesize the relationship to distress in the current research.
Measuring Identity Valence: Anticipated Stigma, Internalization, and Outness
People with CSIs are likely to have a variety of different experiences and beliefs related to their identity. Anticipated stigma is the extent to which a person believes it is likely that others will devalue or distance themselves from the person with the CSI if the identity becomes known. Previous work has shown that anticipated stigma is a strong predictor of psychological distress among college samples with a variety of different CSIs [1]. Anticipated stigma within the health care system predicts people avoiding or underutilizing needed health care services [8]. Whether people anticipate stigma or not, they may have internalized the negative stereotypes about their CSI simply by living in a society that denigrates them. Stigma internalization occurs when people believe the negative stereotypes about their identity to be true of the self and/or wants to reject and distance the self from the identity. Internalized stigma has been related directly to psychological distress for people living with HIV-AIDS [21][22], for people with mental illness [23][24], and for LGB individuals [25]. Thus it has been a strong predictor of distress throughout the literature, and we, too, predict that greater internalization will be related to greater psychological distress.
Whereas most research on stigma focuses on negative experiences, there are identity related constructs that may be more positive and predictive of less distress. One such construct is outness or the extent to which other people in the environment know about the identity. Research on outness with LGB and HIV+ samples generally shows that being more out is related to less psychological distress [26][27], although there can be important moderators, such as the perceived supportiveness of the environment [28] and individual level of rejection sensitivity [29]. A recent review of strategies to reduce self-stigma of mental illness promotes being more out about one's mental illness history as a way to increase self-esteem and decrease self-stigma [30]. A pilot study supports the positive effects of outness for people with mental illness [31]. Thus, we predict that greater levels of outness will be related to lower levels of psychological distress.
Measuring Identity Magnitude: Centrality and Salience
Centrality is the extent that an identity is considered central and important to one's self-definition. Given the variability in the types of CSIs -rape, substance abuse, childhood abuse -the centrality of the identities is also likely to vary widely. Some people may think of the CSI as a single life experience that is just a small part of their identity whereas others may find the same identity to be largely self-defining. Centrality has been a key construct in work on visible stigmatized identities, particularly racial identity. Research on centrality and racial identity among ethnic minorities has shown inconsistent results with some evidence that greater centrality is associated with less psychological distress [32], more psychological distress [33], or is unrelated to distress [34]. This pattern has been reconciled to some extent by research showing that identification with a racial identity is a personal resource that people can use to deflect perceived devaluation of their group [35]. When centrality of racial identity is perceived as a positive selfresource, it is correlated with feelings of solidarity, similarity, and satisfaction with the identity [36]. We do not think this positivity can be assumed for the concealable stigmatized identities we are examining because people with CSIs often lack access to similar others and positive group-level identities [37]. Indeed, previous work on concealed identities found greater centrality was related to more distress [1]. Thus, in the current work we expect greater centrality of the CSI to predict greater reported distress.
As an additional measure of identity magnitude, we include salience, or the frequency with which a person is thinking about the identity. Salience is not meant to capture whether the thoughts about the identity are positive or negative, but rather the frequency of thoughts. In essence, a person could conceptualize a CSI as not being defining of the self (low centrality), but may nonetheless spend a lot of time thinking about the identity (high salience). In previous work, salience predicted psychological distress and it did so even when controlling for levels of anticipated stigma and centrality [1]. Salience may capture the cognitive burden of thinking about an identity that is kept secret [38]. In the current work, we hypothesize that increased salience will predict increased distress.
Examining 5 Specific Concealable Stigmatized Identities
Work on CSIs has largely proceeded by examining separate identities in isolation. There is a body of research, for example, on mental illness stigma, both in the sociology and psychology literatures [23,39]. Separate from that, often using completely different terminology and measures, is stigma work on minority sexual identity [2][3]; on HIV+/AIDS stigma [40][41][42]; on epilepsy stigma [43], and domestic violence [10,44], amongst others. In our work we have tried to formulate a general model that incorporates key constructs from these various literatures as well as the large body of work with visible identities [1,20,45]. Despite working to build a general model, we recognize that the model may operate differently for different identities but we have been heretofore unable to examine this question. In the current research we collected data from people with five specific CSIs, with the goal of being able to conduct analyses both with the full group and with each individual group, allowing further insight into how stigma variables may (or may not) predict distress within specific identities.
In the current research project we focus on five CSIs: mental illness, experience of childhood abuse (physical, emotional, and sexual), experience of domestic violence, experience of sexual assault, and substance abuse. We chose these five identities for four reasons. First, they are very common, affecting a large part of the population, thus the research is maximally useful. For example, 14-32% of adults report a history of childhood sexual abuse [46], 8.8% of adults meet criteria for substance dependence or abuse (alcohol and illicit drug) in the past year [47], rates of domestic violence range from 10-30% of married couples [48] and are reported at nearly equal rates for men and women [49], and 26% of adults have experienced some type of mental illness [11]. Second, they are stigmatized identities, both in the sense that having such an identity is seen as a mark of failure or shame as well as being experienced as something that devalues the self in the eyes of others and should be hidden (e.g., for mental illness [39,50], for rape [51][52][53][54], for domestic violence [55][56], for substance abuse [57][58][59][60][61], for childhood sexual abuse [62][63][64]). Third, they decrease the likelihood of having a disproportionate representation of one gender. Although we expect to see a higher percentage of men reporting substance abuse and women reporting rape; mental illness, child abuse, and domestic violence occur with a high degree of frequency for both genders. Fourth, these 5 identities have all been related to increased psychological distress, yet variability exists in how people are affected. The risk of depression, anxiety, trauma related symptoms, poor health outcomes, reduced quality of life, disability burden, co-morbidity and dual diagnosis, and interpersonal difficulties is substantially increased for these stigmatized identities [65][66][67]. In addition, lack of effective treatment and impaired functioning over time are associated with increased distress, poorer outcomes, and risk for revictimization [68]. Yet, despite the increased risk of psychological distress and negative consequences, there is significant variability in how these outcomes are experienced between, across, and within individuals [69][70]. In short, although we are testing our theoretical predictions, we are also attuned to the clinical impact of our findings.
Regardless of whether people with CSIs believe negative stereotypes about their groups, they are aware of societal stigma and devaluation [71]. The five identities in the current work do vary in the extent to which they are culturally devalued and this may impact the mean levels of anticipated stigma, internalization, centrality, salience, and outness that people report. Research from an attributional perspective finds that people whose stigmatized identities are considered controllable -either in their onset or in their continuation -elicit more negative affect and blame from others compared to identities considered less controllable [72]. Research has found that substance abuse and mental illness are considered personally controllable [72][73][74], although blaming the victims for sexual assault and domestic violence also occurs [10,75]. Mental illness and substance abuse are also more likely to elicit dispositional attributions because they do not include the ''perpetrator/victim'' context associated with child abuse, sexual assault, and domestic violence. In addition to attributions of blame, evolutionary psychology suggests that certain people are stigmatized because they would make poor exchange partners or members of cooperative groups [76]. People considered dangerous or unpredictable are such poor exchange partners. Here again, a consistent stereotype of people with substance abuse and mental illness is their dangerousness and unpredictability [74,77]. Thus, from both attributional and evolutionary perspectives, substance abuse and mental illness are more culturally stigmatized than the other three identities. Given these differences, we expect that participants with mental illness and substance abuse identities will report increased anticipated and internalized stigma. There is, however, no a priori reason to predict differences in centrality, salience, or outness across the 5 groups.
Overview
In the current research, we collected data from an adult community sample. We sought a sample that was quite different from the university samples used in previous work. Participants report on one of five CSIs: Mental illness, substance abuse, domestic violence, sexual assault, or experience of childhood abuse. Using the full sample, we will first examine whether we replicate that anticipated stigma, centrality, and salience each uniquely predict psychological distress. We will then add internalized stigma and outness to examine if they account for additional variance in distress. As noted above, we predict that anticipated stigma, centrality, salience, and internalized stigma will uniquely predict increased distress whereas outness will predict decreased distress.
Next, we will examine interactive effects. We hypothesize that the magnitude of the identity should moderate the effects of the valenced content. Thus, for people whose identity is relatively small in magnitude (low centrality or salience), the effects of negative valenced content (anticipated stigma or internalization) on distress should be attenuated; whereas for people whose identity is larger in magnitude (greater centrality or salience), the effects of negatively valenced content should be greater. In addition, because anticipated stigma is a worry about being rejected if others find out about the identity and previous research has shown that the benefits of outness for gay men are moderated by level of rejection-sensitivity [29], we will examine whether outness and anticipated stigma interact such that for people high in anticipated stigma, the relationship between outness and reduced distress is attenuated.
Once we have examined the direct and interactive effects of the stigma variables on distress for the full sample of CSIs, we will examine the mean level differences in the stigma variables for each of the 5 types of CSI. As noted above, we expect that people reporting on mental illness and substance abuse will report greater levels of anticipated and internalized stigma because these identities are more culturally devalued. There is no a priori reason, however, to expect that there would be differences in centrality, salience, or outness across the five identities. Finally, we will examine whether the direct effects model (with all 5 stigma variables predicting distress) differs across the 5 identities. Because we developed the model to be general, we do not expect it to be moderated by identity type.
Method
Data were collected over three years (2009-2011) from three locations in and around Hartford, Connecticut. Locations were chosen to maximize the probability of reaching participants with one of the target concealed identities. Locations included (1) a state run agency offering mental and behavioral health care counseling; (2) a private community-based agency offering a range of social services (e.g., housing and employment assistance, counseling) to predominately African American and Latino communities; and (3) a community college that serves a diverse student population with higher representation of racial/ethnic minorities, veterans, and transitioning students than other local colleges.
Ethics statement
All study procedures and measures, including consent and debriefing forms, were approved by both the institutional review board of the University of Connecticut and the institutional review board of the Department of Mental Health and Addiction Services (DMHAS) of the State of Connecticut.
Because our study focused on identities that are both stigmatizing and concealable, considerable time was devoted to training and supervising research assistants to ensure that data were collected in a sensitive and consistent manner and that participant identities were not inadvertently exposed. To read a full description of the experimenter training see Text S1.
Procedure
At each location one or more trained research assistants approached people in public areas using a prepared script and wearing badges identifying their affiliation with the university conducting the research. Potential participants were first asked if they were at least18 years old. If they were not 18, they were not able to participate in the study. However, when the data were reviewed it was found that some participants wrote in response to the open-ended age question that their age was 17. On advice from the Institutional Review Board, data from these participants were deleted from the dataset. If participants were interested in the study, the assistant led them to a room set aside for the study to complete the survey on a mini-laptop using MediaLab software [78]. Use of the laptop and mouse was explained and the consent form was read aloud by the experimenter while the participant viewed it onscreen. Participants were given a choice of completing the survey in English or Spanish and at least one bilingual research assistant was available at all times. Because there was not an option to have the full survey read aloud, participants needed to be literate. To check for the necessary literacy level, there were several example questions in the beginning of the survey devised so that participants had to be able to read the directions in order to answer the question correctly. These questions also served to explain the way the scales worked. If a participant could not answer the questions correctly, they were asked to stop the survey and thanked for their interest. This occurred for 2 participants. Upon completion of the survey, participants were given a sheet explaining the purpose of the study that also included resources for mental health, substance abuse, and shelter services if needed or desired. They were paid $15-20 for survey completion.
Participant Demographics. We surveyed 735 people total. The first prescreening question asked participants if they had any of the 5 target CSIs. If participants clicked on any of the CSIs to signify they had it, they were taken to a second question that asked them to choose the CSI that was most important to them. The computer program entered this specific identity into all of the questions pertaining to the concealed identity, thus the survey was personalized to each participant's identity. Because participants did not have the option of choosing multiple stigmatized identities, we do not have a count of how many participants had more than 1 of the 5 target identities. In total, 394 participants signified they had one of the target identities. The sample of CSIs included N = 105 people reporting on mental illness, N = 103 on substance abuse, N = 74 on experience of childhood abuse (physical, sexual, or emotional), N = 65 on domestic violence (experiences of physical abuse from a partner), and N = 47 on experience of sexual assault. Demographic information for the CSI sample is included in Table 1.
Measures: Concealed Stigmatized Identity
Anticipated Stigma captures people's concerns about mistreatment and devaluation from others if their concealed identity becomes known. Starting with the stem ''If others knew about your experiences of {specific CSI inserted here}, how likely do you think the following would be to occur?'' 15 items were presented, 9 of which were taken from the ''day-to-day'' discrimination scale of Kessler, Mickelson, and Williams [79] (e.g. ''People acting as if you are not smart,'' ''You are threatened or harassed''), as well as 6 additional items focusing on more relational concerns (e.g. ''Friends avoiding or ignoring you,'' ''People not wanting to date you''). Each question is on a scale of 1 (Very Unlikely) to 7 (Very Likely). This 15-item scale was used previously in Quinn and Chaudoir [1]. The scale has high internal reliability, with an alpha of. 95.
Centrality or the importance of the identity to the self was initially measured with 8 items -4 items from the Identity subscale of the Collective Self-Esteem scale [80] plus 4 additional items created for this study. Two items from the Collective Self-Esteem scale are reverse worded (e.g., ''My {CSI} is not important to my sense of what kind of person I am.''), and these items were confusing to participants (item to total scale correlations after being reversed were 2.06 and 2.21) and reduced reliability for the 8-item scale to.62. Thus, a 6-item scale with all questions asked in the same direction, with an alpha of.81 was retained. The items are as follows, all on a 7 point scale of strongly disagree (1) to strongly agree (7) Salience is the frequency with which people are thinking about their CSI. We used 3 items to capture salience. The first item was used previously [1]: ''How often do you think about your {CSI}?'' with a scale ranging from almost never (1) to many times each day (7). We included two additional items ''I spend a lot of time thinking about my {CSI}'' and ''My {CSI} often crosses my mind for no reason.'' Both were measured on a 7 point scale from strongly disagree (1) to strongly agree (7). This 3-item scale has a Cronbach's alpha of. 78.
Internalization of the negative beliefs about the self was measured with 4 items, based on Link's Devaluation-Discrimination scale for mental illness [71] with modification [81]. The scale had a Cronbach's alpha of. 78 Although sums rather than averages are often reported in the literature for the CES-D, we had a programming glitch for the first 74 participants in the study where one of the scale items did not appear. Because these participants only answered 19 items instead of 20, reporting sums would result in errors. As a result, we use averages rather than a sum score. Using an alternative scoring method for the CES-D has been employed by other researchers [84]. For the STAI, participants are asked to report the frequency of symptoms based on how they ''generally feel.'' Each item is on a 1 (almost never) to 4 (all the time) scale. Each scale is high in internal validity. For this study, Cronbach's alpha is.90 for the CES-D and.89 for the STAI-T. The two scales are correlated at.79. In order to create the composite measure of psychological distress, all items are standardized and then aggregated. The 40item scale has an alpha of. 94.
Neuroticism/Emotional Stability was measured in order to control for a personality variable that is associated with reporting increased distress. We used the 1 item emotional stability from the Single-Item Measures of Personality (SIMP; [85]). The SIMP has shown good convergence with longer measures of personality. The bipolar measure ranges from 1 to 9 with higher numbers indicating greater neuroticism. Mean level of neuroticism is 5.63 (SD = 2.32).
Results
Predicting Distress for the Full Sample of CSIs Table 2 displays the zero-order bivariate correlations between all of the stigma variables and distress (as well as means and standard deviations for each variable). As would be predicted, anticipated stigma, centrality, salience, and internalization are each correlated positively with distress; whereas outness is negatively correlated with distress. The effect sizes of the relationship between each of the stigma measures and distress range from small (outness and centrality) to medium (anticipated stigma, salience, and internalization). Although the stigma variables are correlated with each other, the correlations range from.05 to.53, demonstrating construct validity but no problems with discriminant validity. Nonetheless, it is only by examining the variables simultaneously in regression that we will be able to ascertain whether each of these constructs accounts for unique variance in distress.
Using hierarchical linear regression, we first examine a replication of the Quinn and Chaudoir [1] finding that showed anticipated stigma, centrality, and salience each accounted for unique variance in psychological distress. Including all participants with a CSI, we regress psychological distress first on the demographic factors: Income, education, and sex. Table 3 (Step 1 column) shows that together these demographic factors account for 5% of the variance in distress in this sample, with people with higher incomes and educational level showing lower levels of distress and women tending to report more distress. In the next step, we entered anticipated stigma, centrality, and salience. With anticipated stigma, centrality, and salience entered simultaneously in the model (see Step 2 in Table 3) we see a partial replication of Quinn and Chaudoir's [1] findings with college students: Anticipated stigma and salience account for unique, significant amounts of variance. Centrality however, is not significant. Thus, in this very different, diverse, low SES, community sample, the model largely replicates and accounts for 25% of the variance in psychological distress-roughly equal to the amount of variance accounted for in the college student sample [1].
We now turn to two additional stigma variables-internalization and outness-to test for further variance accounted for in distress. In Step 3 (Table 3), we added stigma internalization. The addition of internalization accounts for 1% of additional variance, but it is not statistically significant. Anticipated Stigma and salience remain significant when entered simultaneously with internalization. In Step 4, we added level of outness, or how open people are about their CSI with others in their environment. We expected outness to offer a level of protection, and, indeed, greater outness was related to less psychological distress, accounting for an additional 3% of the variance in distress. Notably, in this step anticipated stigma, salience, and outness are each unique significant predictors of distress. Taken together, the model accounts for 29% of the variance in psychological distress, a large effect size.
Finally, we address a critique of the stigma literature that perhaps what we are detecting is that the same people who are anticipating or perceiving stigma in the world are also more likely to report distress because of an underlying personality trait-not because of stigma, per se. In this case the relationship between stigma and distress would be spurious. In order to test this possibility, we added a measure of neuroticism in Step 5 (Table 3). People who report more dispositional emotionality (neuroticism) do report more distress. However, as can be seen by comparing the betas in steps 4 and 5, controlling for neuroticism had little effect on the relationships between the stigma variables and distress.
Interactions. Thus far we have been examining unique direct effects of the stigma variables. We also predicted that the magnitude and emotionally valenced content would interact. People who feel the identity is more central or salient and report more negatively valenced stigma (i.e., increased anticipated stigma or internalization) may experience more distress than those whose identity is lower in magnitude (less central or salient). Thus, magnitude may be a moderator of the relationship between valenced content and distress. In order to test these possible interactive effects, we first centered each of the stigma variables, and then we created product terms for each of the 2-way interactions. We ran a new regression analysis with distress regressed on the demographic variables in Step 1, the 5 stigma variables in step 2, and all 2 way interactions in step 3. Results showed that only one 2-way interaction was significant: Centrality by Internalization, b = .14, p = .04.
In order to explore the interaction, we used the PROCESS program [86] where moderation can be examined while including the 3 demographic factors (education, income, and sex) and the other stigma variables as controls. Figure 1 shows that as hypothesized, when the magnitude of the identity to the self is low -in this case when centrality is low-the level of internalization does not predict distress. However, as the level of centrality rises, the positive relationship of internalization to distress also rises. Another way to describe this result is that the relationship between internalization and distress does not become significant until centrality reaches the 75 th percentile (t = 2.30, p = .02). Thus, internalization of negative stereotypes about one's CSI is particularly distressing to the extent that the identity is also highly central to one's self-definition. We did not find any other significant two-way interactions, including between outness and anticipated stigma as we hypothesized.
Examining the 5 CSI Groups
Thus far we have focused on examining a prediction model for the full sample of participants with CSIs. We now turn to examining the 5 specific identities. First, we will examine whether there are between group differences in the experience of the identities. We predicted that people with the more culturally stigmatized identities -mental illness and substance abuse -would anticipate and internalize more stigma than those with less Table 3. Predicting Psychological Distress for all Concealable Stigmatized Identities.
Step 1 Step 2 Step 3 Step 4 Step culturally stigmatized identities -domestic violence, sexual assault, and childhood abuse. We made no a priori predictions for centrality, salience, or outness. Statistical significance of Bonferroni corrected post hoc tests are reported in the legend under each figure of means. Anticipated stigma. The means and standard errors for anticipated stigma for each CSI are shown in Figure 2. As hypothesized, participants in the mental illness and substance abuse groups reported the greatest amounts of anticipated stigma. A 1-way ANCOVA, covarying income, education, and sex was conducted with type of CSI (childhood abuse, sexual assault, domestic violence, mental illness, and substance abuse) as the between-subjects factor. The ANCOVA was significant, F (4, 383) = 10.59, p,.001, g p 2 = .10. Post-hoc comparisons revealed that the mean for the substance abuse group differed significantly from all other groups except the mental illness group. Similarly, the mental illness group differed from child abuse group and the sexual assault group but not the domestic violence group. The childhood abuse, sexual assault, and domestic violence groups did not differ from each other in level of anticipated stigma. The mental illness and substance abuse groups are above the midpoint of the scale, signifying they believe it to be likely that others will denigrate and socially distance from them if their CSI becomes known. The domestic violence mean is just under the midpoint of the scale, showing relative uncertainty about what might happen if they tell others.
Internalization. We hypothesized that the mental illness and substance abuse groups would report greater internalized stigma than the three other groups. Again, we conducted an ANCOVA, controlling for education, income, and sex, to examine between group differences. The ANCOVA was significant, F (4, 383) = 5.71, p,.001, g p 2 = .06. Post hoc tests gave partial support of our hypotheses. As can been seen in Figure 3, the substance abuse group reported the highest levels of internalization and they differed significantly from the two groups reporting the lowest levels of internalization: childhood abuse and sexual assault. The substance abuse group did not, however, differ from either the mental illness group or the domestic violence group. Indeed, there were no other differences between the groups. It is also notable that internalization is relatively low across the groups, signifying that people do not necessarily endorse negative stereotypes and beliefs about their identities.
Centrality and salience. Examining centrality using AN-COVA with education, income and sex covaried, we find that the test of between group differences is significant F (4, 383) = 3.27, p = .01, g p 2 = .03, although the effect size is small. As can be seen in Figure 4, the group with the lowest level of centrality for the identity is the substance abuse group, which differed significantly from the mental illness group. There were no other differences between the groups. Thus despite being very different identities, with varying times of onset and continuation, the mean levels of centrality are similar and around the midpoint of the scale. It is of interest that the group that has the highest rates of internalization and anticipated stigma also reports that the identity is least selfdefinitional. Figure 5 shows the means and standard errors for salience. The pattern here is different from centrality and more similar to anticipated stigma. Participants in the substance abuse and mental illness groups are reporting greater salience -more frequent thoughts about their identities -than participants in the childhood abuse, sexual assault, and domestic violence groups. As above, an ANCOVA covarying education, income, and sex shows a significant between groups effect, F (4, 383) = 8.74, p,.001, g p 2 = .08. Post-hoc comparisons show that the mental illness group reports significantly higher salience than the childhood abuse, sexual assault, and domestic violence groups, but no difference from the substance abuse group. Likewise, the substance abuse group differs from the childhood abuse and domestic Figure 2. Means and Standard Errors for Anticipated Stigma for each CSI group. Analyses controlling for income, education, and sex found significant differences in mean levels of anticipated stigma reported across the groups. Bonferroni corrected post-hoc comparisons show that the substance abuse groups reported greater levels of anticipated stigma than the child abuse (p,.001), sexual assault (p,.001), and domestic violence groups (p = .03) but not more than the mental illness group. Similarly, the mental illness group reported more anticipated stigma than the child abuse (p,.001) and sexual assault groups (p = .003) but not the domestic violence group. doi:10.1371/journal.pone.0096977.g002 violence groups although it is only marginally different from the sexual assault group. There are no significant differences in mean salience level between the childhood abuse, sexual assault, and domestic violence groups.
Outness. Figure 6 shows the means for outness by group. Surprisingly, the group that reported the most anticipated and internalized stigma also reported being the most out-the substance abuse group. An ANCOVA with the same covariates as above was significant, F (4, 383) = 6.68, p,.001, g p 2 = .07. Post hoc comparisons show that the substance abuse group was significantly more out than all other groups except the mental illness group. There were no other significant between group comparisons. In summary, there were group differences in the mean level of the stigma variables, reflecting the different experiences and beliefs that people have surrounding their stigmatized identities. The effect size of group type on stigma variable was small, with group type accounting for between 3 and 8 percent of the variance in the stigma variables. As predicted, the substance abuse and mental illness groups had higher levels of anticipated stigma and internalization, but contrary to predictions, the substance abuse group reported being the most out but with the lowest level of identity centrality. Moreover, the domestic violence group had relatively high levels of internalization. The childhood abuse and sexual assault groups report the lowest levels of anticipated stigma, internalization, salience, and outness, but levels of identity centrality were on par with the other groups. Analyses controlling for income, education, and sex found significant differences in mean levels of salience reported across the groups. Bonferroni corrected post-hoc comparisons show that the mental illness group reports significantly higher identity salience than the childhood abuse (p,.001), sexual assault (p = .002), and domestic violence groups (p = .001), but no difference from the substance abuse group. Likewise, the substance abuse group differs from the childhood abuse (p = .003) and domestic violence groups (p = .03) although it is only marginally different from the sexual assault group (p = .07). doi:10.1371/journal.pone.0096977.g005 Figure 6. Means and standard errors for Outness for each CSI Group. Analyses controlling for income, education, and sex found significant differences in mean levels of outness reported across the groups. Bonferroni corrected post-hoc comparisons show that the substance abuse group reported being significantly more out about their identity than the childhood abuse (p,.001), sexual assault (p = .001), and domestic violence (p = .001) groups but not different from the mental illness group (p = .12). There were no other significant between group differences. doi:10.1371/journal.pone.0096977.g006
Examining Moderation of the Prediction Model by Identity Type
We hypothesized that although the mean levels of the stigma variables might differ by identity type, the predictive model of distress would be similar across the identities. In order to examine this hypothesis, a nested, multi-group path model was tested to examine potential moderating effects of CSI group (utilizing AMOS 17). In this approach, the multivariate regression model is estimated separately for the five groups and the magnitude of the regression coefficients can be compared using a critical ratios z test [87]. Five models were tested against a default model. In the default model, all of the parameters of interest (i.e., regression paths between each of the independent variables and distress) were freely estimated for the five groups. This is identical to the regression model described above in Table 3, Step 4. Then, the default model was compared to models in which the path from one of the independent variables (e.g., anticipated stigma R distress) was constrained to be equal across the five stigmatized groups; this is repeated for each predictor variable. In this approach, the difference in chi square values between the constrained and unconstrained models indicates if the particular path is equivalent or different across the five groups. In cases when the chi-square differential (CMIN) was significant-signifying a moderation of the path by CSI group-follow-up pairwise contrasts were made using critical ratio z tests to see which specific groups differed on that path.
As would be expected given the linear regression above, the default model has excellent model fit in predicting distress, with a non-significant chi-square (df = 10) = 14.32, p = .16; the comparative fit index (CFI) = .991, and RMSEA = .033. Next, we examined the nested model comparisons. A significant chi-square differential (CMIN) indicates that the path between predictor variables and distress should not be constrained to be equal, i.e., were not moderated by CSI group. In Table 4 we list the standardized regression weights by CSI group. Examination of the regression weights makes it is clear that one group -the sexual assault group -is quite different from the others and is the cause of the significant moderation effects. The regression weights for anticipated stigma, centrality, and outness are all in the opposite direction for the sexual assault group compared to the other 4 CSI groups, although only the centrality coefficient is significant (anticipated stigma is non-significant, and outness is marginal). Specifically, for the sexual assault group only, greater centrality of the identity is related to less distress, but greater outness is (marginally) related to more distress. Follow-up pairwise comparisons between each of the standardized regression weights support this finding. Using the critical ratio z-tests (at p,.05), for the anticipated stigma R distress path, the path for the sexual assault group is significantly different from each of the other groups, which do not differ from each other. For centrality R distress path, the sexual assault group differs from each of the other groups except substance abuse; none of the other groups differ from each other. And, for outness R distress, the sexual assault group differs from each of the other groups, which again do not significantly differ from each other.
To summarize, using a nested, multi-group path model to test for moderation by CSI group, we found that internalization and salience predict similarly across the 5 groups: greater salience is strongly related to greater distress; greater internalization is related to greater distress but the effect is relatively weak. Greater anticipated stigma is related to greater distress for the substance abuse, mental illness, childhood abuse, and domestic violence groups and these paths do not significantly differ across the 4 groups. However, for the sexual assault group, greater anticipated stigma is related to less distress (albeit not significantly). For outness, again, for the substance abuse, mental illness, childhood abuse, and domestic violence groups, more outness was related to less distress and the path weights did not significantly differ by group. For the sexual assault group, more outness was related to more distress. Finally, for centrality, level of centrality had weak to null predictive effects for substance abuse, mental illness, childhood abuse, and domestic violence groups but it was a strong negative predictor for the sexual assault group with greater centrality predicting lower levels of distress for this group only. Thus, for 4 of the CSI groups -substance abuse, mental illness, domestic violence, and childhood abuse -the prediction model works similarly, with no group moderation. However, the model works quite differently for the sexual assault group.
Discussion
Using a diverse, low SES, urban sample of adults, we found that greater anticipated stigma from others, greater salience of the concealed stigmatized identity, greater internalization of negative beliefs about the identity, higher centrality of the stigmatized, and decreased outness were each correlated with greater distress. Salience, anticipated stigma, and outness each predicted a unique portion of variance in distress when entered simultaneously in regression. The full model accounted for just under 30% of the variance in distress, and the effects were not explained or weakened by the personality trait neuroticism/emotional stability. Moreover, we predicted that the magnitude of the identity might moderate the effects of negative beliefs and experiences around stigma -anticipated stigma and internalization -on distress. We did find a significant interaction between centrality of the identity and internalization such that when centrality was low -the identity was not considered self-definitional -internalization of negative beliefs about the identity were not related to distress. As the centrality of the identity to the self increased, greater internalization predicted greater distress. Thus, as predicted, to the extent that an identity has a greater magnitude within the self, negative beliefs related to the identity had a greater effect on outcomes. We also predicted, however, that anticipated stigma would moderate the positive effects of outness, similar to earlier work that found gay men who were the most rejection sensitive were hurt (rather than helped) by being more out about their identity [29]. However, we found no support for this prediction.
Because we targeted five specific CSIs -substance abuse, mental illness, domestic violence, sexual assault, and childhood abuse -we were also able to look for similarities and differences between these identities. Participants with a history of substance abuse reported the greatest levels of anticipated and internalized stigma. They also reported the greatest level outness and the lowest level of centrality. Although more work needs to be done, this seems like a particularly psychologically protective combination. That is, individuals with a history of substance abuse recognize that their identity may be devalued by others and may, in fact internalize that devaluation. And yet, the identity is not selfdefinitional and is less likely to be kept hidden from others. This pattern of response is consistent with the most common treatment modality for substance abuse- 12 Step Fellowship. The 12-Step program emphasizes a certain degree of outness (e.g., attending meetings, public acknowledgement that one is an addict or alcoholic, seeking forgiveness for past wrongs, and working with a sponsor) but at the same time, there is a de-emphasis on attributing one's addiction to flawed character (e.g., addiction is a disease).
Participants with mental illness history show a slightly different pattern: they were relatively high in all of the stigma measuresanticipated stigma, internalization, centrality, salience, and outness. This pattern may make individuals with a history of mental illness particularly vulnerable to distress in that they are expecting stigma from others about an identity that they feel is selfdefinitional and for which they are more likely to endorse the negative stereotypes associated with it. People who experienced childhood abuse showed the lowest mean levels of anticipated and internalized stigma, but they did not differ from the other groups in terms of centrality of the identity. Finally, sexual assault and domestic violence showed similar mean levels of the stigma variables although the domestic violence group reported slightly (but not significantly) higher levels of anticipated and internalized stigma. Although we have focused on a general prediction model, we do not want to minimize the differences in the experiences of people with different identities.
We examined whether our prediction model was moderated by CSI group. We hypothesized that the prediction model would work similarly across the five groups. What we found was that for four of the groups -substance abuse, mental illness, domestic violence, and childhood abuse -the model did work the same, with anticipated stigma, internalization, and salience predicting more distress and outness predicting less distress (and centrality being non-significant). However, for the sexual assault group, analyses showed that internalization and salience continued to predict increased distress, but that greater anticipated stigma and greater centrality predicted less distress but greater outness predicted more distress. These results were unexpected and need to be replicated. The sexual assault group was our smallest group with only 49 participants and thus, we are cautious in interpreting these findings. A case could be made that the greater centrality reflects an integration of the sexual assault identity that is positive for mental health, but this does not easily concur with the idea that being more out to others would be psychologically risky. Certainly more work needs to be done to follow up and replicate these unexpected findings.
Implications for Reducing Psychological Distress
Testing a model of concealable stigmatized identities has several important clinical implications. First and foremost is the opportunity to provide an integrated model of psychological vulnerability that takes into account individual, social, clinical, and sociocultural factors. Based on lifetime prevalence rates, it is likely that most people will either experience a stigmatized identity or care for or about someone who does. Thus, it is critical to understand how stigmatization affects psychological well-being on both an individual and societal level. On a societal level, stigmatization may increase discriminatory behaviors, victimization, isolation, and alienation and continue to facilitate myths and stereotypes regarding mental illness, abuse, and addiction. On an individual level, stigmatizing identities may result in unnecessary suffering as they tend to limit help seeking behaviors [88] and reduce positive treatment effects [89]. More than a decade ago, the Surgeon General (1999) identified stigma as the single greatest barrier to addressing mental health issues and as a primary factor in contributing to racial/ethnic disparities in care, and yet most of the evidence-based interventions give scant attention to the role of stigma. For some individuals, concealing a stigmatized identity may be functional (i.e., situationally protective) even though the consequences of doing so may be maladaptive (i.e., increased symptomatology, limited support). Such issues may be particularly complicated for individuals managing multiple concealed stigmas (e.g., substance abuse and sexual assault), in conjunction with chronic or acute stressors (e.g., HIV positive status and poverty, depression and job loss), or both visible and concealed stigmatized identities (e.g., physical handicap and mental illness). Some recent interventions have begun to address ways to reduce self-stigma for people with serious mental illness [31,[90][91]. Specifically addressing stigma, particularly the degree to which is it selfdefinitional, internalized, salient, and frames expectations, may have a direct impact on treatment engagement and effectiveness.
The current work, however, has a number of limitations. We did not conduct formal diagnostic interviews and thus are not able to define the types of mental illness participants are experiencing. Also, we included only one personality variable as a control, but it is possible that there could be others that influence the perceptions of stigma and distress [92]. Because we are focused on creating a general model that can be used across multiple identities, we created or adapted measures that would work for multiple identities. Thus, we cannot test for content belief that is specific to a particular identity, such as knowledge of specific stereotypes about mental illness [93][94]. Also, because we asked participants to focus on one particular identity, we are not able to examine how they might feel about possessing multiple stigmatized identities or whether possessing multiple stigmatized identities affects the impact of the stigma variables. People with a stigmatized identity often have multiple identities and research examining how these identities intersect is very important although methodologically difficult (e.g., [95][96]). Finally, the sample sizes for our groups of sexual assault, domestic violence, and childhood abuse are relatively small. A larger, more nationally representative sample would strengthen conclusions to be drawn.
Implications for Stigma Research
Replication and Generalization. Although university samples are convenient, it is important for stigma researchers to test their ideas on more diverse samples. College students have incredible resources in terms of access to healthcare and mental health professionals compared to most adults in the United States. Our sample was ethnically diverse and very low SES. Replicating research with different samples allows the researchers to see similarities and differences. In the current research we found partial replication of the research with college samples: In both samples, anticipated stigma and salience of the identity predict greater psychological distress. However, in the college sample, centrality of the identity also had a direct effect on distress. Although this direct effect did not replicate in our community sample, centrality did interact with internalization to predict distress. Another interesting comparison is that the mean anticipated stigma levels of the community sample are a full scale-point higher than the college samples. We believe this points to the reality of discrimination in the lives of members of the community sample. In summary, replication with multiple samples can increase the generalizability and reliability of stigma research, making researchers and clinicians more confident about the utility of the research.
Measuring Stigma. The current work is a step forward into specifying and testing five specific stigma constructs but more work needs to be done. We think it is particularly important to differentiate between internalized stigma, which is a belief that the negative stereotypes about the identity apply to the self, and anticipated stigma, which is a worry about being devalued once the identity is revealed. The primary stigma measure used in most research on concealed identities (e.g., mental illness stigma, HIV+ stigma, minority sexual orientation stigma) has been internalized stigma. Not surprisingly, internalized stigma correlates highly with low self-esteem and lower self-efficacy [23]. Anticipated stigma does not assume that people believe any negative stereotypes about their identity, yet in the current work anticipated stigma is a stronger predictor of psychological distress than internalized stigma.
Understanding the role of salience, separate from centrality, is an important future direction. In the current work, salience of the identity was the strongest predictor of distress. Future research could focus on whether heightened salience is due to frequent life disruptions due to such things as identity-related symptoms, required medication use, or treatment utilization; or whether salience is capturing the cognitive burden of holding an identity secret [38]. Continued refinement of the measurement of stigma constructs will help to determine which aspects of stigma are the most important as points of intervention.
Conclusion
Understanding how possessing a concealable stigmatized identity impacts psychological distress is crucially important to the development of methods to alleviate distress and increase resilience. The current work highlights how identity beliefs and experiences at the individual level relate to distress. The individual level, however, must be combined with research examining ways to reduce discrimination at the institutional and interpersonal levels. Only then will the burden of stigma be lightened. | 2017-04-13T17:12:24.799Z | 2014-05-09T00:00:00.000 | {
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237561399 | pes2o/s2orc | v3-fos-license | A global long-term (1981–2019) daily land surface radiation budget product from AVHRR satellite data using a residual convolutional neural network
. The surface radiation budget, also known as all-wave net radiation (R n ), is a key parameter for various land surface processes including hydrological, ecological, agricultural and biogeochemical processes. Satellite data can be effectively used 10 to estimate R n , but existing satellite products have coarse spatial resolutions and limited temporal coverage. In this study, a point-surface matching estimation (PSME) method is proposed to estimate surface R n using a residual convolutional neural network (RCNN) integrating spatially adjacent information to improve the accuracy of retrievals. A global high-resolution (0.05°), long-term (1981–2019), and daily mean R n product was subsequently generated from Advanced Very High-Resolution Radiometer (AVHRR) data. Specifically, the RCNN was employed to establish a nonlinear relationship between globally distributed ground measurements from 523 sites and AVHRR top of atmosphere (TOA) observations. Extended triplet collocation (ETC) technology was applied to address the spatial scale mismatch issue resulting from the low spatial support of ground measurements within the AVHRR footprint by selecting reliable sites for model training. The overall independent validation results show that the generated AVHRR R n product is highly accurate, with R 2 , root-mean-square error (RMSE), and bias of 0.84, 26.77 Wm -2 (31.54%), and 1.16 Wm -2 (1.37%), respectively. Inter-comparisons with three other R n products, i.e., the 5 km Global Land Surface Satellite (GLASS), the 1° Clouds and the Earth’s Radiant Energy System (CERES), and the 0.5°
Introduction
Net radiation (Rn), which characterizes the surface radiation budget, is the difference between downward and upward radiation 30 across the shortwave (0.3-3.0 μm) and longwave (3.0-100 μm) spectra. The surface radiation budget links the atmospheric climate system to the land surface. Rn is thus a critical variable for studying Earth-atmosphere interactions, including meteorological, hydrological, and biological processes, and is also responsible for the redistribution of surface available energy (Sellers et al., 1997). Accurate characterization and quantification of spatial-temporal variations in surface Rn are essential for both scientific and industrial applications, such as hydrological modeling and water resource management (Hao et al., 2019).
35
However, because the spatial and temporal dynamics of surface Rn are affected by multiple surface features (e.g., albedo, emissivity, and land surface temperature) and atmospheric parameters (e.g., clouds, aerosols, ozone, and water vapor) (Wang et al., 2015b), existing surface Rn data suffer from large uncertainties Jiang et al., 2018;Yang and Cheng, 2020). Therefore, there is an urgent need for long-term, high-resolution surface Rn dataset to more properly understand its spatial pattern and temporal dynamics (i.e., seasonal and inter-annual variability).
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Traditionally, historical Rn and surface radiative components have been measured at ground meteorological stations. These ground-based measurements are widely used to study spatiotemporal variations in regional surface radiation and to evaluate gridded products Zhang et al., 2020;Zhang et al., 2015). Nevertheless, the high cost of maintaining radiometers means that stations are sparely distributed, severely hindering our ability to study and understand the spatiotemporal variability of surface Rn at global scale. 4 many of these discrepancies are attributed to two aspects: first, the spatial representation mismatch between satellite data and ground-based measurements and, second, the neglect of spatial adjacent effects on surface radiation estimation.
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The spatial scale mismatch between surface radiation for domain averages and ground point measurements with insufficient spatial representativeness (Jiang et al., 2019b;Barker and Li, 1997) has attracted attention for a long time in the development of ML and in the evaluation of gridded products (Huang et al., 2016;Yang, 2020;Romá n et al., 2009).
However, many current studies still use matched point-surface sample datasets to train ML model regardless of the difference in spatial representativeness of matched point-surface data. The triple collocation (TC) technique (Stoffelen, 1998) was 95 considered as an appropriate upscaling approach for the impact of random measurement error on ground-based measurements in comparison to other complicated upscaling methods (e.g., the time stability approach and the block kriging algorithm (Crow et al., 2012)) . Furthermore, an extended triple collocation (ETC) method was proposed by Mccoll et al. (2014) and then applied to the validation activities of the Soil Moisture Active Passive (SMAP) level-2 surface soil moisture (SSM) product (Chen et al., 2017) and satellite surface albedo products (Wu et al., 2019). Therefore, the ETC technology is 100 employed to limit the effect of upscaling errors of ground measurements on the final surface Rn estimates at the satellite footprint scale.
Spatial adjacent effects should also be considered in the development of ML methods. With an increase in spatial resolution, horizontal inhomogeneities in the atmosphere have become increasingly important and reduce accuracy of surface radiation retrievals at higher spatial resolutions, especially in conjunction with high solar and viewing angles (Wyser et al., 2002); the 105 correlation between satellite TOA observations and surface radiation measurements weakens, and surface radiation cannot be accessed directly from satellite TOA data for individual pixels. Convolutional neural networks (CNNs) were initially designed to perform image recognition tasks; they can be readily used to extract various high-level, hierarchical, and abstract spatial pattern features from original multispectral or hyperspectral satellite images Ball et al., 2017). Using this approach, multiple environmental parameters and their spatially adjacent effects can be accounted for in the estimation of 110 surface Rn (Jiang et al., 2019b). Therefore, CNN represents a promising method for integrating potential spatially adjacent effects in surface radiation estimation Jiang et al., 2019b).
Several studies have successfully employed CNNs and other deep neural networks to retrieve surface parameters, such as global solar radiation (Jiang et al., 2019a), precipitation , and land surface temperature (Yin et al., 2020), with varying success rates. However, no study has yet attempted to retrieve global surface Rn using a CNN model. In this study, a 115 residual CNN (RCNN) based point-surface matching estimation method (PSME) is proposed for estimating global land surface Rn. Specifically, the RCNN model links ground-based Rn measurements with multiple image blocks of AVHRR TOA observation data, including reflectance in visible channels and brightness temperature in thermal infrared channels, along with other additional auxiliary variables. These auxiliary variables include angular information, i.e., solar zenith angle (SZA), viewing zenith angle (VZA), and relative azimuth angle (RAA), and daily Modern-Era Retrospective analysis for Research 120 and Applications, Version 2 (MERRA2) Rn data Zhang et al., 2016). Before training the RCNN, ETC technology is applied to select reliable sites at a global level to generate representative training samples, making the established statistical relationship more representative of surface Rn variation at the AVHRR footprint scale. After validation and comparison, the best-trained model was subsequently implemented through the proposed PSME scheme to generate a globalscale 39-year daily surface Rn dataset (1981-2019) with 0.05° resolution from the AVHRR data and MERRA2 reanalysis.
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The remainder of this paper is organized as follows. Section 2 summarizes the characteristics of the data used for the reconstruction of the ETC triplet and PSME method development. Section 3 describes the ETC method for the selection of reliable sites and the PSME process for surface Rn estimation using the RCNN model. The results for selected sites based on the ETC, the performance of the RCNN model and the long-term spatiotemporal variation of the Rn dataset are presented in Sect. 4. The discussion is presented in Sect. 5. The data availability is described in Sect. 6. Finally, the conclusions are 130 presented in Sect. 7. at 523 globally distributed stations from 15 observation networks/programs, as shown in Fig. 1. Detailed information about 6 these observation networks/programs is listed in Table 2, and specific information about these sites is shown in Table S1.
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These stations are maintained by multiple global and regional observation network organizations, such as Global Fluxnet, the Greenland Climate Network (GCNET) and the Programme for Monitoring of the Greenland Ice Sheet (PROMICE) . These stations vary in elevation from 4 to 5,063 m above sea level and are located in areas with different land covers, including forest, grassland, shrubland, wetland, cropland, ice/snow, and urban areas. The collective in situ measurements, therefore, represent an accurate and comprehensive dataset that is capable of accounting for surface Rn spatial-temporal 145 variation on a global scale.
AVHRR data
AVHRR TOA observations at five spectral channels (a visible band (0.55-0.68 μm), a near-infrared band (0.75-1.1 μm), a 180 middle-infrared band (3.55-3.93 μm), and two thermal bands (10.5-11.3 and 11.5-12.5 μm) were utilized for their comprehensive surface and atmospheric electromagnetic information. The National Aeronautics and Space Administration's (NASA) Land Long-term Data Record (LTDR) project produced a consistent long-term dataset by reprocessing Global Area Coverage (GAC) data, which were obtained from AVHRR sensors onboard the National Oceanic and Atmospheric Administration (NOAA) satellites (Pedelty et al., 2007). The primary reprocessing improvements included radiometric in-185 flight vicarious calibrations for the visible and near infrared channels, along with inverse navigation to relate a specific Earth location to each sensor's instantaneous field of view (Vermote and Kaufman, 1995;Pedelty et al., 2007). Multiple Climate Modeling Grid (CMG) data from AVHRR and MODIS instruments have been created for land climate studies (Xiao et al., 2017;Pedelty et al., 2007). In this study, we utilized a daily AVHRR TOA data product (AVH02C1) with a resolution of 0.05° from 1981 to 2019 to retrieve surface Rn estimates. Additionally, solar/viewing geometry data (i.e., SZA, VZA, and RAA)
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were also incorporated into the model as the amount of solar radiation incident on the Earth's surface varies greatly under different geometric observation conditions. A summary of these gridded products and their attributes is presented in Table 3.
GLASS product
The GLASS daily surface Rn product from MODIS data, one part of the GLASS product suite , was produced using two sets of algorithms. The main algorithm primarily uses the well-documented conversion relationships between downward shortwave radiation and all-wave Rn combinations (Wang and Liang, 2009;Jiang et al., 2015). It also incorporates a combination of other meteorological variables under various environmental conditions, such as different 200 daytime lengths and land cover characteristics, which are designated based on the albedo and normalized difference vegetation index (NDVI). Multiple MARS learners were employed to establish efficient statistical relationships using GLASS downward shortwave radiation and MERRA2 meteorological variables, allowing land surface Rn to be estimated from these inputs across most spatial domains Jiang et al., 2015). Conversely, when surface solar radiation data were not available, the backup algorithm created a function that separately employed MODIS TOA observations to retrieve surface all-wave Rn 205 using the length ratio of daytime (LRD) classification method, which was accomplished by the genetic algorithm-artificial neural network (GA-ANN) . By using these two algorithms, the GLASS Rn product can provide seamless global land surface Rn estimates with a 0.05° resolution. Several studies have used in situ measurements to conduct evaluation studies, illustrating high accuracy performance as well as good application potential Guo et al., 2020). Thus, we used the GLASS daily Rn product covering 2000 to 2018 as a reference to help select reliable sites and validate the results 210 from this study.
CERES-SYN product
The CERES instruments onboard the Terra, Aqua, and Suomi National Polar-Orbiting Partnership (Suomi NPP) satellites observe the TOA global energy budget by measuring shortwave reflected radiation, longwave Earth-emitted radiation and all wavelengths of radiation at a spatial resolution of 20 km at nadir (Wielicki et al., 1996). The CERES Synoptic (CERES-SYN)
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product combines CERES and MODIS observations with geostationary (GEO) data to provide hourly broadband TOA radiant flux and cloud properties (Doelling et al., 2013). The CERES-SYN product also contains computed TOA and in-atmosphere and surface fluxes based on the Fu-Liou radiation transfer model. Aerosol and atmospheric data were included as inputs to calculate the radiation flux. CERES-SYN fluxes were provided as a 1° gridded product with an hourly temporal resolution.
CERES-SYN surface Rn data have been evaluated in many studies, which indicate that the product has high accuracy, although 220 systematic overestimation exists in the surface net radiation flux data Jiang et al., 2016;. Thus, the CERES-SYN surface Rn obtained from four surface radiative components was used as a reference for comparison.
MERRA2 reanalysis
MERRA2, produced by the NASA Global Modeling and Assimilation Office (GMAO), is the latest global atmospheric product and employs satellite observation data from 1980 to the present. The MERRA2 reanalysis assimilates space-based observations 225 of aerosols and represents their interactions with other physical processes in the climate system. The goals of MERRA2 are to provide a regularly gridded, homogeneous record of the global atmosphere, and to incorporate additional climatic variables and conditions, including trace gas constituents (stratospheric ozone), improved land surface representation, and cryospheric processes (Gelaro et al., 2017b). The MERRA2 products have a 0.5° × 0.625° spatial resolution and hourly temporal resolution.
Previous studies Delgado-Bonal et al., 2020) have confirmed that the MERRA2-calculated 230 surface Rn and its radiative component provide outstanding accuracy and a reasonable spatial-temporal distribution compared to other reanalysis data. Therefore, MERRA2 Rn data calculated from four surface radiative components were also used in this study to help retrieve accurate high-resolution surface Rn estimates by providing average atmospheric information.
Methods
The entire workflow of the RCNN-based PSME method is shown in Fig. 2. First, the ETC technology was applied to the triplet 235 system constructed from ground-, satellite-, and model-based Rn data to identify reliable sites at which measurements can well represent the dynamic variation of surface Rn at a 5 km scale. Then, AVHRR TOA reflectance, brightness temperature, angular information and MERRA2 Rn were matched with the ground-based Rn measurements, both spatially and temporally.
Specifically, the site-measured Rn data were collocated with the 5 km AVHRR grid product covering the site. If one grid in the AVH02C1 product covered multiple sites, the mean values from these sites' measurements were used to match the grid 240 data. Subsequently, the matched input-output training samples were fed into the RCNN to train the model. Reference Rn measurements taken from reliable sites were used to evaluate the model's performance and, subsequently, identify the best option to produce surface Rn by tuning the hyper-parameters of the RCNN. Finally, surface Rn retrievals were generated using the best-trained model for the global scale, and CERES-SYN and GLASS Rn products were applied to perform intercomparisons to illustrate the accuracy and spatiotemporal variation of the surface Rn retrievals.
Extended triple collocation (ETC)
To address the spatial scale mismatch issue owing to the small spatial support of sparse ground measurements in comparison to gridded satellite data, which introduces large uncertainty into the collaborative inversion process, triplet collocation (TC) technology was employed (Stoffelen, 1998;Yuan et al., 2020a). Specifically, based on the availability of three collocated, 255 independent measurement systems describing the same geophysical variable, TC was designed to estimate the unknown error standard deviations (or RMSEs) of three mutually independent measurement systems, without treating any one system as perfectly observed "truth" (Stoffelen, 1998;Gruber et al., 2016). To perform the TC, the following assumptions were made: 1) each of the triplet is related with the unknown truth of the geophysical variable in the linear form; 2) zero cross-correlation across each of the triplet; 3) zero error cross-correlation between the triplet and the true signal state (T); and 4) the signal and 260 error statistics are stationary (Chen et al., 2017). Following the first assumption, the independent triplet systems (Xi, Xj, and Xk) are related to the unknown true quantity in a linear error model: where and are the additive and multiplicative bias terms, respectively; and is the mean-zero random error. Similar calibration constants ( , , , and ) and random error terms ( and ) are also defined for Xj and Xk.
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The objective of TC is to find a solution that individually estimates the variance of the random error term ( ) for each of the triplet based on the listed assumptions (Stoffelen, 1998;Yuan et al., 2020a). However, to obtain the calibration constants, one dataset is chosen from the three collocated measurement systems as the reference dataset, and the other two are rescaled into the same reference data space. This results in a dependency of the error variance of the other two datasets on the climatology of the scaling reference (Draper et al., 2013;Yuan et al., 2020a). To deal with this issue, ETC technology was 270 proposed by Mccoll et al. (2014), based on the same assumptions as TC, to estimate an additional evaluation metric independent of the reference dataset, i.e., a correlation coefficient ( ( , ) ) of each measurement system with respect to the unknown target variable as formulated below: where ( , ) is correct up to the sign ambiguity, as the measurement systems will almost always be positively correlated to 275 the unobserved truth.
Following the ideals of Chen et al. (2017) and Yuan et al. (2020a), ETC was applied to determine the reliable site measurements over the AVHRR data footprint scale (i.e., 5 km grid). Specifically, the triplet dataset was first constructed using ground-based measurements (i.e., site measurements), satellite-derived retrievals (GLASS Rn) and downscaled model-based simulations (MERRA2 Rn) depending on the conversion ratio of GLASS Rn between original (0.05°) and aggregated (0.5°) spatial 280 resolutions, as they belong to different measurement systems and are not dependent on each other. Then, ( , ) was calculated using Eq. (2) for individual site, illustrating the fraction of 5 km satellite footprint-scale Rn dynamics captured by point-scale ground-based measurements. An appropriate threshold of ( , ) was determined to select reliable sites with the greatest representativeness within a 5 km footprint to obtain sample datasets for the model training. After testing a series of thresholds between 0.2 and 0.9 at intervals of 0.1, a threshold of 0.9 was selected, above which the sites were assigned as 285 'reliable' (also see Sect. 5). Based on these reliable sites, errors of upscaling reliable site-based measurement to a 5-km scale can be weakened to a certain degree due to its better representativeness within the AVHRR footprint.
RCNN
As the spatial resolution of satellite sensors increases, the spatial adjacent effects induced by spatially inhomogeneous 290 atmospheric constitutes (or clouds) fields become more significant, for example, clouds affect the distribution of surface radiation in a region larger than the resolution of an individual pixel. One spatial adjacent effect is the diffusion of radiation that removes part of radiation from an atmospheric column and transfer it to neighboring columns. Two other effects are related to the solar and viewing geometry, such as a shift of the apparent position of clouds and their shadows. Surface Rn is no longer accurately estimated with retrieval algorithms based on the individual pixel approximation (IPA). Comprehensive information 295 within a certain spatial extent centered at reliable sites needs to be considered to help retrieve surface Rn.
Loosely inspired by the human visual cortex, CNNs were originally applied to analyze common visual imagery using convolution instead of general matrix multiplication (Ball et al., 2017). CNN model can extract features hierarchically from input multi-channel images using multiple filters. Therefore, the most important feature information regarding reliable sitebased Rn measurements can be effectively extracted by CNN within a certain spatial extent rather than on IPA, to help retrieve 300 Rn, which weakens the spatial adjacent effects to a certain extent. A general CNN consists of multiple layers of operations, such as convolution, pooling, normalization, and nonlinear activation functions. In the convolutional layers, a series of convolution (Conv) operations are performed using convolutional kernel weights and biases on the input images within the receptive field. The result of the locally weighted sum (feature map) is then passed through a nonlinear layer, such as a rectified linear unit (ReLU), which increases the nonlinear properties of the decision function and the overall network (Romanuke, 305 2017). The pooling layer, a form of non-linear down-sampling, merges semantically similar features into one, thereby reducing the amount of computation in the network (Gé ron, 2019). Additionally, a batch normalization layer is placed between the convolutional layers and nonlinearities to speed up the training of the CNN and reduce the sensitivity to network initialization.
By stacking two or three stages of convolution, nonlinearity, and pooling, followed by more fully connected layers, a typical CNN architecture is built.
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To improve network performance in complicated tasks, a deeper CNN architecture is needed; however, a deeper neural network is difficult to train well because a degradation problem occurs with deeper networks converge (He et al., 2016). Specifically, as the network depth increase, the training accuracy becomes saturated and then degrades rapidly. To address the degradation problem, He et al. (2016) proposed a residual block to improve the gradient flow through the network , which enables the training of deeper networks. Residual blocks were employed in our CNN architecture to construct the RCNN. The structure of 315 the RCNN proposed in this study is shown schematically in Fig. 3. Table 4 lists the detailed configurations of the proposed RCNN.
The input signals of the RCNN included AVHRR TOA reflectance and brightness temperature, angular information (SZA, VZA, and RAA), and daily MERRA2 Rn with a spatial size of 15×15 pixels (these specifications are further discussed in Sect.
5).
To avoid introducing new errors, nearest-neighbor interpolation was used to resample MERRA2 Rn to 0.05° to match the 320 spatial resolution of the other predictors. The output signal was ground-based Rn from the reliable sites. Essentially, the RCNN model uses a convolution operation taken at stages of the feature map and residual learning block to recognize spatial patterns centered on a reliable site. Then, the multiple layer perceptron links abstract spatial patterns with ground-based measurements to construct a strong non-linear relationship to reproduce the spatial and temporal variation of surface Rn. This approach had been carried out in previous studies (Jiang et al., 2019b;Jiang et al., 2020a;Jiang et al., 2019a).
RCNN model training and evaluation
Sample data from the reliable sites in the training site group (red circles as shown in Fig. 1) were used to train the RCNN model; datasets from reliable sites in the independent validation site group (blue circles shown in Fig. 1) served as test datasets
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to independently evaluate the model's performance. Specifically, in the training process, 10-fold cross-validation (CV) was used to test the model's predictive power. All of the sample datasets from the reliable training sites were randomly shuffled and divided into ten groups. One group of these data was then removed as a hold-out or validation dataset and the remaining nine groups of data were treated as the training datasets. The training datasets were used to fit the RCNN model, and the validation datasets were applied to evaluate the trained model's performance to fine-tune the model's parameters. The process 340 was repeated ten times to ensure that each group of data validated the model, and the remaining nine groups of data were trained. Finally, the evaluation results were presented by summarizing and averaging the ten evaluation scores. After determining the hyper-parameter settings using the CV, the model was trained again using datasets from all the reliable training sites, which was then independently evaluated using the test datasets from the reliable validation sites.
The following five evaluation metrics were used to evaluate the performance of the RCNN model and the Rn retrievals: bias, 345 relative bias (rbias), RMSE, relative RMSE (rRMSE) and the coefficient of determination (R 2 ). Detailed information regarding the application of these metrics can be found in Yang et al. (2018).
Identification of reliable sites
The number of reliable and unreliable sites for each observation network, identified by a threshold of 0.9 for the ETC-derived 350 correlation coefficient, is listed in Table 5. A total of 262 sites could be considered reliable, accounting for ~50% of the sites.
Furthermore, no site was considered reliable for some observation networks/programs, namely ChinaFlux, GCNET, GAME.ANN, HiWATER, IMAU-Ktransect, and LBA-ECO. It is not surprising that sites from the ChinaFlux network were assigned as unreliable; several studies have revealed that the reliability of site measurements from China is questionable and should be examined carefully before use Tang et al., 2013;Tang et al., 2011). The GCNET network is 355 located in inner Greenland, where systematic measurements errors are common due to difficulties in instrument maintenance and operation-related failures. Sites from the GAME.ANN network are located in the Tibetan Plateau (TP) region, where abnormal climate and complex terrain make it difficult to accurately measure variations in Rn. Similar issues also exist in in situ measurements from the IMAU-Ktransect, HiWATER and LBA-ECO networks. In contrast, some of the international observational networks, such as BSRN (Ohmura et al., 1998) and FluxNet (Wilson et al., 2002), provide many ground-based 360 measurements with sufficient spatial representativeness for Rn at 5 km resolution. In addition, the ARM (Stokes and Schwartz, 1994) and SURFRAD (Augustine et al., 2000) networks were classified as containing reliable sites. In situ measurements from the SURFRAD (Augustine et al., 2000) network were well known in surface radiation budget studies because of their high data quality, and have been widely utilized as a result (Wang et al., 2015b;Hao et al., 2019;Wang et al., 2015a;Qin et al., 2012). Overall, compared to other networks, the sites from ARM, BSRN, SURFRAD, and FluxNet networks were mostly 365 identified as reliable sites, illustrating the superiority of these observation networks.
The spatial and proportion distributions of the reliable training and validation sites for different surface types are presented in Fig. 4. The most reliable sites are distributed in the United States, Europe, and East Asia. In turn, many sites located in South America, Africa, Eurasia, and the Polar Regions were identified as unreliable. The reasons that these sites were classified as unreliable are closely related to the complex surface and atmospheric environment and poor instrument maintenance in their 370 corresponding regions. Most grassland and cropland sites were classified as reliable (~66% and ~62%, respectively), whereas the fewest reliable sites were classified in ice/snow-covered areas (~14%). In addition, sites neighboring the sea were mostly identified as unreliability due to the presence of large water bodies within the satellite footprint. Thus, the processing of identifying reliable sites highlights the need to pay more attention to such areas for surface radiation estimations.
Assessment of the RCNN model
Ten-CV was used to evaluate the performance of the RCNN model at reliable and all training sites, respectively, and the evaluated performances are summarized in Table 6. Note that the model fitting result represents the model with the best fitting accuracy over the 10 CV rounds, while the cross-validation results are the averages of the 10-round combination. 2 , the RCNN model trained using ground-based Rn measurements obtained at the reliable sites is considered as the best-trained model, which was selected for the subsequent analysis.
Figure 5: Scatterplots of (a) mode training (fitting) accuracy and (b) model test accuracy for the reliable training and independent
validation sites. The color bar illustrates the normalized density of samples. and 20.9% for day and night, respectively) over the sun-glint ocean and polar regions both during daytime and nighttime (Xi et al., 2018;Xu et al., 2020). Additionally, when the aerosol optical depths (AODs) used to calculate CERES-SYN surface 415 solar radiation are compared with the ground-based observations, the calculated shortwave radiation is 1-2% higher (Fillmore et al., 2021). Therefore, large uncertainties in these atmospheric input parameters may lead to serious overestimations of the CERES-SYN Rn. In addition, the MERRA2 Rn shows the lowest accuracy, with an RMSE of 20.87 Wm -2 , reflecting the reanalysis model's inability to accurately describe the evolution of cloud properties (Betts et al., 2006). Comparatively, the AVHRR retrievals show a better accuracy than the other three products. In addition, our estimates have a higher spatial 420 resolution compared to the CERES-SYN and MERRA2 data.
Evaluation of the RCNN-based AVHRR Rn retrievals
Compared to the validation results at the reliable sites, the accuracy evaluation at all sites shows the ability of the RCNN to accurately capture Rn variation at a global scale, even though some measurements from unreliable sites added large uncertainties to the final evaluation. Figure S2 shows a comparison of results for the four datasets at all of the sites. Overall, the AVHRR and GLASS Rn retrievals were still better than those of CERES-SYN and MERRA2; however, the accuracy of 425 AVHRR Rn decreases slightly, with R 2 , RMSE, and bias values of 0.85, 26.51 Wm -2 (35.50%) and 1.32 Wm -2 (1.76%), which is comparable to the GLASS Rn retrievals, with values of 0.85, 26.56 Wm -2 (35.38%) and -0.83 Wm -2 (-1.10%), respectively.
Therefore, even if the RCNN model is trained using measurements from less reliable sites, it still accurately reproduces surface Rn distributions at global scale. In the following analysis, the GLASS Rn retrievals were used as the main comparison because of their high accuracy and reasonable spatiotemporal variation Jiang et al., 2018).
Figure 6: Scatterplots of product validation for (a) AVHRR, (b) CERES-SYN, (c) MERRA2, and (d) GLASS at the reliable sites.
Inter-comparison results for the AVHRR and GLASS Rn retrievals against the ground-based measurements over each network are displayed in Fig. 7 To further improve understanding of the temporal variations of the AVHRR Rn retrievals, coincident time series from all the Rn datasets were inter-compared over seven sites representing different surface types, as shown in Fig. 8
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Rn values only capture the general Rn trend in the snow/ice areas. The GLASS Rn retrievals are also most consistent with the in situ measurements among the four datasets, although small biases still exist.
The overall evaluation results of the AVHRR and GLASS Rn retrievals for different surface types are displayed in Table 7.
Generally, both datasets achieved high accuracy, with RMSEs ranging from 20 to 25 Wm -2 . The AVHRR Rn retrievals show the better performance for most of the surface types, except for snow/ice, as previously discussed; however, the difference in 465 the RMSEs between the AVHRR and GLASS Rn retrievals for the snow/ice cover type is small (0.5 W/m -2 ). Together, these results further indicate that the RCNN model can generate accurate Rn estimates for different land cover types.
Analysis of influencing factors
Variation in surface Rn is mainly affected by atmospheric conditions, but also, to a lesser degree, by surface characteristics (He et al., 2015). Under clear-sky conditions, AOD and column water vapor (CWV) are the main atmospheric constituents that modulate surface shortwave and longwave radiation, and further affect spatiotemporal variations of surface Rn. In contrast, 480 clouds and CWV control surface Rn dynamics under cloud-sky conditions, especially clouds that have significant impacts on shortwave and longwave radiation. Therefore, AOD, CWV, and cloud optical thickness (COT, as a surrogate for cloud optical properties) derived from MERRA2 were employed to analyze the sensitivity of the accuracy of the AVHRR and GLASS Rn retrievals to variations in these influencing factors. In addition, Rn retrieval performance at different elevations was also evaluated.
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All the evaluation results are displayed in Figure 9. The AVHRR Rn retrievals were always better than the GLASS Rn retrievals under all conditions of the four influencing factors, except for elevations ranging 800-1000 and 1200-1500 m, which demonstrates the superiority of our algorithm. Specifically, as the COT increases (i.e., increasing cloud thickness), the AVHRR and GLASS Rn RMSE values increase accordingly but still remain relatively low for both datasets (< 27 Wm -2 ). Note that the differences in RMSE between the two datasets also increased with increasing COT (Fig, 9(a)). A small COT indicates relative 490 clear-sky conditions, which results in surface total solar radiation dominated by direct solar radiation. Therefore, the performance of the RCNN model and the MARS models used for the GLASS Rn product is comparable with regard to the accuracy of their Rn retrievals. However, when the absorption and scattering effects are enhanced for direct solar radiation from TOA, depending on the IPA, it is difficult to retrieve the total surface Rn accurately using the MARS model because the spatially adjacent effects (i.e., 3-D effects from clouds) are not considered. Although the RMSEs of the 495 AVHRR retrievals also increase, the rate of increase is lower than that of the GLASS Rn retrievals. This is because the RCNN model recognizes spatial textural and contextual information and comprehensively considers atmospheric conditions within a certain area rather than on IPA, which to some extent addresses the spatially adjacent effect on the accuracy of AVHRR Rn retrievals.
Aerosols also have absorption and scattering influences on solar radiation, and therefore, a similar conclusion can be drawn.
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For example, when AOD increases from 0.3 (a non-clean atmosphere), the difference in the performance of the two retrieved model becomes more pronounced. The AVHRR Rn retrievals maintain a stable level of uncertainty (RMSEs = 22-24 Wm -2 ), while the errors GLASS Rn retrieval errors increase dramatically (up to 29 Wm -2 ). This illustrates the importance of integrating the spatial adjacent effect into the inversion model under hazy atmospheric conditions.
In the case of CWV, which has a strong influence on longwave radiation, when the condition is < 50 kgm -2 , the accuracy 505 differences between the AVHRR and GLASS Rn values are small (< 1 Wm -2 ). However, as CWV increases, the RMSEs of the GLASS Rn retrievals increase dramatically, while the AVHRR Rn estimates maintain a high level of accuracy (RMSEs = ~23 Wm -2 ).
With respect to elevation, there was no notable difference between the two datasets; our estimates were better than GLASS Rn under different elevation ranges, except for the 800-1,000 and 1,200-1,500 m bins, although these differences were less than 510 1 Wm -2 . The lower accuracy of AVHRR Rn values for these two elevation ranges is attributable to the less reliable sites used for the RCNN training. In addition, the AVHRR Rn retrievals show steady and very low (close to zero) biases under different conditions, while the biases of the GLASS Rn retrievals show a high degree of variation. This illustrates that the RCNN model has a greater capability for unbiased surface Rn estimation.
Overall, the RCNN-derived Rn retrievals show a high accuracy under different atmospheric and surface conditions relative to 515 the GLASS Rn retrievals and especially for thick-cloud and hazy atmospheric conditions. In such cases, spatially adjacent information is important for accurately estimating the surface Rn retrievals. Although previous studies have proposed several methods for integrating spatial information to retrieve surface and atmospheric variables, such as PM2.5 Wang et al., 2020a), ozone (Li and Cheng, 2021), and nitrogen dioxide , these methods only considered discrete surrounding points within a certain area to train the model using IPA. This artificially destroys the natural correlation between 520 the target and the surroundings. Our RCNN model can automatically recognize complete spatial information centered at an interesting location and, thus, is a more reasonable and effective method.
Figure 9: Accuracy changes in AVHRR and GLASS Rn retrievals under different conditions for (a) cloud optical thickness (COT), (b) aerosol optical depth (AOD), (c) column water vapor (CWV), and (d) elevation. The values in parentheses on the left-axis
525 correspond to the RMSE differences denoted by bar charts. The shaded area show the variance ranges of the biases.
Spatiotemporal analysis
The global-scale spatial distributions of mean AVHRR and GLASS surface Rn for January and July 2008 are displayed in Fig. 10. The missing values in the Polar Regions reflects the unavailability of valid data at the five bands in the case of the AVH02C1 product. The overall distribution of surface Rn for the two datasets is very similar, although slight differences exist 530 in some regions, such as the TP region, the Sahara Desert, and Greenland. AVHRR Rn retrievals are notably larger than the GLASS Rn retrievals in the TP region. Based on the results shown in Fig. 9(d), greater confidence can be placed in the AVHRR Rn retrievals for high-elevation regions relative to GLASS Rn retrievals; however, the AVHRR Rn retrievals are relatively lower in Greenland. Because few sites from the GCNET and PROMICES networks were classified as reliable for the model training, the RCNN model has less knowledge about the spatiotemporal variations of Rn in Greenland compared to other 535 regions. The validation results in Fig. 8 and Table 7 for the ice/snow surface cover type further confirm that GLASS Rn product may offer a better performance in Greenland region. Therefore, new algorithms and data are required for the Polar Regions to address this problem.
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The spatiotemporal consistency of the AVHRR and GLASS daily Rn retrievals against COT was examined at a global scale in January and July 2008, respectively, as shown in Fig. 11. Overall, the spatial consistency is high for the two datasets.
Specifically, in January, as the COT increases, the daily mean Rn values and the absolute differences between the two datasets also increase. As shown in Fig. 9(a), when COT increases, the AVHRR Rn retrievals are more accurate. Thus we believe that 545 the large discrepancies under high COT conditions are mainly attributed to the uncertainty of GLASS Rn retrievals. In July, surface daily mean Rn remained relatively stable under different COT conditions, and the absolute differences between the two datasets also remain steady, with a mean absolute difference of about 20 Wm -2 .
Based on the previous analysis, spatially adjacent information is important for surface Rn estimation when COTs values are large; however, if the cover of the entire cloud layer is small compared to the scale of the AVHRR footprint, the spatial adjacent 550 effects will be significantly weakened in the inversion process, even if the corresponding COT is large. IPA-provided information includes the properties of the entire cloud layer. Figure S3 shows the spatial distribution of the monthly mean cloud cover fraction (CF) at the global scale in January and July, and the corresponding differences in CFs (Jan.-July). In January, the CFs are higher than in July over most land regions except in Central Africa, Southern Asia, Southern Australia and Antarctica. However, most regions had smaller CFs in July. The differences in CFs for the two months are also marked; 555 the positive differences demonstrate that more than 72% of the land pixels had a higher CFs in January than in July. The spatial adjacent effects induced by clouds are more significant on surface Rn in January than in July. Therefore, when large and thick cloud layers exist, such as in the Polar Regions, CNN is a better choice for surface Rn estimation, especially for downward longwave radiation (DLR) because the temperature of cloud-base, which is an essential variable in the parameterized calculation of DLR, is difficult to retrieve from multispectral remote sensing (Yang and Cheng, 2020).
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To examine the temporal reliability of the generated AVHRR Rn dataset, a long-term analysis of surface Rn for the four datasets was carried out, the results of which are shown in Fig. 12
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Rn values are overestimated against ground-based measurements. GLASS Rn temporal profile is more consistently correlated with the AVHRR Rn retrievals.
Note that the LTDR project only uses afternoon satellite to generate the AVHRR product to do with the high uncertainty of the atmospheric correction algorithm when applied to low sun elevation pixels present in morning (am) satellites. Afternoon satellites include NOAA-7, NOAA-9, . The use of 575 these satellites alone inevitably leads to small gaps in the data in exchange for a higher accuracy in the atmospheric correction.
The time series is not fully complete and presents some observational gaps. Specifically, some large discrepancies occur, during some periods including 1994-1995, 1999-2000, 2007-2008, and 2018-2019. These periods correspond to the alternative update times of the NOAA-series satellites. For example, NOAA-11 was successfully succeeded by NOAA-14 from 1994 to 1995. Important gaps and noise were found in the images from March to September and empty data from 580 September to December, due to NOAA-11 orbital degradation. NOAA-16 replaced NOAA-14 in 2000 for monitoring of the Earth's surface and atmosphere. During these periods of satellite replacement, the corresponding AVHRR data also contain large gaps. Similarly, NOAA-20 was launched on November 18, 2017, yet the quality of the AVHRR TOA observations from this platform was poor due to important gaps in the images and the presence of artefacts. This explains the abnormal temporal variations in the AVHRR Rn profile in these years shown in Fig. 12(a). Therefore, effective multi-source data fusion algorithms 585 and spatial gap-filling technology are urgently needed to improve the quality and coverage of the AVHRR Rn dataset.
The temporal variations in monthly Rn anomalies for the four datasets are shown in Fig. 12 (Vermote and Kaufman, 1995), which enables a temporally reliable Rn dataset to be produced. Following the approach, overall, our AVHRR Rn dataset is more accurate and shows reasonable spatiotemporal variations compared to the other three datasets. This dataset will play an important role in climate change study. Fig. 13. Overall, the average R increases from 0.61 to 0.708, and RMSE decreases from 50.12 to 46.17, respectively, for the MLR model. As the valid spatial extent increases, essential and complete spatial features are exposed and incorporated into the MLR model, which helps to continuously improve the model's retrieval accuracy. The spatial extent of B13 (approximately 65 × 65 km 2 ) is the smallest size that exhibits convergent R and RMSE values, and the spatial extent at the B15 reaches a more stable state 610 for surface Rn estimations. This finding is in line with the results of Jiang et al. (2020b), showing that scale effects have a considerable impact on solar radiation retrieval accuracy, and distances of approximately 20 to 40 km from the central point (corresponding to areas of 40 × 40 km 2 to 80 × 80 km 2 ), are optimal spatial scale. In addition, previous studies (Hakuba et al., 2013;Huang et al., 2016) recommended a threshold distance of approximately 30 km, equal to a 13 × 13 grid region with a spatial resolution of 0.05°, for shortwave radiation estimation. Therefore, a 15 × 15 grid area was selected for the input sub-615 images to generate AVHRR Rn retrievals.
620
The RCNN model uses instantaneous satellite-sensed signals to directly estimate daily mean Rn retrievals. Though some previous studies Wang et al., 2015a;Wang and Liang, 2017;Xu et al., 2020) directly estimated dailyaveraged surface radiation from instantaneous satellite observations, like MODIS, the idea is theoretically flawed because the AVHRR sensor only offers instantaneous "snapshots", which cannot capture daily mean information about the diurnal cycles of the atmosphere and clouds. King et al. (2013) acknowledged that the frequency of cloud variations is high at different times 625 and locations based on twin MODIS cloud products. In view of the wide satellite overpass times over a particular location, e.g., equatorial crossing time generally ranges from 1300 to 1730 in local time, representing different instantaneous atmospheric conditions for different AVHRR sensors, daily mean MERRA2 Rn is incorporated into the input collection to provide daily mean information about the surface, atmosphere and clouds for the RCNN model. Figure 14 shows the effect of the daily mean MERRA2 Rn on the final AVHRR Rn retrievals at different AVHRR overpass 630 times in local time. The improved effect is slightly more significant during the afternoon than in the morning when more overland clouds are present (King et al., 2013). This improvement is also more pronounced during the night. The AVHRR Rn retrievals can only be obtained when solar radiation is available (Fig. 10) because of the missing values in the AVH01C1 product; therefore, the results during the night are based on the validation results for high latitudes, which demonstrate that daily mean information about the diurnal cycles of the atmosphere and clouds is more important for daily surface radiation 635 estimation at high latitudes than that at middle and low latitudes. Shupe et al. (2011) found annual cloud occurrence fractions are 58%-83% at the Arctic observatories, with a clear annual cycle wherein clouds are least frequent in the winter and most frequent in the late summer and autumn.
Additionally, MERRA2 downward shortwave radiation (DSR) was used as a replacement for MERRA2 Rn to test its contribution to daily mean surface Rn estimations when using instantaneous satellite data. The results presented in Fig. S4
640
show that the improved effect of daily MERRA2 DSR is not comparable with that achieved using daily MERRA2 Rn, and the former is closer to the results obtained when only instantaneous AVHRR observations are used. Therefore, MERRA2 Rn is a meaningful input for the RCNN model. Moreover, the AVHRR Rn retrievals could also be further improved by using more accurate daily mean Rn data, such as GLASS Rn, or other parameters that accurately represent daily mean atmospheric and cloud variations. The bars indicate RMSE and lines indicate absolute biases. The shading shows the variation range of absolute bias.
Determination of a threshold for the ETC-derived correlation coefficient
The threshold for the ETC-derived correlation coefficient between in situ measurements and the unknown truth within the 5 650 km AVHRR grid in Eq.
(2) affects the selection of reliable sites and the subsequent PSME process. A series of thresholds for the ETC-derived coefficients were considered, ranging from 0.2 to 0.9 with an interval of 0.1. In each case, the corresponding measurements from the selected reliable sites were fed into the RCNN model to train and subsequently generate AVHRR Rn retrievals. Then, the training and test accuracies of the RCNN were calculated over all of the reliable sites for comparison.
Another important consideration is the representativeness of the RCNN for global Rn estimation, given that the number of 655 reliable training sites decreases with higher thresholds. Thus, the trained RCNN model was again evaluated at all sites, including reliable and unreliable sites, to examine the global representativeness. The number of reliable sites (training and test accuracies) and the associated global accuracy are presented in Fig. 15. As the threshold increases, the number of reliable sites decreased. The training and test relative RMSEs of the RCNN model showed a general decreasing trend, especially above a threshold of 0.5, which illustrates that the selection of reliable sites and the measurements from these sites have better 660 representativeness for the AVHRR footprint scale using ETC. This helps address the spatial scale mismatch issue and improve the accuracy of AVHRR retrievals at a 5 km resolution. In addition, a trade-off between the RCNN's fitting accuracy at the reliable sites and the global accuracy at all sites needs to be considered. Even when a threshold of 0.9 was used, the global accuracy of the RCNN was only slightly lower, which explains why this threshold was applied in Sect. 3.1.
Orbital drift of the NOAA-series satellites
The orbital drift problem of the NOAA-series satellites has attracted the attention of users applying AVHRR-derived highlevel remote sensing products, such as land surface temperature (LST) (Ma et al., 2020;Liu et al., 2019) and TOA albedo 670 (Song et al., 2018). As shown in Fig. 16
Conclusions and outlook
A long-term (1981-2019) global daily surface Rn product with spatial resolution of 0.05° was generated from historical NOAA-695 series AVHRR data using a RCNN-based PSME method. The specific steps employed were as follows: (1) selecting reliable sites from all sites based on ETC to generate the sample dataset; (2) training and independent testing of the proposed RCNN model; (3) evaluating the AVHRR Rn retrievals against in situ measurements and performing inter-comparisons with three other Rn products (GLASS, CERES-SYN, and MERRA2); and (4) generating and evaluating the long-term AVHRR Rn product.
700
ETC was first applied to select reliable sites to prepare a sample dataset with better spatial representativeness at the AVHRR footprint scale (i.e., 5 km). In total, 262 sites were classified as reliable sites from a total of 523 sites and used as a sample dataset for the RCNN model. The proportions of the selected reliable sites representing cropland and grassland surfaces were highest (~66% and ~62%, respectively), while those representing ice/snow surface were lowest (~14%). The sample dataset from the 262 sites ensured that the trained RCNN model had both a good fitting accuracy for the reliable sites and global 705 accuracy across all sites.
A simple MLR model was used to examine the spatial adjacent effects on surface Rn estimation, and a spatial extent of 15 × 15 pixels (75 × 75 km 2 ) was then determined as the input size of the RCNN to provide sufficient spatial information. Based on 10-fold CV, the trained RCNN model achieved an R 2 of 0.90, with an RMSE of 20.76 Wm -2 (25.93%), and a bias of -0.11 Wm -2 (-0.14%); the corresponding independent validation values were 0.84, 26.77 Wm -2 (31.54%), and 1.16 Wm -2 (1.37%) at 710 the reliable sites, respectively. These results demonstrate the overall ability of the RCNN model to accurately predict surface Rn.
The results of an inter-comparison between the AVHRR Rn retrievals and three other products illustrated that our retrievals show a better accuracy against in situ measurements, with an R 2 of 0.90, RMSE of 20.99 Wm -2 (26.18%), and bias of -0.04 W/m 2 (-0.05%) at the reliable sites, and an R 2 of 0.85, RMSE of 26.51 Wm -2 (35.30%), and bias of 1.32 Wm -2 (1.76%) across 715 all sites. At the same time, the AVHRR Rn retrievals show better performance for different observational networks and surface cover types, except for the snow/ice surface cover. Under different elevations and atmospheric conditions, the AVHRR Rn retrievals performed better than the GLASS Rn equivalent, especially in the presence of thick clouds and hazy atmospheric conditions because of the integration of spatially adjacent information into the inversion process in the RCNN model. In addition, the spatiotemporal variation of the AVHRR Rn retrievals is similar to that of the GLASS Rn values, demonstrating 720 the ability of the RCNN model to generate a long-term global Rn product.
The long-term global Rn dataset generated by the RCNN model displays high accuracy and reasonable spatiotemporal variation at the global scale, which is suited to many applications including, for example, studies to understand the radiation budget and global climate change. Besides, compared to current satellite-derived Rn products, e.g., CERES-SYN and GLASS (2000present), a more long record of the AVHRR Rn dataset shows its value in climate change studies. However, 725 further research is needed to solve some problems to further improve the data quality of the AVHRR Rn dataset. First, new algorithms and satellite data are needed to estimate surface Rn in the Polar Regions, such as MODIS data .
Second, an effective data gap-filling method or multi-source data-fusion algorithm is required to fill the data gaps over land, especially during periods of satellite replacement work. Third, coupled with spatially adjacent information, real-time temporal information, or historical information should be incorporated to further improve the accuracy of the Rn retrievals.
730
As a type of machine learning, deep learning involves using data-driven models to find potential relationships and patterns, and offers high adaptability to training data sample inputs. The predictive ability of a data-driven model completely depends on the limitations of the training dataset and in the case of Rn, the ability of the model to accurately portray spatiotemporal dynamics in areas where the availability of training data is relatively poor, such as for AVHRR Rn retrievals for ice/snowcovered surfaces. To address this problem, more physical knowledge is needed to fully utilize data-driven modeling to estimate 735 surface Rn under different atmospheric and surface conditions. In particular, more attention should be paid to understanding inherent physical processes in addition to obtaining optimal estimation by coupling physical process models with the versatility of data-driven machine learning (Reichstein et al., 2019). | 2021-09-18T23:47:03.433Z | 2021-09-17T00:00:00.000 | {
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9084268 | pes2o/s2orc | v3-fos-license | Necessary Optimality Conditions for Optimal Control Problems with Equilibrium Constraints
This paper introduces and studies the optimal control problem with equilibrium constraints (OCPEC). The OCPEC is an optimal control problem with a mixed state and control equilibrium constraint formulated as a complementarity constraint and it can be seen as a dynamic mathematical program with equilibrium constraints. It provides a powerful modeling paradigm for many practical problems such as bilevel optimal control problems and dynamic principal-agent problems. In this paper, we propose weak, Clarke, Mordukhovich and strong stationarities for the OCPEC. Moreover, we give some sufficient conditions to ensure that the local minimizers of the OCPEC are Fritz John type weakly stationary, Mordukhovich stationary and strongly stationary, respectively. Unlike Pontryagain's maximum principle for the classical optimal control problem with equality and inequality constraints, a counter example shows that for general OCPECs, there may exist two sets of multipliers for the complementarity constraints. A condition under which these two sets of multipliers coincide is given.
Introduction
We are given a time interval [t 0 , t 1 ] ⊆ IR, a multifunction U mapping [t 0 , t 1 ] to nonempty subsets of IR m , and a dynamic function φ : [t 0 , t 1 ] × IR n × IR m → IR n . A control or control function u(·) is a measurable function on [t 0 , t 1 ] such that u(t) ∈ U (t) for almost every t ∈ [t 0 , t 1 ]. The state or state trajectory, corresponding to a given control u(·), refers to a solution x(·) of the following controlled differential equation: x(t) = φ(t, x(t), u(t)) almost everywhere (a.e.) t ∈ [t 0 , t 1 ], (1.1) where E is a given closed subset in IR n × IR n andẋ(t) is the first-order derivative of the state x(·) at time t. The differential equation (1.1) linking the state x(·) and the control u(·) is referred to as the state equation. In optimal control, one looks for a state and control pair (x(·), u(·)) satisfying the state equation (1.1) and the boundary condition (1.2) so as to minimize an objective function J(x(·), u(·)). In practice, there are generally extra constraints to be satisfied by the state and control pair. Such constraints are called mixed state and control constraints (mixed constraints for short).
Motivated by the studies for the DVI, we consider a class of controlled differential complementarity system where in addition to the state equation (1.1) and the boundary condition (1.2), the state and control pair (x(·), u(·)) satisfies some mixed equality and inequality constraints, as well as a mixed equilibrium system formulated as a complementarity system: 0 ≤ G(t, x(t), u(t)) ⊥ H(t, x(t), u(t)) ≥ 0 a.e. t ∈ [t 0 , t 1 ], (1.5) where G, H : [t 0 , t 1 ]×IR n ×IR m → IR l . We say that an index i is degenerate if G i (t, x(t), u(t)) = H i (t, x(t), u(t)) = 0. It is obvious that such a system includes DCPs (1.3) and (1.4) as special cases. Correspondingly, it is natural to determine what is the "best" control (or the "best" state and control pair) satisfying such a system to achieve some given objective. A simple example is to find the best control from such a system so that the final state x(t 1 ) will reach some prescribed target from a given initial state x(t 0 ). In this paper, we introduce a class of optimal control problems with equilibrium constraints (OCPEC) in which one looks for a state and control pair (x(·), u(·)) from such a system so as to minimize an objective function J(x(·), u(·)). Mathematically, the OCPEC considered in this paper is of the form (OCPEC) min J(x(·), u(·)) s.t.ẋ(t) = φ(t, x(t), u(t)) a.e. t ∈ [t 0 , t 1 ], g(t, x(t), u(t)) ≤ 0, h(t, x(t), u(t)) = 0 a.e. t ∈ [t 0 , t 1 ], (1.6) 0 ≤ G(t, x(t), u(t)) ⊥ H(t, x(t), u(t)) ≥ 0 a.e. t ∈ [t 0 , t 1 ], where g : [t 0 , t 1 ] × IR n × IR m → IR l 1 and h : [t 0 , t 1 ] × IR n × IR m → IR l 2 .
The OCPEC can be considered as a dynamic version of the mathematical program with equilibrium constraints (MPEC) that has been an active area of research in recent years (see, e.g., the monographs [26,28]). The OCPEC provides a powerful modeling paradigm for many practical problems such as the dynamic optimization of chemical processes with changes in the number of equilibrium phases [31]. A large part of source problems of the OCPEC comes from bilevel optimal control problems (see, e.g., [1,15,16,46,47]), Stackelberg differential games (see, e.g., [17,43]), and dynamic principal-agent problems (see, e.g., [25,34]) when there exist inequality constraints in the lower-level problem. For those problems, if the lowerlevel problem, which is a parametric optimal control problem, is replaced by Pontryagain's maximum principle (see [30,38]) which is the well-known first-order necessary optimality condition for optimal control problems, then an OCPEC results; see, e.g., [15,Section 6.1].
It is desirable to know whether there exists an optimal control before solving the OCPEC. The Filippov's existence theorem for Mayer's problem that is due to Filippov [11] (see also [3,Theorem 9.2.i]) requires the convexity of the velocity set φ(t, x, U (t, x)) where The velocity set is in general nonconvex due to the existence of the complementarity constraints. Thus, the classical existence theorem may not be applicable and one may need to look for new ways to prove the existence of optimal controls for the OCPEC or use the existence theorem in a relaxed control setting ( [39,50]). We leave these challenging questions for future research.
In this paper, we assume that an optimal control exists for the OCPEC and focus on deriving its necessary optimality conditions. To the best of our knowledge, there is no such result in the literature so far. Although deriving necessary optimality conditions for optimal control problems with mixed constraints is a highly challenging problem, some progresses have been made; see, e.g., [5, 7-10, 20, 21, 24]. Unfortunately, none of these results are applicable to the OCPEC and its reformulations. The constraint (1.5) is obviously equivalent to that for almost every t ∈ [t 0 , t 1 ], G(t, x(t), u(t)) ≥ 0, H(t, x(t), u(t)) ≥ 0, G(t, x(t), u(t)) ⊤ H(t, x(t), u(t)) ≤ 0, (1.8) where ⊤ denotes the transpose, which is clearly a system of inequalities. However, all the inequalities in (1.8) never hold strictly at the same time. This means that the Mangasarian-Fromovitz constraint qualification (MFCQ) is violated at any point satisfying (1.8). The classical necessary optimality conditions for optimal control problems with mixed equality and inequality constraints generally require the linear independence constraint qualification (LICQ) (see, e.g., [21]) or the Mangasarian-Fromovitz condition (MFC) (see, e.g., [7]) over some neighborhood of the local minimizer. But both LICQ and MFC are stronger than MFCQ. Thus, the classical necessary optimality conditions for optimal control problems with equality and inequality constraints cannot be applied to the OCPEC with the complementarity constraint (1.5) reformulated as inequality constraints (1.8). In the MPEC literature, by using the so-called "piecewise programming" approach (see, e.g., [26,48]), the feasible region of an MPEC is locally reformulated as a union of finitely many pieces where each piece is expressed as a system of equality and inequality constraints, and then it can be shown that the strong (S-) stationarity holds under the so-called MPEC LICQ. It is obvious that such an approach fails for the dynamic complementarity system (1.5). A well-known technique to derive a necessary optimality condition for an MPEC called the Clarke (C-) stationarity is to use the equivalent nonsmooth reformulation min{G, H} = 0 (" min " denotes the componentwise minimum) to replace the complementarity system 0 ≤ G ⊥ H ≥ 0 (see, e.g., [35,45]). This technique, however, is also not applicable to the OCPEC since such an approach leads to an optimal control problem with a nonsmooth mixed equality constraint for which there does not exist any applicable necessary optimality conditions in the control literature. Another equivalent reformulation of the complementarity constraint is G, H ∈ C l where is called the complementarity cone. It is known that this reformulation is useful to obtain a necessary optimality condition in the form of Mordukhovich (M-) stationarity in the MPEC literature; see, e.g., [45]. Using this reformulation, the OCPEC can be equivalently reformulated as An optimal control problem in the form of (P s ) with an abstract mixed constraint S(t) was recently studied by Clarke and De Pinho [7]. In this paper, we first derive a slightly sharper necessary optimality condition for (P s ) than [7, Theorem 2.1] and then apply it to the problem with S(t) defined as in (1.11). We hope that we would get the M-stationarity as in the MPEC literature. Unfortunately, for the OCPEC, no sign information on the multipliers associated with the degenerate indices can be derived and, consequently, we can only obtain a weak stationarity. In order to get more sign information on the multipliers associated with the degenerate indices, we further utilize the Weierstrass condition to obtain the second set of multipliers. A counter example shows that in general these two sets of multipliers may be different in measure. However, under the MPEC LICQ, since the multipliers corresponding to the weak stationarity are unique, these two sets of multipliers coincide almost everywhere and then we can obtain the S-stationarity with one set of multipliers.
The rest of this paper is organized as follows. In Section 2, we give some preliminaries and preliminary results. In Section 3, we develop the necessary optimality conditions for the OCPEC. Section 4 illustrates our derived results with a simple example.
Preliminary and preliminary results
Throughout this paper, · denotes the Euclidean norm and B δ (x) := {y : y − x < δ} the open ball centered at x with positive radius δ. The boundary, closure, convex hull, and closed convex hull of a subset Ω ⊆ IR n are denoted by bd Ω, cl Ω, co Ω, and clco Ω, respectively. Moreover, dist Ω (x) denotes the Euclidean distance from x to Ω. For any a, b ∈ IR n , a + := max{a, 0} denotes the nonnegative part of vector a and a, b the inner product of vector a and vector b. Given a mapping ψ : IR n → IR m and a point x ∈ IR n , ∇ψ(x) stands for the transposed Jacobian of ψ(·) at x and I ψ (x) := {i : ψ i (x) = 0} the active index set of ψ(·) at x. The Minkowski sum of a singleton {a} and an arbitrary set A is denoted by a + A.
Background in variational analysis
In this subsection, we review some basic concepts and results in variational analysis that will be used later on; see, e.g., [6,27,33] for more details. Given a subset Ω ⊆ IR n and x ∈ cl Ω, the proximal normal cone to Ω at x is defined as the limiting normal cone to Ω at x is defined as and the Clarke normal cone to Ω at x is defined as N C Ω (x) := clco N L Ω (x), which also holds true even if the space is not finite dimensional but a more general Asplund space [27]. We can easily obtain the following inclusions: Both the limiting normal cone mapping N L Ω (·) and Clarke normal cone mapping N C Ω (·) are closed in the sense that their graphs are closed.
The following expression for the limiting normal cone of the complementarity cone C l is well-known (see, e.g., [44,Proposition 3.7]) and will be used in Section 3.
Proposition 2.1 For any (a, b) ∈ C l where C l is defined in (1.9), Given a lower semicontinuous function ϕ : IR n → IR ∪ {+∞} and a point x with ϕ(x) finite, the limiting subdifferential of ϕ at x is defined as If ϕ(·) is Lipschitz continuous near x, then the Clarke subdifferential of ϕ(·) at x can be defined as ∂ C ϕ(x) := clco ∂ L ϕ(x), which also holds true even if the space is not finite dimensional but a more general Asplund space [27]. Both the limiting subdifferential mapping ∂ L ϕ(·) and Clarke subdifferential mapping ∂ C ϕ(·) are closed in the sense that their graphs are closed.
Given a point (x, u) ∈ cl gphΞ for a multifunction Ξ : IR n ⇒ IR m , the coderivative D * Ξ(x, u) : IR m ⇒ IR n of Ξ(·) at (x, u) is defined as The symbol D * Ξ(x) is used when Ξ(·) is single-valued at x and u = Ξ(x
Local error bound condition and constraint qualifications
In this subsection, we consider the following constrained system: where D is a closed subset in IR d , and g : IR d → IR l 1 , h : IR d → IR l 2 , G, H : IR d → IR l are all strictly differentiable. We say that the local error bound condition holds (for the constrained system representing the set Ω as in (2.1)) atz ∈ Ω if there exist τ > 0 and δ > 0 such that It is well-known that the local error bound condition atz is equivalent to the calmness of the perturbed constrained system 2) at (0, 0, 0, 0,z) (see, e.g., [19]). The local error bound condition is very weak and there exist many sufficient conditions for it to hold; see, e.g., [14,19,[40][41][42]49]. The following constraint qualifications are such sufficient conditions. Definition 2.1 (MPEC constraint qualifications) Letz ∈ Ω where Ω is defined in (2.1). When D = IR d , we say that the MPEC LICQ holds atz if the family of gradients We say that the MPEC linear condition holds if all the functions g(·), h(·), G(·), H(·) are affine and D is a union of finitely many polyhedral sets.
We say that the MPEC quasi-normality holds atz if there is no nonzero vector (λ, υ, µ, ν) such that • there exists a sequence {z k } ⊆ D converging toz such that for each k, It should be noted that the MPEC quasi-normality is a weak condition which holds automatically when the MPEC linear condition holds with D = IR d and is also implied by the MPEC LICQ.
Proposition 2.2
The local error bound condition holds atz ∈ Ω if the MPEC linear condition or the MPEC quasi-normality holds atz.
Proof.
If the MPEC linear condition holds, then it is easy to see that the perturbed constrained system Ω(y g , y h , y G , y H ) defined in (2.2) is a polyhedral multifunction and hence the local error bound condition holds [32]. Moreover, by [14,Theorem 5.2], the local error bound condition follows from the MPEC quasi-normality immediately.
Optimal control problem with an abstract set constraint
In this subsection, we consider the optimal control problem (P s ) where Here F : [t 0 , t 1 ]×IR n ×IR m → IR and f : IR n ×IR n → IR. The basic hypotheses on the problem data, in force throughout this subsection, are the following: F (·), φ(·) are L × B measurable, S(·) is L measurable, and f (·) is locally Lipschitz continuous, where L × B denotes the σalgebra of subsets of appropriate spaces generated by product sets M × N where M is a Lebesgue (L) measurable subset in IR and N is a Borel (B) measurable subset in IR n × IR m .
We refer to any absolutely continuous function as an arc. An admissible pair for (P s ) is a pair of functions (x(·), u(·)) on [t 0 , t 1 ] for which u(·) is a control and x(·) is an arc that satisfies all the constraints in (P s ). Given a measurable radius function R : [t 0 , t 1 ] → (0, +∞], as in [7], we say that an admissible pair (x * (·), u * (·)) is a local minimizer of radius R(·) for (P s ) if there exists ǫ > 0 such that for every pair (x(·), u(·)) admissible for (P s ) which also satisfies . Note that the so-called W 1,1 local minimizer in the control literature is actually the case where the radius function R(·) is identically +∞.
Let (x * (·), u * (·)) be a local minimizer of radius R(·) for (P s ). For every given t ∈ [t 0 , t 1 ], a radius function R(·), and a positive constant ǫ, we define a neighborhood of the point (x * (t), u * (t)) as follows: Other than the basic hypotheses on the problem data, we also assume that the following assumptions hold for (P s ).
(ii) There exists a positive measurable function k S (·) such that for almost every t ∈ [t 0 , t 1 ], the bounded slope condition holds: ] are integrable and there exists a positive number η such that R(t) ≥ ηk S (t) a.e. t ∈ [t 0 , t 1 ]. Assumption 2.1(i) can be seen as a local Lipschitz condition in variable (x, u) with measurable Lipschitz constants. This condition is automatically satisfied with time independent Lipschitz constants when u * (·) is bounded over [t 0 , t 1 ], the radius function R(·) is a positive constant function, and the functions F (·), φ(·) are locally Lipschitz continuous in variable (t, x, u). Assumption 2.1(ii) is a key condition proposed in [7] to derive the necessary optimality conditions. We will investigate some sufficient conditions for such an assumption to hold in our problem setting in Section 3.
For a general optimal differential inclusion problem where F t : IR n ⇒ IR n is a multifunction, Clarke [5] has derived a new state of the art necessary optimality conditions in the optimal control literature. These conditions are stratified in that both the hypotheses and the conclusions are formulated relative to a given radius function. However, it should be noted that for a point v lying on the boundary Theorem 2.1 Let (x * (·), u * (·)) be a local minimizer of radius R(·) for (P s ) and Assumption 2.1 hold. Then there exist a number λ 0 ∈ {0, 1} and an arc p(·) such that (a) the nontriviality condition holds: (λ 0 , p(t)) = 0 ∀t ∈ [t 0 , t 1 ]; (b) the transversality condition holds: (c) the Euler inclusion holds: For almost every t ∈ [t 0 , t 1 ], Proof. First we consider the case where F (·) ≡ 0. Similarly as in the proof of [ the transversality condition holds: the Euler inclusion holds: For almost every t ∈ [t 0 , t 1 ], ; and the Weierstrass condition holds with radius The Euler inclusion (c) of this theorem for the case where F (·) ≡ 0 can be obtained by estimating the limiting normal cone of the above formula as in the last paragraph of Page 4521 in [7].
The general case in which a nonzero F is present is reducible to the already treated one by augmentation as explained at the end of the proof of [7, Theorem 2.1].
Note that in the proof of Theorem 2.1, we are unable to prove that the Weierstrass condition holds with full radius R(·) as claimed in the proof of [7, Theorem 2.1]. The reason is that for a given u lying on the boundary of the set in the case where F (·) ≡ 0, we would need to find u M ∈ {u : (x * (t), u) ∈ S(t), u − u * (t) < R(t)M/(M + 1)} such that u M → u as M → ∞ and take limits in (2.6) to derive the desired inequality (2.7). But this may not be always possible if Ω is disconnected.
then the conclusions of Theorem 2.1 hold with λ 0 = 1. Such a condition is automatically satisfied in the case of free initial or final point, that is, E = E 0 × IR n or E = IR n × E 1 with closed subsets E 0 , E 1 in IR n . Supposing λ 0 = 0, the result (b) in Theorem 2.1 yields that p(t 1 ) = 0 or p(t 0 ) = 0, respectively, which contradicts the result (a) of this theorem. Throughout the paper, all the derived necessary optimality conditions are FJ type conditions. The desired case where λ 0 = 1 can be obtained provided that there is no nonzero abnormal multiplier, which is always true if the initial or final point is free.
Necessary optimality conditions for OCPEC
In this section, we develop necessary optimality conditions for the OCPEC under the following basic hypothesises.
and G, H : [t 0 , t 1 ] × IR n × IR m → IR l are L measurable in variable t and strict differentiable in variable (x, u), U : [t 0 , t 1 ] ⇒ IR m is a L measurable multifunction, f : IR n × IR n → IR is locally Lipschitz continuous, and E is a closed subset in IR n × IR n .
In fact, we can easily extend our results to the case where the mappings g(·), h(·), G(·), H(·) are only Lipschitz continuous in variable (x, u) and strictly differentiable at (x * (t), u * (t)). But for simplicity of exposition, we assume that they are strictly differentiable in variable (x, u) as in Assumption 3.1.
In Definition 3.1, there are two sets of multipliers. The ideal situation is when these two sets of multipliers are identical almost everywhere. In the case where the multipliers λ g (·), λ h (·), λ G (·), λ H (·) and η g (·), η h (·), η G (·), η H (·) can be chosen as the same almost everywhere, (x * (·), u * (·)) being C-, M-, S-stationarities becomes that (x * (·), u * (·)) is W-stationary with multipliers satisfying the following extra sign conditions: respectively. Although we hope that these two sets of multipliers can be chosen as the same almost everywhere, the following example shows that it is not always possible.
where x, u : IR → IR. Since x(·) is absolutely continuous and x(t) ≥ 0 for almost every t ∈ [t 0 , t 1 ], we must have x(t) ≥ 0 for every t ∈ [t 0 , t 1 ]. Then it is easy to see that (x * (·), u * (·)) ≡ (0, 0) is a minimizer of the above problem. Moreover, it is not hard to verify that for the system Ω := {u : F (u) ∈ C 1 } with F (u) := (−u, x * (t) − u 2 ) T and C 1 defined as in (1.9), where T Ω (u * (t)) o stands for the polar of the tangent cone to Ω at u * (t). It has been shown in [13,Theorem 3.2] that this condition T Ω (u * (t)) o ⊆ ∇F (u * (t))N C 1 (F (u * (t))) is a constraint qualification for M-stationarity at u * (t). Thus, for almost every t ∈ [t 0 , t 1 ], u * (t) = 0 is M-stationary to the problem By solving the M-stationarity condition at (x * (·), u * (·)), we have Since p(·) is absolutely continuous, by virtue of (3.11), there must exist an This together with (3.12)-(3.13) implies We now show that the FJ type M-stationarity for the OCPEC in Definition 3.1 is necessary for optimality under certain constraint qualifications. Note that problem (3.6) is an MPEC with respect to variable u. In the following theorem, we will assume that some MPEC constraint qualifications for M-stationarity, which are qualifications to derive M-stationarity for optimality, are satisfied. The reader is referred to [13,23,45,49] and the references within for MPEC constraint qualifications for M-stationarity.
For problem (P s ), if S(t) = IR n × U (t) for almost every t ∈ [t 0 , t 1 ] (which corresponds to the case of standard optimal control problem without mixed constraints), then the bounded slope condition (2.4) holds automatically for almost every t ∈ [t 0 , t 1 ] since in this case, (2.4) becomes then (2.4) at time t ′ never hold since there exists 0 = α ∈ N P X(t ′ ) (x * (t ′ )) by [33,Exercise 6.19]. If the set of such a point t ′ ∈ [t 0 , t 1 ] is not of measure zero, then the bounded slope condition in Assumption 2.1 does not hold. As a consequence, the bounded slope condition can hardly hold for the case of the pure state constraint S(t) = X(t) × IR m . Generally speaking, the bounded slope condition is a strong condition and is also hard to verify. In the rest of this section, we will investigate sufficient conditions for the bounded slope condition to hold in our problem setting. Proposition 3.1 Assume that the local error bound condition for the system representing S(t) as in (1.11) holds at every (x, u) ∈ S ǫ,R * (t) and x, u; λ, υ, µ, ν) + ζ . (3.14) Then the bounded slope condition (2.4) holds.
A sufficient condition for condition (3.14) to hold is the following stronger condition that is similar to the M ǫ,R In the same way as in [7,Proposition 4.2], we can have that condition (3.14) holds with k S (t) := κ(t)(k g . Consequently, the bounded slope condition follows from Proposition 3.1 immediately. The proof is complete. In general, it is not easy to guarantee the integrability of the measurable function k S (·) in the bounded slope condition (2.4). We next consider a special case where the mappings g(·), h(·), G(·), H(·), U (·) are all autonomous (i.e., independent of t). In this case, we will give some sufficient conditions to ensure that the function k S (·) is a positive constant function which is clearly integrable. We denote U (t) ≡ U , S(t) ≡ S, S(x) := {u : (x, u) ∈ S}, and Note that C ǫ,R * may be unbounded since u * (·) may be unbounded on [t 0 , t 1 ].
The following condition that is stronger than condition (3.16) is similar to the so-called MFC proposed in [7]: t (x, u), =⇒ (λ, υ, µ, ν) = 0, (3.17) which clearly implies the MPEC quasi-normality defined in Theorem (3.2)(c) and hence the local error bound condition for the system representing S(t) as in (1.11) holds at (x, u) by Proposition 2.2. Thus, by Proposition 3.3, we can have the following result immediately.
In Proposition 3.3 and Corollary 3.1, conditions (3.16) and (3.17) are both required to hold over some neighborhood of the optimal process (x * (·), u * (·)). In order to weaken this requirement, Clarke and De Pinho [7,Definition 4.7] introduced the following concept.
Definition 3.2 We say that (t, x * (t), u) is an admissible cluster point of (x * (·), u * (·)) if there exist a sequence {t k } ⊆ [t 0 , t 1 ] converging to t and corresponding points (x k , u k ) ∈ S(t k ) such that lim k→∞ x k → x * (t) and lim Based on Definition 3.2, we have the following sufficient condition for the bounded slope condition to hold with certain positive constant. Proposition 3.4 Let R(·) ≡ r > 0 be a positive constant function and all the mappings g(·), h(·), G(·), H(·), U (·) autonomous. Assume that for all admissible cluster points (t, x * (t), u) of (x * (·), u * (·)), condition (3.16) and the local error bound condition for the system representing S(t) as in (1.11) hold at (x * (t), u) or the stronger condition (3.17) holds at (x * (t), u). Then for every t ∈ [t 0 , t 1 ], the bounded slope condition (2.4) holds with some radius η ∈ (0, r) and k S (t) = π for some constant π > 0.
Although all the mappings g(·), h(·), G(·), H(·), U (·) are assumed to be autonomous in Propositions 3.3-3.4 and Corollary 3.1, their results can be applied to the non-autonomous case if U (t) ≡ U is autonomous and we treat the time variable t as a state variable. We now illustrate how this can be done. Since 18) it is clear that the OCPEC is equivalent to the following optimal control problem: It is easy to see that (σ(·), x * (·), u * (·)) is a local minimizer of radius R(·) for the above problem if (x * (·), u * (·)) is a local minimizer of radius R(·) for the OCPEC and σ(·) is defined in (3.18). Thus, the results in Propositions 3.3-3.4 and Corollary 3.1 can be applied to the above problem to get the desired result.
We close this section by noting the equivalence of the S-stationarity for the OCPEC and the classical necessary optimality condition for the OCPEC treated as an optimal control problem with mixed inequality constraints (1.8). The proof for the following result is similar to [12,Proposition 4.1] and we omit the proof here. Proposition 3.5 (x * (·), u * (·)) is an FJ type stationary solution of the OCPEC treated as an optimal control problem with mixed inequality constraints (1.8) if and only if (x * (·), u * (·)) is an FJ type S-stationary solution of the OCPEC for which those two sets of multipliers can be chosen as the same.
A simple example
In this section, we consider a simple class of the OCPEC in which the "best" control needs to be chosen from the DCP (1.3) so as to make the final state x(t 1 ) reach some prescribed target T from a given initial state x(t 0 ). Mathematically, the problem considered in this section is where E 0 ⊆ IR n is a closed subset. In this case, S(t) := {(x, u) : (u, Υ(t, x, u)) ∈ C m } and S ǫ,R * (t) is defined as in (2.3). For simplicity, we assume that the functions φ(·), Υ(·) are L measurable in variable t and strictly differentiable in (x, u). Moreover, there exist measurable functions k φ x (·), k φ u (·), k Υ x (·) such that for almost every t ∈ [t 0 , t 1 ], In the following, we apply the derived results in Section 3 to problem (4.1). The following result follows immediately from Proposition 3.2 and Theorems 3.1-3.3. Note that since the final point x(t 1 ) in problem (4.1) is free, λ 0 can be chosen as 1 by Remark 2.1. Moreover, in this case, and since (x * (·), u * (·)) is feasible to problem (4.1), we have Theorem 4.1 Let (x * (·), u * (·)) be a local minimizer of radius R(·) for problem (4.1). Assume that there exists a measurable function κ(·) such that for almost every t ∈ [t 0 , t 1 ], (x, u) ∈ S ǫ,R * (t), µ i = 0 ∀i ∈ I +0 t (x, u), ν i = 0 ∀i ∈ I 0+ t (x, u), µ i > 0, ν i > 0 or µ i ν i = 0 ∀i ∈ I 00 t (x, u), =⇒ (µ, ν) ≤ κ(t) µ + ∇ u Υ(t, x, u)ν . | 2016-04-30T20:01:52.000Z | 2016-04-30T00:00:00.000 | {
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15440347 | pes2o/s2orc | v3-fos-license | Resistance Potential of Bread Wheat Genotypes Against Yellow Rust Disease Under Egyptian Climate
Yellow rust (stripe rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive foliar diseases of wheat in Egypt and worldwide. In order to identify wheat genotypes resistant to yellow rust and develop molecular markers associated with the resistance, fifty F8 recombinant inbred lines (RILs) derived from a cross between resistant and susceptible bread wheat landraces were obtained. Artificial infection of Puccinia striiformis was performed under greenhouse conditions during two growing seasons and relative resistance index (RRI) was calculated. Two Egyptian bread wheat cultivars i.e. Giza-168 (resistant) and Sakha-69 (susceptible) were also evaluated. RRI values of two-year trial showed that 10 RILs responded with RRI value >6 <9 with an average of 7.29, which exceeded the Egyptian bread wheat cultivar Giza-168 (5.58). Thirty three RILs were included among the acceptable range having RRI value >2 <6. However, only 7 RILs showed RRI value <2. Five RILs expressed hypersensitive type of resistance (R) against the pathogen and showed the lowest Average Coefficient of Infection (ACI). Bulked segregant analysis (BSA) with eight simple sequence repeat (SSR), eight sequence-related amplified polymorphism (SRAP) and sixteen random amplified polymorphic DNA (RAPD) markers revealed that three SSR, three SRAP and six RAPD markers were found to be associated with the resistance to yellow rust. However, further molecular analyses would be performed to confirm markers associated with the resistance and suitable for marker-assisted selection. Resistant RILs identified in the study could be efficiently used to improve the resistance to yellow rust in wheat.
Wheat is the most widely cultivated cereal crop in the world. In Egypt, it is considered as the major winter cereal crop and the third major crop in terms of area planted. Severe losses due to different wheat diseases including yellow rust, also known as stripe rust, have been reported (Kissana et al., 2003). Wheat yellow rust is one of the most devastating diseases of wheat worldwide. It is caused by the basidiomycete fungus Puccinia striiformis Westend. f. sp. tritici Eriks (Eriksson, 1894;Hassebrauk, 1965;Stubbs, 1985 andHovmoller et al., 2010) and continues to cause severe damage worldwide (Chen et al., 2013). Biotrophic plant pathogens such as rust pathogens secrete an array of proteins, known as effectors, to modulate plant innate immunity and enable parasitic infection (Hogenhout et al., 2009). Yellow rust is a highly destructive disease threatening wheat production and quality worldwide. This is mainly due to the pathogen's ability to mutate and multiply rapidly as well as to use its air borne dispersal mechanism from one field to another (Brown and Hovmøller, 2002;Watson and De Sousa, 1983).
Slow rusting, a form of quantitative resistance, prolongs the latent period of fungal infection and decreases disease severity (Rashid, 1997;Wang et al., 2000), can slow the incidence and development of stripe rust in the field, thus reducing yield losses and being of practical value. Slow rusting is thought to be a non-race-specific resistance that effectively controls the epidemic spread of stripe rust in a stable, sustained, and durable manner (Das et al., 1992). Among all the control measures of this disease, genetic resistance is the only economic and practical control measure, causing no additional cost to the farmer . Therefore, breeding for resistance to yellow rust Resistance to Yellow Rust in Bread Wheat 403 and developing new resistant cultivars became the main target in wheat breeding programs and considered as the most economical and effective way to eliminate the use of fungicides and reducing crop losses caused by the disease. Afshari (2004) and Singh et al. (2004) reported that a new stripe rust race can attack wheat cultivars which possessing Yr27 resistance gene in India, Yemen, Egypt, Ethiopia, Eritrea, Tajikistan, Uzbekistan and Kyrgyzstan during previous years Thus, it is of great importance to develop wheat cultivars possessing new resistance genes for yellow rust.
A number of genes controlling yellow or stripe rust resistance in wheat has been identified (McIntosh et al., 2011). Moreover, genetic associations of various microsatellite or simple sequence repeats (SSR) and random amplified polymorphic DNA (RAPD) markers with stripe rust resistance genes have been reported in wheat (Akfirat et al., 2010;Bariana et al., 2002;Bariana et al., 2006;Chague et al., 1999;Khlestkina et al., 2007;Robert et al., 2000;Sun et al., 2002;Tabassum 2011;Wang et al., 2002;William et al., 2003;Wang et al., 2008). Identification of molecular markers associated with yellow rust resistance has facilitated the markerassisted selection of the resistance genes in wheat breeding program. Furthermore, to ensure optimal costeffectiveness, molecular markers used for marker-assisted selection should permit efficient screening of large populations (Huang and Röder, 2004). Bulked segregant analysis (BSA) is a highly efficient method developed firstly by Michelmore et al. (1991) for rapidly identifying markers linked to any specific gene or genomic region. The use of BSA in combination with PCR-based markers such as RAPD, SSR and SRAP markers has proven to be a very powerful technique for identifying molecular markers associated with a quantitative trait locus (QTL) or a gene of interest (Avila et al., 2003;Bakhit andAbdel-Fatah, 2013 andEl-Sayed et al., 2013;Cho et al., 1996;Diaz-Ruiz et al., 2010;Nakamura et al., 2001;Rostoks et al., 2002;Shen et al., 2003;Torres et al., 2010).
In Egypt, a large number of wheat landraces have been preserved, which potentially possess many yellow rust resistance genes. Thus, it is of great importance to identify resistance genetic resources from the landraces and use them in wheat breeding programs aiming to develop improved varieties. In the present study, a population of fifty F 8 recombinant inbred lines (RILs) derived from a cross between resistant and susceptible Egyptian bread wheat landraces was used to identify genotypes resistant to yellow rust, and to develop molecular markers associated with the disease resistance.
Materials and Methods
Plant material and greenhouse trials. The plant material utilized in the present study consisted of a population of fifty F 8 recombinant inbred lines (RILs) derived from a cross between resistant and susceptible bread wheat landraces to yellow rust collected from farmers' fields in Upper Egypt in 1993. Two Egyptian bread wheat cultivars i.e. and Sakha-69 (susceptible) were used as controls.
The trials pertaining to screening different genotypes for their resistance against yellow rust were conducted at the greenhouse of Plant Pathology Department, Faculty of Agriculture, Assiut University, Egypt during 2011 and 2012. During this investigation, fifty two entries (fifty RILs and two Egyptian wheat cultivars) were sown at the greenhouse to observe the yellow rust response. A 6 seeds of each entry was planted in a sterilized pot (No.8) containing sterilized soil, supplemented with NPK at the ratio of 1%. Three replicates were made for each genotype. The plants were irrigated when necessary and daily observed for infection.
Collection of yellow rust samples and inoculation.
Diseased leaf samples were collected from different cultivars and breeding lines from three different locations in Assiut Governorate, Egypt, namely Assiut, Manfalout and Abuteeg during 2011 and 2012 wheat growing seasons. Artificial inoculations were carried out using a spores mixture of the most prevalent yellow rust of the field collections in 2011 and 2012. Diseased leaf samples were placed on moist filter paper in a Petri dish that was kept at 10ºC overnight. Urediniospores were collected from diseased leaf samples with the help of small brush. The inoculums were prepared immediately prior to use by suspending urediospores in a solution of diH 2 O and Tween-20, which 1 to 2 drops were added to break the surface tension. Spore suspension was prepared 0.6 ml per plant at concentration of 6 × 10 5 spores/ ml. Spray were apply on the plants on front and back from 6 inch away with one hand behind the plants to catch most of the inoculum on the plant. Inoculations were carried out in the early evening (after sunset).The inoculation of all plants was carried out at booting stage according to the method of Tervet and Cassell (1951).
Disease observation.
Observations were recorded at the first appearance of stripe rust infection on the susceptible wheat lines. Observations on response and severity of stripe rust were recorded according to Loegering (1959) and Hussain (1997). Yellow rust severity (%) was recorded for each cultivar from the time of rust first appearance then every seven days until the early dough stage (Large, 1954). Estimates of severity were measured according to Modified Cobb Scale (Paterson et al., 1948), which is used to determine the percentage of possible tissue rusted and was evaluated from 1% to 100%. The severity was recorded as percent of rust infection on the plants (Fig. 1). As severity is determined by visual observation, readings cannot be absolutely correct. Therefore, below 5% severity, the intervals used are trace (T) to 2. Usually, 5 percent intervals are used from 5 to 20 percent severity and 10 percent intervals for higher readings.
Readings of severity and reaction are recorded together with severity first as follow: TR = Trace severity of resistant type infection 10MR = 10 percent severity of a moderately resistant type infection 30MS = 30 percent severity of a Moderately Susceptible type infection 50S = 50 percent severity of a susceptible type infection Calculation for ACI and RRI. The Coefficient of Infection (CI) for stripe rust has been calculated according to (Akhtar et al., 2002) as shown in Table 1. Coefficient of Infection was calculated by multiplying the response value with the intensity of infection in percent. Average Coefficient of Infection (ACI) was derived from the sum of CI values of each entry divided by the number of tested years.
The highest ACI of a candidate line is set at 100 and all other lines are adjusted accordingly. This gives the Country Average Relative Percentage Attack (CARPA). The '0' to '9' scale previously designated as Resistance Index (R.I) has been re-designated as RRI (Relative Resistance Index). From CARPA, RRI was calculated on a 0 to 9 scale, where 0 denotes most susceptible and 9 denotes highly resistant (Akhtar et al., 2002). The RRI was calculated according to the following formula: Bulked segregant analysis (BSA). In order to identify molecular markers associated with the resistance against yellow rust in specific genomic regions, the F 8 RILs population was subjected to BSA with three molecular marker systems including simple sequence repeat (SSR), sequencerelated amplified polymorphism (SRAP) and random amplified polymorphic DNA (RAPD) markers. The BSA was performed at the Department of Genetics, Faculty of Agriculture, Assiut University in 2013.
Based on RRI value of the two-year trial for each RIL, seven resistant and seven susceptible contrasting genotypes selected from the RILs population were used to construct resistant and susceptible DNA bulks. DNA extraction from young and fresh leaves of each RIL was carried out according to the cetyltrimethylammonium bromide (CTAB) method for isolation of total genomic DNA from plants (Murray and Thompson, 1980) with some modifications. Aliquots of DNA from the two extreme groups of seven resistant and seven susceptible RILs were mixed to produce resistant and susceptible DNA bulks for BSA.
Bulked segregant analysis (BSA).
For identification of molecular markers associated with yellow rust resistance, resistant and susceptible DNA bulks were screened for differences using sixteen 10-mer RAPD primers (Operon, USA), eight SSR and eight SRAP makers.
RAPD markers:
Out of 16 RAPD primers tested, 6 RAPD primers (37.5%) showed polymorphic amplification patterns which distinguished resistance from susceptible bulks (Fig. 6). Individual primers produced bands in a range of 10 (OPC-07) to 17 (OPG-10), with an average of 13 bands per marker. Of the 78 bands amplified with 6 primers, 17 bands (21.8%) were polymorphic (Table 3) with an average of 2.8 polymorphic bands per marker. Polymorphic bands for individual primers ranged from a unique band (9.1%), which was present only in the resistance bulk, with OPC-18 to 7 bands (41.2%) with OPG-10. These bands could be considered as specific markers for yellow rust resistance in wheat.
Discussion
Tolerance to yellow rust is one of the most important objectives of wheat breeding programs in all wheat growing regions of the world (Akfirat et al., 2010). Wheat landraces from diverse geographic regions are a potential source of novel rust resistance genes for developing new and diverse resistant germplasm (Sthapit et al., 2014). A large number of wheat landraces have been preserved in Egypt, which possessed abundantly genetic diversity including many yellow rust resistance genes. Therefore, it is of great importance to identify resistance genetic resources from the landraces to be used in wheat breeding programs aiming to develop improved varieties. In the present study, a popula-tion of fifty F 8 RILs derived from a cross between resistant and susceptible Egyptian bread wheat landraces was evaluated for yellow rust resistance under greenhouse conditions during two growing seasons. RRI values of two-year trial ( hypersensitive type of resistance against the pathogen and 4 RILs (i.e. RIL-27, RIL-28, RIL-44 and RIL-47) had expressed moderately resistant type of reaction. These genotypes were having highest RRI which exceeded the resistant cultivar Giza-168, suggesting that resistant genotypes are expected to possess diverse resistance genes and could be efficiently used as parents to improve resistance to yellow rust in breeding programs. Bulked segregant analysis in combinations with three molecular markers systems revealed that out of eight SSR and eight SRAP and sixteen RAPD markers tested, three SSR, three SRAP and six RAPD markers distinguished resistance from susceptible bulks and were found to be putatively associated with the resistance to yellow rust. Therefore, these markers could be useful as a base for markerassisted selection program aiming to improve yellow rust resistance in wheat. In accordance with these results, genetic associations of various SSR and RAPD markers with stripe or yellow rust resistance genes have been reported in wheat (Akfirat et al., 2010;Bariana et al., 2002;Bariana et al., 2006;Chague et al., 1999;Khlestkina et al., 2007;Robert et al., 2000;Sun et al., 2002;Tabassum, 2011;Wang et al., 2002;Wang et al., 2008;William et al., 2003). When utilizing BSA in segregating populations with minimal gene distortion, the likelihood of falsely identifying linked markers to the target gene is minimized; therefore, fewer individuals are required per bulk (Lin et al., 2006). Thus, BSA can provide fast detection of molecular markers linked to genes of interest. RAPD analysis in combination with BSA has been long used to identify molecular markers linked to genes of interest (Bakhit and Abdel-Fatah, 2013;Chague et al., 1997;Mackay and Caligari, 2000;Lin et al., 2006;Michelmore et al., 1991;Zhang et al., 1994). SRAP is an efficient molecular technique with the marker behaves as codominant and more reproducible than RAPD (Li and Quiros, 2001). However, unlike RAPD and SRAP, SSRs reported firstly in plants by Condit and Hubbel (1991) are PCR-based markers characterized by a high level of polymorphism that permits to discriminate among cultivars and even among closely related wheat breeding lines (Maccaferri et al., 2007;Mantovani et al., 2008). Moreover, SSRs are locus-specific, codominant markers evenly distributed over the genome and require only small amounts of genomic DNA for analysis which are particularly useful for mapping and genetic analysis. A large number of SSR markers are already available for several important agricultural crops including wheat (Gupta and Varshney, 2000;Mantovani et al., 2008;Röder et al., 1998;Somers et al., 2004;Sourdille et al., 2004;Wang et al., 2007). The 2013 Catalogue of Gene Symbols for Wheat (McIntosh et al., 2013) and the 2013-2014 Supplement include 67 officially named Yr genes (Yr1 to Yr67) designated for the resistance to stripe rust and 42 with temporary Yr designations. Furthermore, over 140 QTLs for resistance to yellow rust in wheat have been published and through map ping flanking markers on consensus maps, 49 chromo somal regions are identified (Rosewarne et al., 2013). In the present study, we identified three SSR markers associated with the resistance to yellow rust in bread wheat; namely Xgwm339, Xgwm493 and Xwmc398 located on chromosomes 2AS, 3BS and 6BS, respectively. Accordingly, many of previously reported Yr genes, QTLs and SSR markers associated with the resistance to yellow rust in wheat were located on chromosomes 2A, 3B and 6B (McIntosh et al., 2013;Rosewarne et al., 2013). Chromosome 2A has one region associated with the resistance on the short arm (2AS) and another region on the long arm (2AL). The region around the 2AS QTL (QRYr2A.1) are associated with the major, race-specific seedling resistance gene Yr17 (Rosewarne et al., 2013), while a major QTL for adult-plant resistance (QYr.osu-2A) was located on chromosome 2AS by Fang et al., 2011. The 3B chromosome appears to have at least three regions associated with stripe rust resistance. The majority of QTLs identified on 3B are on the short arm (3BS) and it is interesting that Xgwm493 was mapped into QRYr3B.1 region on 3BS which is known to be extremely important as it is the location of Yr30 gene, and it has a consistent intermediate effect on stripe rust and is fairly consistent across environments (Rosewarne et al., 2013). The temporarily designated gene Yrns-B1 on 3BS, which identified as non-race specific adult-plant resistance gene against stripe rust, was located in a 3 cM interval between Xgwm493-3B and Xgwm1329-3B (McIntosh et al., 2013). Recently, Zhou et al., 2014 reported that, the recessive gene Yrwh2 is located on chromosome 3BS and it is different from previously reported stripe rust resistance genes Yr30, QYr.ucw-3BS, Yrns-B1, YrRub and QYrex.wgp-3BL previously mapped on chromosome 3B. They also suggested that Yrwh2 and its closely linked markers are potentially useful for developing stripe rust resistance wheat cultivars if used in combination with other genes. Lowe et al., 2011 reported that the gene underlying QYr.ucw-3BS appears to be different from Yr30 and Yrns-B1 genes. The QYrucw.3BS was mapped 3.6 cM distal of Xgwm493 by Lowe et al., 2011, whereas the adult plant resistance gene Yrns-B1 was mapped 2.5 cM proximal of Xgwm493 (Borner et al., 2000;Khlestkina et al., 2007). However, Zhou et al., 2014 could not determine the distance between Yrwh2 and Xgwm493 as the marker was not polymorphic. Similarly, chromosome 6A had three clearly defined regions associ ated with stripe rust resistance and these are likely to be conferred by three distinct genes (Rosewarne et al., 2013). The first region (QRYr6B.1) is at the telomere of 6AS. Generally, molecular markers tightly linked to target genes can provide a useful tool in plant breeding since they can used to detect tolerant genes of interest without the need of carrying out field evaluations. Moreover, the use of molecular markers can increase the efficiency of conventional plant breeding by identifying markers linked to the trait of interest which are difficult to evaluate and/ or are largely affected by the environment. Screening a large number of breeding materials or populations at early growth stages and in a short time could be also performed using molecular markers. Therefore, it is urgent to identify new genes for resistance to yellow rust and to develop molecular markers for efficient incorporation and pyramiding of new genes into wheat cultivars (Zhou et al., 2014). Consequently, SSR markers identified in the present study (i.e. Xgwm339-2AS, Xgwm493-3BS and Xwmc398-6BS) which showed the highest polymorphism (43.6%) than SRAPs (36.4%) and RAPDs (21.8%) could be efficiently used for marker-assisted selection of yellow rust resistance in wheat breeding programs. Moreover, it is likely that resistant RILs identified in the present study contain potentially some adult plant resistance genes. For example, the non-race specific adult-plant resistance gene (Yrns-B1) which was identified previously as located on 3BS and linked to Xgwm493-3BS could be present within the resistant RILs. Furthermore, some seedling and/or adult plant resistance genes on 2AS and 6BS may also be present. In addition, diseased leaf samples were collected from different cultivars and breeding lines across different locations, thereby there is a possibility of the presence of more than one pathotype. Therefore, it is likely that interaction between disease resistance genes could also be present. Subsequently, resistant RILs identified in the current study could be efficiently used to develop improved varieties in wheat breeding programs. However, further molecular analyses of the whole RIL population with co-segregating markers would be performed to confirm markers associated with the resistance and suitable for marker-assisted selection.
In conclusion, resistant RILs identified in the present study are expected to possess diverse resistance genes and could be efficiently used as parents to improve resistance to yellow rust in wheat breeding programs. Molecular markers identified in the study using BSA, once verified by genotyping the whole RILs population, could allow implementation of marker-assisted selection for incorporating desirable resistant genotypes in wheat breeding programs. Furthermore, SSRs are likely to be the most effective markers used in the study. | 2016-05-12T22:15:10.714Z | 2015-12-01T00:00:00.000 | {
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248831709 | pes2o/s2orc | v3-fos-license | Garlic (Allium sativum) feature-specific nutrient dosage based on using machine learning models
Brazil presents large yield gaps in garlic crops partly due to nutrient mismanagement at local scale. Machine learning (ML) provides powerful tools to handle numerous combinations of yield-impacting factors that help reducing the number of assumptions about nutrient management. The aim of the current study is to customize fertilizer recommendations to reach high garlic marketable yield at local scale in a pilot study. Thus, collected 15 nitrogen (N), 24 phosphorus (P), and 27 potassium (K) field experiments conducted during the 2015 to 2017 period in Santa Catarina state, Brazil. In addition, 61 growers’ observational data were collected in the same region in 2018 and 2019. The data set was split into 979 experimental and observational data for model calibration and into 45 experimental data (2016) to test ML models and compare the results to state recommendations. Random Forest (RF) was the most accurate ML to predict marketable yield after cropping system (cultivar, preceding crops), climatic indices, soil test and fertilization were included features as predictor (R2 = 0.886). Random Forest remained the most accurate ML model (R2 = 0.882) after excluding cultivar and climatic features from the prediction-making process. The model suggested the application of 200 kg N ha-1 to reach maximum marketable yield in a test site in comparison to the 300 kg N ha-1 set as state recommendation. P and K fertilization also seemed to be excessive, and it highlights the great potential to reduce production costs and environmental footprint without agronomic loss. Garlic root colonization by arbuscular mycorrhizal fungi likely contributed to P and K uptake. Well-documented data sets and machine learning models could support technology transfer, reduce costs with fertilizers and yield gaps, and sustain the Brazilian garlic production.
Introduction who allowed setting up the experiments and collecting samples. The meeting was organized by Santa Catarina Association of Garlic Producers (ACAPA); local producers willing to collaborate with the research volunteered to it and also authorized the experiments. All producers participating in the research are members of ACAPA, which is the local authority that approved the research.
Climate in the investigated region is temperate and humid, with mild Summer; according to Köppen's classification, it is classified as Cfb [40]. Landscape was moderately flat to slightly undulated. Soils were classified as Typic Hapludox [41]. Soil cation exchange capacity (CEC) at soil surface (0-20 cm) ranged from 13 to 23 cmol c kg -1 . Base saturation of CEC, computed as the sum of exchangeable cations, divided by CEC, ranged from 20% and 94%.
Fertilizer trials were conducted with nitrogen (N), phosphorus (P) and potassium (K). There were 34 fertilizer trials in 2015 (5 N, 14 P and 15 K); 28, in 2016 (6 N, 10 P and 12 K); and 4, in 2017 (4 N) ( Table 1). There were three additional observational 'Chonan' sites located in one farm in 2018, and in two farms in 2019. There were 1,023 observations, in total, including data about the 61 growers, which were collected in 2018-2019. History of preceding crops and fertilization gathered during the 1,023 observations are presented in S1 Table in S1 File.
In order to compare data sets, ML and broken curve regression models were run across 78 experiments in USA to measure corn (Zea Mays) response to P addition [42] and across 370 experiments in Canada [35] to model potato (Solanum tuberosum) response to fertilization (273 field trials, 5,913 lines). A study with 198 fertilizer experiments carried out in Midwest US modeled N response in corn [32]. Data from 1,504 experimental wild blueberry data were collected for 11 years [38]. The size of other data sets to run ML models ranged from 60-240 to nearly 8,000 observations-smaller numbers correspond to more difficulties and higher costs with data gathering [32,[42][43][44][45][46][47][48][49][50].
Experimental setups followed the randomized block design with three repetitions. Fertilizers were broadcast-applied and incorporated into the 0.20 m layer with the aid of a rotary tiller. The experimental sites received five N doses (0, 100, 200, 300 or 400 kg N ha -1 ) as ammonium nitrate, 1/3 of them were applied in the rows at planting time; 1/3 was used as top-dressing, 30 days after planting; and 1/3 was used as top-dressing at bulb formation time (approximately 95 days after planting, when plants differentiated into bulbs). Five (5) P doses in the form of triple superphosphate were applied on the soil, as well as 5 K doses in the form of potassium chloride -all doses were applied before planting. Sites presenting N variation were subjected to P and K application at equal doses of 175 kg P ha -1 and 333 kg K ha -1 , respectively. Sites presenting P [14]. They were weighed after *40 days of natural drying. Marketable bulbs corresponded to the sum of #2 (< 32 mm ;), #3 (32-37 mm ;), #4 (37-42 mm ;), #5 (42-47 mm ;), # 6 (47-56 mm ;) and #7 (> 56 mm ;) bulbs, which were classified according to [51]. Bulbs showing secondary growth, or damage, were considered nonmarketable. Maximum yield ranged from 7.8 and 13.3 Mg ha -1 over the years; it was comparable to yield of 9.2-13.4 Mg ha -1 -which was recorded by [52] in the same region.
Soil analyses
Eight (8) soil subsamples were collected per plot in the 0-0.20 m soil layer, at harvest time. Soil samples were air-dried and ground to lesser than 2-mm in size, before chemical analyses were performed based on [53], as follows: pH 1:1 soil:water volumetric ratio; clay, based on the densimeter method; Mehlich-1 extraction method for P, K, Cu, Zn, and Fe; KCl method for Mn; and hot-water extraction for B. Elements were quantified through plasma-emission spectroscopy (ICP-OES). Total carbon was quantified based on dichromate oxidation (Walkley-Black) and multiplied by 1.724 to find the organic matter content [54]. Clay content was determined based on sedimentation. Cation exchange capacity was computed as the sum of exchangeable cations and total acidity (SMP buffer pH). Values recorded during soil P and K tests applied to the control treatment were selected for modeling purposes. The Brazilian fertilizer recommendation system [55] adjusts P and K recommendations to soil clay content (P) and to cation exchange capacity (K). Variation in soil properties in experimental sites is shown in S2 Table in S1 File.
Climatic indices
Annual crops' performance in comparison to that of the perennial ones is little affected by offseason climatic conditions. Only adverse temperatures, or extreme meteorological events, such as the excessive rainfall recorded in 2015, can have impact on crop yield, in places subjected to irrigation. The following meteorological data were collected in the closest [56] meteorological station to study area: daily minimum and maximum temperatures, daily rainfall between planting and harvest time, and growing season duration. All experiments were located within the radius of 50 km away. The meteorological station used for data collection was located within this radius. The yearly distribution of experimental and observational data in the Brazilian garlic data set is shown in S3 Table in S1 File.
Plantation dates went from May 7 th to July 26 th, depending on site and year. Minimum, median and maximum Julian days for plantation were 152, 180, and 238, respectively. Harvest dates went from November 3 rd to December 5 th . Minimum, median and maximum Julian days for harvest were 306, 322, and 342, respectively. Growing period duration was computed based on the difference between Julian day at planting and Julian day at harvest. Minimum, median and maximum growing days were 102, 143, and 162, respectively. Heat accumulation was measured as cumulated degree-days above critical temperature during the growing season. Critical temperature for cold crops was 5˚C [57].
Statistical analyses
Traditional models. Crop yield was associated with N dose in corn or soybean crops used as preceding crops. Crop response to N fertilization could be low when N credits are high preceding crops (soybean). Soil organic matter also provides N credits [55].
RY of P and K was calculated at each experimental site as %Yield = 100 × (Y control /Y maximum ), wherein Y control is yield in control treatment zero and Y maximum is maximum yield [19]. Numbers of points are maximized in opposite quadrants in order to compute classification accuracy. Critical soil test separates high from low crop response to P or K addition. Machine learning models. Machine learning models can predict phenomenological outcomes from key features [58]. Random Forest, k-nearest neighbors (KNN), support vector machine (SVM), gradient boosting, stochastic gradient descent (SGD), Adaboost, linear regression and Neural Networks were the ML models available in Orange data mining freeware vs. 3.23. Decision trees were commonly used in plant nutrition studies [35,39].
Marketable yield, the logistic partition between marketable and non-marketable yield were the target variables. The logistic partition was computed as ln(Yield marketable /Yeld rejected ). Predictor features were set as follows: N, P and K dosage, soil tests, cultivar, cropping system (preceding crop, seeding or planting), and climatic indices (growing season duration, cumulated degree-days, total seasonal rainfall).
Models were calibrated through stratified cross-validation (k = 10). The data set was divided into 979 experimental and observational data for calibration, and into 45 experimental data (2016) to test ML models and compare results to 'universal' state recommendations. The training data set excluded one P trial in 2016, one K trial in 2016, and one N trial in 2017, in cultivar 'Ito', which was used as testing data set. The most accurate ML regression models were selected by using the training data set. Accuracy was measured as root mean-square error (RMSE), mean absolute error (MAE) and R 2 statistics. Predictors contribution to model accuracy was assessed by removing predictors and examining any change in model accuracy. Excluded trials were used for model testing. Garlic response predicted by the models were compared to the observed response.
Traditional crop-response models
The association between garlic yield and N dosage in the experimental data set was different between corn and soybean (Fig 1). Maximum marketable garlic yield reached 20 Mg ha -1 after corn in comparison to 9 Mg ha -1 after soybean. However, soybean was only used a preceding crop in 2015, when climatic conditions were adverse due to excessive rainfall. The association between RY and soil test P showed classification accuracy of 0.708. Critical soil test P was �14 mg P dm -3 (Fig 1); this number is close to the separation between low and medium soil test P (CQFS RS/SC, 2016). The association between RY and soil test K was partitioned at �160 mg K dm -3 , at classification accuracy of 0.815 (Fig 1); this number was close to the separation values (135-180 mg K dm -3 ) between medium and high soil test K.
Machine learning models
Random Forest, Gradient Boosting and Adaboost were the most performing ML regression models ( Table 2). Because climate can be hardly used in predictions, and cultivar effect was negligible (data not shown), such features can be excluded to make predictions. Still, R 2 values reached 0.832-0.882 when the top three ML models were used (e.g., Fig 2). Coefficient of variation (RMSE divided by mean) was 0.19 across the top ML models, for mean garlic yield of 7.3 ton ha -1 . Random Forest also showed the highest model accuracy for the logistic partition between marketable and non-marketable yield (RMSE = 0.792; MAE = 0.615; R 2 = 0.787).
Fertilization treatments' contribution to model accuracy was tested by fully, or sequentially, removing N, P and K fertilization from Model 2. R 2 of Random Forest dropped to 0.797 after all fertilization treatments were removed from it. Nitrogen (N) was the most yield-impacting nutrient. R 2 of Random Forest dropped from 0.882 to 0.812 after N fertilization effect was removed from the model. R 2 values of Random Forest were little changed after P (0.873) or K (0.881) treatments were removed from the model, and this finding indicates the small effects of P and K fertilization on garlic yield.
Model evaluation
Garlic crops' response to N, P and K fertilization treatments were predicted at three sites in 2016 and 2017 by using the calibration dataset. Models presented non-linear trajectory for N and stationary trajectories for P and K (Fig 3). Random Forest Model 2 predictions for garlic yield response to N addition were close to the observed yield. Based on model predictions for N, the dosage of 200 kg N ha -1 would be enough to maximize marketable yield across sites. The model did not predict response to K or P addition, despite apparent observed response, at 250 kg P 2 O 5 ha -1 .
Early regional guidelines
The 'universal' state recommendations based on preceding crops and soil organic matter for N, as well as the association between RY and soil test P and K [55], proved to change in ML predictions. Nutrient recovery coefficients must vary due to water supply, soil test level, root competition in constrained rooting volume, and fertilizer dosing and placement [2,59,60]. The study by [26] challenged the belief that tropical soils' ability to supply P mostly depends on crop recovery coefficients. Trials in tomato fields conducted in São Paulo State [26] showed that irrigated tomato plants responded much less to P addition than the predictions for it, which were based on crop removal and on an assumed P recovery coefficient. There was great potential to reduce costs with fertilization obtained with predictive models, without agronomic loss. Nevertheless, it is safe to compare the model's predictions to state recommendations, or grower's predictions to secure knowledge transfer.
Model accuracy
Total N offtake ranged from 108 and 292 kg N ha -1 (avg. 207 kg N ha -1 ), P offtake ranged from 17 to 37 kg P ha -1 (avg. 29 kg P ha -1 ), and K offtake ranged from 67 to 167 kg K ha -1 (avg. 123 kg K ha -1 ) for garlic yields ranging from 9.1 to 24.2 Mg ton ha -1 (avg. 17.3 ton bulb ha -1 ) [6]. Mean garlic offtake was 12 kg N ton -1 , 1.7 kg P ton -1 and 7.1 kg K ton -1 . Nitrogen (N) requirements were found to be much lower (�200 kg N ha -1 ) in case study (Fig 3), than the 'universal' state recommendations of 300 kg N ha -1 [55]. For yield of 13 ton ha -1 , crop N of take would be 156 kg N ha -1 (Fig 3). The chosen response curve, and range and number of doses, may also have impact on the optimum nutrient dosage [24,25,61]. Results presented by [52] showed total and marketable garlic yield non-linear response pattern under greenhouse conditions-it reached 320 kg N ha -1 at yield level of 9 Mg ha -1 . Maximum marketable yield of 12.9-15.8 Mg ha -1 , at 200 kg N ha -1 , was recorded under field conditions in Mid-Eastern Santa Catarina State, where soil organic matter ranges from 3.9% to 4.9%, [14]. Mean fertilizer dosage of 222±30 kg N ha -1 was recorded in garlic farms, in Minas Gerais State, Brazil [8]. Nitrogen (N) applied by growers (2018-2019) ranged from 130 to 180 kg N ha -1 in the present collaborative study, and this finding reflects growers' caution about the potential negative effects of N over-fertilization on crop quality. Optimum N dosage could be reassessed at local scale by testing N treatments close to optimum experimentally derived N [24,25]. This finding emphasizes the need of customizing fertilizer recommendations in order to reach factor-specific conditions, at local scale, save money to the grower, improve crop quality and reduce environmental footprint.
There is little information on garlic response to P and K addition under field conditions, in Brazil. Phosphorus (P) placement can increase garlic yield in low-P soils [60]. Significant yield difference between zero and 200 kg P 2 O 5 ha -1 was reported in low-P soils (4.3 mg P-Mehlich-1 dm -3 ) recording total bulb yield of 11-14 Mg ha -1 [62]. On the other hand, a small yield response to 100-150 kg K 2 O ha -1 was reported by [63], when total yield levels were low (3.5 to 7 Mg ha -1 ) in high-K soil (47 mg K dm -3 ). Mean fertilizer dosage of 371±39 kg P ha -1 , and 417 ± 62 kg K ha -1 in garlic farms, was recorded in Minas Gerais State, Brazil [8]. Growers (2018-2019) followed state recommendations for P and K, or over-fertilized soil-already showing very high test values. Hence, every grower made different decisions based on their own knowledge and perception about local conditions.
On the other hand, based on Random Forest results, it is recommended to significantly reduce N, P and K doses. Garlic can successfully grow even in low-P soils due to root colonization by arbuscular mycorrhizal fungi (AMF) [64,65]. Plant Growth Promoting Rhizobacteria (PGPR) can also improve N and P supply to garlic in Southern Brazil soils [27]. Disregarding soil biological quality effects can have great impact on N and P prescriptions only based on soil test N and P, alone. Based on the "Biological Buffering Capacity-BBC", crop yield and N uptake are closely linked to soil/crop interactions affected by the weather during the growing season, since plants are the path to transfer weather effects to changes in soil/crop interactions" [32]. However, BBC can be hardly measured under all combinations of factors and environmental conditions. Very wet, dry or favorable climatic conditions were recorded during the 3-year experiment. Nonetheless, the data set did not cross over the numerous combinations of factors influencing garlic yield and quality in Southern Brazil. Acquiring more observational and experimental data from a larger number of predictors combinations, in collaboration with growers, could help updating ML models and improving nutrient management predictions Experimental and observational data can be turned into large datasets in order to support decision-making based on combining research results, growers' results and knowledge, as well as to feed and run machine learning models to reach high crop yield based on the numerous combinations of site-specific factors [37].
Conclusion
Based on the pilot study, ML models can be more accurate than state recommendations at local scale, as shown by the three case studies. Predictions have shown that growers can significantly reduce nutrient applications and yet reach potential marketable yield. However, ML models remain sensitive to the tested number of doses and to undocumented features in the data set, such as biological quality indices. Nevertheless, learners have the potential to support trustful technology transfer from research to the field, as well as to contribute to sustain the Brazilian garlic production. Customized fertilizer recommendations to site-specific conditions are promising tools to reach (i) high yield with economic viability; (ii) adequate plant mineral nutrition, (iii) high fertilizer-use efficiency. These achievements are possible by sharing larger and more diversified data sets among stakeholders. | 2022-05-18T06:23:39.514Z | 2022-05-17T00:00:00.000 | {
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213433716 | pes2o/s2orc | v3-fos-license | Increasing the efficiency of vibration treatment of complex surfaces
The article is devoted to improving the quality and reliability of high-loaded parts that ensure the service life of responsible products. It is recommended to apply vibro-impact hardening treatment in combination with nano-coatings of working tool elements. It is revealed that calculations based on classical ideas about the elastic properties of coatings of materials lead to overestimated values of allowable tangential stresses on the treated surface. According to the results of an experimental study of the process, the interrelation of the parameters of the vibro-impact and possible instrument defects that have a direct impact on the surface quality of the workpiece was established. The analysis of the effect of various nanocoatings of working elements on the surface quality of a complex configuration is given. It has been established that when using epilams the number of defects in the instrument decreases sharply.
Introduction
Improving the quality, reliability and durability of aviation equipment, shipbuilding, power plants for many decades and now is an important economic problem. This problem has become particularly relevant in connection with the creation of new generations of helicopters, airplanes, ships, engines and heightened competition in the global market. The designs of these types of products include groups of high-loaded parts, the reliability and durability of which greatly determine the service life and reliability of the entire product. A significant amount of this kind of parts has a complex shape, large size, limited stiffness and high demands on the quality parameters of the surface and surface layer.
These parts, depending on the type of product and its purpose, have different structural shapes and sizes, are made, as a rule, from structural alloy steels or aluminum alloys.
High requirements are imposed on the surface quality due to harsh operating conditions -high level of alternating loads, high speed and contact loads, fluctuations in the temperature gradient, corrosion and erosion processes. In this regard, the surface roughness is limited -Ra = 0.5 ÷ 1.25 μm, the structure and direction of the microrelief is specified; the surface layer is subjected to hardening treatment by surface plastic deformation (PPD). The noted features require an unconventional approach to solving technological problems of hardening processing: the development and improvement of vibro-impact methods, new technological schemes for vibro-impact processing of parts, the use of nano-coatings of working parts of tool surfaces [1,2].
Theory
Epilation implies the formation of multimolecular layers with regularly oriented molecules on the surface of a solid. The degree of orientation and thickness of the oriented layer depend on the nature of the solid and the surfactant molecules. Within the homologous series of the deposition of molecules on the surface, the degree of orientation increases with increasing length of the epilam molecules.
The surface of the metal base of the product has an orienting effect due to the presence of a nearsurface electric field with intensity м / V 10 E 9 -for each specific material this value can be estimated by physical methods. When the coating material is connected with the base its atoms or molecules continue the atomic lattice of the base. This is equivalent to the fact that the same atoms are rotated by the orienting field of the base material distributed along the contact surface at a certain angle ω, while doing work against the internal forces of the coating material. Because of this, in this material, even in the absence of external influences in the surface layer, a stress state is developed, a certain energy ∆ of elastic deformations is concentrated. For these reasons, the expression is [3]: Then, when estimating the allowable tangential stress max τ , the inequality is true: This inequality suggests that a calculation based on the classical concepts of the elastic properties of coatings of materials leads to overestimated values of permissible tangential stresses.
To estimate the values µ and ∆ it is proposed to use the micropolar theory of elasticity. It is based on the idea that a particle of a continuous elastic medium has not three, as in the classical case, but six degrees of freedom. Accordingly, to describe its movement, the displacement vector ( ) r u u = and the vector of small rotation angle ( ) r ω are used ( r -radius is the center of mass of the particle's vector) In the case of a plane shift, which is interested when considering the problem raised in this paper, the displacement and micro-rotation vectors take the form: In this case, the axis x O x . As a result, only the following values are considered: The equilibrium equations in stresses are in the general case of the form: In this flat case of pure shear on the basis of (5), writing the second equation in displacements, we can get: The boundary conditions for them are the relations: Taking into account the first of the boundary conditions (7), the first equation of system (6) suggests that, as with the classical model of a linear elasticity, in the framework of the considered model, the tangential stress shifting the coating is uniformly distributed over its thickness h effective shear stress on the surface.
Comparing it with the left side of equality (3), we can verify that It can be seen that the classical estimate of the adhesion energy is less compared to the one that can be made by equating (9) to the value a F . Obviously, for sufficiently large values of α and ε + γ classical estimates max τ can not be used. In the overall assessment machining it is a complex set of mechanical, physical and chemical phenomena that have a significant effect on the state, first of all, of the surface and the surface layer of the workpiece and also technological system. The physical essence of the process is characterized by a complex effect on the workpieces and their surface by a number of factors caused by vibration and by the presence of the working environment of a corresponding characteristic.
The main effect of applying a thin film of epilam on the working surface of the tool is to hold the oil in the contact zone, reduce the friction coefficient, and maintain its long-term performance. An additional advantage of working with an epilaminated tool is the adsorption decrease in the strength of processed material, an increase in the plasticizing effect of the cutting fluid, and a decrease in the deformation forces due to the Rebinder effect.
To assess the effect of epilams on tool life and conduct experimental studies under equal conditions, the 5-row separator was conventionally divided into five zones. The number (balls) of the instrumental environment in each track was 38 pieces: Industrial oil was used as a cutting fluid. The force of separation was measured using a dynamometer.
The reverse movement of the tool was carried out using a special control unit mounted on the power panel of the machine.
For tracking the process of machining, a stroboscope was used. For a comparative assessment of the wear of the tool, measurements of the holes in the tool on the amount of departure of the tool ball relative to the surface of the separator before and after the experimental studies were carried out. Measurements were made five times for each hole with a special device according to divided epilamination zones.
An analysis of the experimental data showed that the greatest wear of the tool was observed in the zone without treatment with epilating compositions. A positive effect on the amount of wear of the tool cage was observed if the balls were treated with epilams. The greatest effect on resistance was achieved when balls and the tool were treated. The ordinate axis is the size of the ball imprint on the holder (d, mm), the abscissa is the position of the ball in the tool. The tests were carried out for 60 hours.
The epilam coating in this case plays the role of boundary cutting fluid and it contributes to the retention of oil in the area of shock loads. At the same time, the technological efficiency of the oil in the area of shock loads is realized through its functional actions [4].
Because of wear, the sizes of the balls and the tool change, the gaps between the rubbing surfaces increase, and all these are cause beating and knocking.
The film on the working surfaces contributes to the penetration and retention of oil in the contact zone. As a result of the violation of the integrity of the epilam coating, the formation of energetic barrier sites which prevents the removal of oil from the friction zone, it helps to reduce friction and adhesion forces in the contact zone [5,6].
From the analysis of experimental data can distinguish several types of wear.
-fatigue failure and chipping of the surface; -minor scratches; The presence of the film on the working surfaces of the balls and the tool contributes to the penetration and retention of oil in the contact zone. As a result of violation of the integrity of the epilam coating, energy barrier sections are formed that prevent oil from being removed from the friction zone, which helps to reduce friction and adhesion forces in the contact zone, while paragraph 3 of the technological efficiency of the oil in the shock load zone is realized through its functional actions.
The smallest wear of the tool's surface was observed when epilating the balls and the separator surface with epilame and EFREN -2.
It should be noted a general decrease in the diametral size of the scatter of the holes of the separator by 1.2 times, which associated with a decrease in the roughness of the friction surfaces.
Conclusions
1. It was established that the introduction of additional parameters in the methods of surface plastic deformation is accompanied by the expansion of their capabilities -increased productivity and improved properties of the surface layer of the workpiece. It is shown that the combination of cutting fluids and surface-active substances provides favorable conditions for the operation of the tool. It is shown that the operation of the tool treated by epilama without any cutting fluids is undesirable, in this case, there is a rapid abrasion of epilame nanofilms.
2. It was established that the calculations of tangential stresses arising in the "tool -epilam" boundary zones, made on the basis of classical concepts, lead to overestimated values. It is proposed to apply the provisions of the micromolecular theory of elasticity, which allow predicting the resistance of surface -active coatings the most reliably. A mathematical model of impact at large angles of effect with a deformable body is given. | 2020-01-09T09:22:31.841Z | 2020-01-03T00:00:00.000 | {
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56085617 | pes2o/s2orc | v3-fos-license | Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells
R. R. Panchuk, L. V. Lehka, J. Rohr © 2016 R. R. Panchuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.22 + 577.181.5 Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells
Introduction
Despite advancement of modern pharmacology in the development of efficient anticancer drugs, the overall ratio of successful results of the metastatic cancer chemotherapy still remains rather low.Compared to the golden chemotherapy standards (anthracyclines and vinca alkaloids), developed in early 1960s, over the last decade the specificity of modern targeted medi-cines became markedly higher and their side effects significantly diminished.However, the average survival time of patients with disseminated cancer did not considerably increase] [1].
Such ineffectiveness of cancer chemotherapy is explained by the rapid development of acquired multi-drug resistance (MDR) of tumor cells to the anticancer drugs, which is found in 30-50% of cancer patients already in a year after the treatment ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 3. P 190-202 doi: http://dx.doi.org/10.7124/bc.000920 start [2].The MDR phenotype is characterized by the simultaneous resistance of cancer cells to the pharmacologically and structurally diverse drugs [3].
It could be a cause of the poor prognosis and lower survival rate in several types of cancer such as gastric cancer, breast cancer, ovarian cancer, pancreatic cancer, and leukemia [4,5] MDR is related to an increased production of the ATP-Binding Cassette (ABC) transport proteins.the membrane-bound proteins that extrude a variety of anticancer drugs from the tumor cells using ATP hydrolysis.Until now, 48 human ABC genes have been identified and classified into seven subfamilies based on the sequence homology and domain structure [1].In addition to their physiological expression in normal tissues, these proteins were found to be over-expressed in the human tumors, which significantly lowers their response to the chemotherapy treatment.Although all types of ABC transporters are involved in drug resistance, most studies on the ABC transporter protein in cancer have focused on three proteins: P-gp (p-glycoprotein), MRP-1 (multidrug-resistance associated protein), and BCRP (breast cancer resistance protein).Each of the above drug transporters is specific to a certain type of substrates.In particular, P-glycoprotein is responsible for resistance to colchicine, dexamethasone, doxorubicin, vinblastine, and etoposide, whereas the over-expression of MRP-1 protein results in resistance to vincristine and doxorubicin [6,7].In turn, bcrp, besides resistance to methotrexate and mitoxantrone, is also responsible for the cancer cell resistance to targeted chemotherapies, such as imatinib and lapatinib, which makes it especially important in modern cancer chemotherapy [8].
Thus, the development of novel anticancer drugs, capable of circumventing the cancer drug resistance in different ways, which could not be inhibited by any of known drug resistance mechanisms, remains an important task of the current pharmacology and medicine.Here, one of promising approaches is searching for natural compounds, the anticancer activities of which could not be affected by the ABC-transporter proteins.Landomycins are a novel group of angucycline antibiotics possessing a strong antineoplastic potential.All natural landomycins identified to date, share the same aglycon (landomycinone) group and vary only in their oligosaccharide chain, a linear glycosidic chain containing only di-and trideoxysugars (β-D-olivose and α-L-rhodinose [9].They show a broad activity against many cancer cell lines, with the general tendency that the compounds with longer saccharide chains possess a higher antitumor potential [10,11].
Previously, we studied the cellular and molecular mechanisms of antitumor activity of one landomycin family member -landomycin E, possessing three sugar residues in its saccharide chain.It was shown [12,13] that this drug could overcome the multidrug resistance of cancer cells to chemotherapy treatment due to the over-expression of ABC-transporters.It wasalso shown by us, that the anticancer activity of another member of this family -landomycin A -is accompanied by massive ROS burst at early timepoints of its action, but the molecular mechanisms of this phenomenon still remain unclear [14].
The main aim of the current study was to investigate the ROS profile in HL-60 leukemia cells and the drug-resistant HL-60/adr and HL-60/vinc sublines under LE treatment in vitro, and to evaluate a possible role of mitochondria in circumvention of cancer drug resistance by LE using specific mitochondrial respiratory chain inhibitors.A well-known anticancer drug doxorubicin was used for the comparison .
Materials
LE-overproducing Streptomyces globisporus 1912 strain was obtained in the laboratory of Prof. B. Matselyukh (D.K. Zabolotny Institute of Micro biology and Virology, National Academy of Sciences of Ukraine, Kyiv).LE (99.5% purity, according to HPLC data) was prepared in the laboratory of Prof. J. Rohr (University of Kentucky, USA) and dissolved in absolute ethanol to obtain a 4 mg/ml stock solution.Doxorubicin hydrochloride was obtained from Pfizer (New York, NY).Diphenylene Iodonium (DPI), antimycin A (AMA), oligomycin A (OM), N-acetylcysteine (NAC) and sodium pyruvate were purchased from Sigma-Aldrich (St. Louis, MO).
Cell culture and treatments
Human leukemia cells of HL-60 line, its drug-resistant sublines HL-60/adr (MRP-1 overexpression) and HL-60/vinc (P-glycoprotein overexpression) were obtained from the cell culture collections at Vienna Medical University, Institute of Cancer Research, from ATCC.Cells were cultured in RPMI medium, supplemented with 10% fetal calf serum (Sigma-Aldrich), 50 µg/ml streptomycin (Sigma-Aldrich), 50 units/ml penicillin (Sigma-Aldrich) in 5% CO 2 -containing humidified atmosphere at 37 °C.For experiments[, the] cells were seeded into 24-well tissue culture plates (Greiner Bio-one, Germany).Short-term (24h) cytotoxic effect of antitumor drugs was studied under the Evolution 300 Trino microscope (Delta Optical, Poland) after cell staining with trypan blue dye (0.1%).DPI (3 mM stock solution) was dissolved in DMSO, while antimycin A and oligomycin A were dissolved in absolute ethanol to obtain 2 mM stock solutions.NAC (0.5 M) and D-mannitol (10 M) were dissolved in 1x phosphate buffered saline (PBS).Modulators were added to cell culture 30 min before addition of anticancer drugs, and final concentration of NAC was 1 mM, sodium pyruvate 10 mM, DPI and Oligomycin A -4 µM.Stock solutions of aforementioned reagents were dissolved in PBS before addition to cell culture.
For long-term (72h) cytotoxicity assays Jurkat cells were plated (5x10 3 ) in 100 µl per well in 96well plates, and allowed to grow for 24 h.Drugs were added in another 100 µl culture medium (final concentration of DPI -0.5 µM, oligomycin A -1 µM, antimycin A -2 µM, sodium pyruvate -10 mM, NAC -1 mM) and cells were exposed for 72 h.The proportion of viable cells was determined by EZ4U assay according to the manufacturer's recommendations (EZ4U, Biomedica, Vienna, Austria).Cytotoxicity was expressed as IC 50 values calculated from full dose-response curves (drug concentration including 50% reduction in cell survival comparing to the control cultured in parallel without drug).
Cellular ROS contents were measured by incubating the control or drug-treated cells with fluorescent dye dihydrodichlorofluoresceindiacetate (H 2 DCFDA, H 2 O 2 -specific) in concentration 10 μM of H 2 DCFDA at 37 °C for 30 min.After incubation with the fluorochrome, cells were washed with PBS and immediately analyzed at FL1 channel of FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) Breakdown of ΔΨm mitochondrial membrane potential was determined by FACS analysis using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide).For this purpose the Mitochondrial Membrane Potential Detection Kit (Stratagene, La Jolla, CA, USA) was used, as described in the manufacturer's instruction.10 6 HL-60 or HL-60/adr cells were treated for 1, 3, 6, 12 and 24 h with the tested drugs.After PBS washing, cells were incubated for 10 min in freshly prepared JC-1 solution (10 mg/ml in culture medium) at 37 °C.Spare dye was removed by PBS washing and cellassociated fluorescence was measured with FACS.
Statistical analysis
If not stated otherwise, data are expressed as mean ± SD.The results were analyzed using GraphPad Prism software.Statistical analyses were performed using t-test or two-way analysis of variance (ANOVA).To examine differences between the drug treatment responses, Bonferroni post-tests were conducted.P values below 0.05 were considered as statistically significant and marked with stars: * p<0.05; ** p<0.01; *** p<0.001.
Results and Discussion
At the first stage of our study, the sensitivity of HL-60 leukemia cells and their drug-resistant sublines to the action of landomycin E (LE) and well-known anticancer drug doxorubicin (Dx) was studied in de-tail (Fig. 1).It was revealed that overexpression of P-glycoprotein in HL-60/vinc cells increased their resistance profile to Dx 100-fold, while HL-60/adr cell line, overexpressing MRP-1, was found to be 200-fold more resistant to the Dx action compared to parental HL-60/wt line (Fig. 1).On [the] contrary, no difference in sensitivity of HL-60/wt, HL-60/adr and HL-60/vinc cells to [the] LE action was observed at 72h drug incubation time, according to MTT tests (Fig. 1).
Short-time drug toxicity experiments (24h incubation), analyzed by trypan blue exclusion test, have revealed similar tendencies -20-fold resistance increase of HL-60/vinc cell line, and 40-fold resistance increase of Hl-60/adr cell line towards Dx (Fig. 1), while for LE the only cells which showed a weak (~2-fold) decrease in sensitivity to this drug, were of HL-60/adr subline.Thus, the cytotoxic activity of LE is almost unaffected by ABC transporters, which makes it a promising compound for treatment of drug-resistant leukemia.
Previously we demonstrated that one of the landomycin family members -LA, possessing 6 sugar residues instead of 3 residues in LE, and thus higher cytotoxic activity, leads to specific two-phase ROS burst in Jurkat T-leukemia cells (Lehka et al, 2015), which consists of two peaks 1h and 12h after the target cell treatment with LA.Mitochondria, being the main energetic stations in the eukaryotic cells, are also considered to be the main source of ROS [15].Thus, for the identification of nature and source of LE-induced ROS, the joint treatment of HL-60/adr/ vinc cells with LE and various ROS scavengers (N-acetylcysteine, sodium pyruvate) and mitochondrial respiratory chain inhibitors (antimycin A, oligomycin A, DPI) was addressed.
For studying potential impact of mitochondria on the cytotoxic activity of LE in vitro several specific inhibitors of mitochondrial respiratory chain were used, namely DPI (NADPH dehydrogenase inhibitor [16]), antimycin A (complex III inhibitor [17]) and oligomycin A (ATP-synthetase inhibitor [18]).However, it was revealed that all these compounds, except antimycin A (2 µM), are toxic to HL-60 leukemia cells, even in very low concentrations (1 µM for oligomycin A and 0.5 µM for DPI) (Fig. 2).The same tendency was observed also for HL-60/adr cells, while HL-60/vinc cell line demonstrated a high internal resistance to the action of all tested mitochondrial inhibitors, thus indicating an insignificant role of mitochondria in the regulation of their proliferation and apoptosis.We did not observe any statistically significant impact of aforementioned compounds on inhibition or stimulation of LE cytotoxic action, mainly due to a high toxicity of oligomycin A and DPI towards the tested cell lines at 72h incubation.However, in HL-60/vinc cell line, where no toxic activity of antimycin A, oligomycin A and DPI was observed, they possessed a little, still statistically insignificant impact on lowering antineoplastic action of LE.Thus, mitochondria seem to play rather secondary role in the LE-induced cell death.
For better understanding a role of specific ROS in LE-involved cell death, specific hydrogen peroxide scavenger sodium pyruvate [19] and wide-scale ROS scavenger N-acetylcysteine [20] were addressed.It was revealed that sodium pyruvate possessed the moderate cytoprotective activity towards the LE action (P<0.05 for HL-60 cells and P<0.01 for HL-60/adr cells), whereas NAC almost completely blocked its activity towards all tested cell lines (P<0.001).Such unexpectedly high efficiency of the NAC action may be explained not only by its ROS scavenging activity, but also by its indirect impact on the glutathione level in the cells [20].
Due to the fact that ROS are short-living molecules, MTT assays for 72h drug incubation and trypan blue tests for 24h drug treatment may not show all the dynamics of cellular processes, taking place immediately after drug entry into the cells.For better understanding a role of specific ROS and mitochondria at early timepoints of LE action, a level of hydrogen peroxide in the cells was measured by flow cytometry using DCFDA dye at 1,3,6,12,24h after LE addition to studied cell lines.As seen in Fig. 3-5, LE leads to the] appearance of typical two-peak H 2 O 2 burst in wild-type HL-60 cells, like previously observed by us in LA in Jurkat T-cells (Lehka et al, 2015).At the beginning (1h) the ROS level increases 3-fold (for 2 µM dose of LE) or 5.5-fold (for 4 µM LE), and this process is partially inhibited by N-acetylcysteine (see Fig. 3) Then at 3h the ROS level under LE treatment rapidly drops, almost reaching a control level at 6h, and then slowly recovers at 12h, slightly decreasing again at 24h (Fig. 3).Mitochondrial respiratory chain inhibitors DPI and oligomycin A have no statistically significant impact on the LE-derived ROS production, as seen in Fig. 3, thus confirming our previous MTT data (see Fig. 2).
However, in the HL-60/adr cell line, which was found 2-fold more resistant to the LE action, the drug leads to a massive ROS production, which is approximately 2-fold higher compared to HL-60/wt cells (Fig. 4), and NAC strongly reduces it almost to a control level.Surprisingly, the ROS level under LE treatment in HL-60/adr cells does not drop as rapidly as observed in parental HL-60 line (Fig. 4).Instead, it slowly decreases from 1h till 24h, so no a "paradox" double peak is observed here.Despite major H 2 O 2 production, neither DPI, nor oligomycin A have significant impact on the ROS production 3. LE leads to significant increase of hydrogen peroxide level in human leukemia cells of HL-60 line already at 1h after addition to cell culture, which is partially inhibited by N-acetylsysteine, but not by mitochondrial respiratory chain inhibitors DPI and oligomycin A. The cells were pre-treated for 30 min with NAC, DPI and OM then treated with either vehicle, LE (2 µM and 4 µM) and then stained with DCFDA for analysis by flow cytometry.Bar graphs show an increase (over vehicle control) in the percentage of cells with positive DCF fluorescence in each sample.Baseline level of hydrogen peroxide in control cells is taken as 100%.DPI -diphenylene iodonium, NAC -N-acetylcyteine, OMoligomycin A * p<0.05, ** p<0.01 relative to control, unpaired t-test.p<0.05, relative to LE, unpaired t-test.Fig. 4. LE leads to over 2-fold increase of hydrogen peroxide level in human leukemia cells of HL-60/adr line, overexpressing MRP-1, in comparison to HL-60 parental line already at 1h after addition to cell culture, which is strongly inhibited by N-acetylsysteine, but not by mitochondrial respiratory chain inhibitors DPI and oligomycin A. Cells were pre-treated for 30 min with NAC, DPI and OM, then treated with either vehicle, LE (2 µM and 4 µM) and then stained with DCFDA for analysis by flow cytometry.Bar graphs show an increase (over a vehicle control) in the percentage of cells with positive DCF fluorescence in each sample.A baseline level of hydrogen peroxide in control cells is taken as 100%.DPI -diphenylene iodonium, NAC -N-acetylcyteine, OMoligomycin A * p<0.05, ** p<0.01 relative to control, unpaired t-test.p<0.05, relative to LE, unpaired t-test.under the LE action, thus indicatin their weak involvement in this process (Fig. 4).
The only slight ROS-inhibitive effect of DPI towards the LE action was observed in the HL-60/vinc cell line, characterized by P-gp overexpression (Fig. 5).However, it was much lower compared to NAC alone and statistically insignificant.Thus, all studied mitochondrial respiratory chain inhibitors have a slight effect on the LE-induced ROS produc-tion, which may indicate the fact that LE leads to the extra mitochondrial ROS production.
For checking this hypothesis, the status of mitochondrial membrane in HL-60/wt and HL-60/adr cells was tested using JC-1 staining.Unfortunately, JC-1, being a mitochondrial stain, was also found by us to be a perfect substrate for P-glycoprotein, thus the measurement of JC-1 fluorescence in HL-60/ vinc cells was technically impossible due to a mas- sive dye extrusion from the cells.As seen in Fig. 6, LE leads to the late depolarization of mitochondria in HL-60 and HL-60/adr cells, reaching its maximum at 12h timepoint -time, which coincides with the second ROS burst observed under the LE action (see Fig. 3).However, no signs of mitochondria depolarization were observed at 1h timepoint in both cell lines, where the major ROS peak was observed by us (Fig. 3-5).This fact further indicates a secondary role of mitochondria in the LE-induced ROS production, and corresponds to the second ROS burst, but not the first one (1h).At a combination of LE and mitochondrial respiratory chain inhibitors it was revealed that ATP-synthase inhibitor oligomycin A had no statistically significant impact on the LE-induced mitochondrial membrane depolarization at 1h, 3h and 6h timepoints, whereas DPI strongly enhanced the LE-induced mitochondrial membrane depolarization, increasing it 2.5-5-fold depending on drug concentration and incubation time (Fig. 6).It is known that DPI, blocking mitochondrial Complex I, can lead to subsequent oxidative stress in the cells [16].The synergistic action of DPI and LE at 6h timepoint on depolarization of mitochondria (Fig. 6) clearly indicates that both compounds induce the mitochondrial-mediated ROS production.It takes place much later (12h) compared to a massive ROS burst, caused by LE in both sensitive, and even higher, -in doxorubicin-resistant HL-60 cells at 1h.As DPI was shown to be a broad NAPDH oxidase inhibitor, blocking activity not only of Complex I, but also of other membrane-bound NADPH-oxidases [16], the LE-induced an early ROS burst cannot be explained by involvement of either mitochondria or of the plasma membrane ROS-producing enzymes.The only possible explanation of this early ROS burst is the hydrogen peroxide production by drug itself during its entry into the target cell.It has been already shown that all compounds containing quinone motif in their structure, may be the source of ROS itself as the catalyzers of the continuous redox cycles [21].These ROS may be inhibited by cellular antioxidant systems, and glutathione system is considered to be one of the most efficient [22].
The GSH level in HL-60/adr cells was shown to be 2-fold lower in comparison to HL-60/wt cells [23].This may explain the nature of a 2-fold higher ROS burst, induced by LE in HL-60/adr cells, as this antioxidant at a lower level inside the cells is unable to quickly scavenge all hydrogen peroxide, produced by LE.
Several studies have already demonstrated a tight connection between MRP-1 pump and intracellular GSH pool, which plays an important role in the functioning of this drug transporter [24].In particular, GSH depletion by buthionine sulphoximine (BSO) increases the accumulation of DNR and Rhodamine 123 (Rh123) in several MRP1-positive cell lines [25], thus partially reverting their resistance to these drugs.
The observed connection between the glutathione antioxidant system and the MRP-1 expression in HL-60/adr cells may explain their higher resistance profile to LE. HL-60/vinc cells, which have the basal GSH level, do not demonstrate any significant resistance to the LE action, even despite P-gp overexpression.Thus, joint targeting of tumor cells by LE together with GSH-depleting compounds (such as BSO) may be a promising approach in the treatment of drug-resistant tumors.
Conclusions
LE effectively circumvents drug resistance of HL-60 leukemia cells in vitro, caused by P-gp overexpression, whereas in HL-60/adr cells, characterized by MRP-1 overexpression, the activity of this drug drops 2-fold compared to the parental cell line.The LEinduced cell death is accompanied by a massive ROS burst, consisting of two peaks of hydrogen peroxide production -major one at 1h and secondary at 12h after drug treatment.The highest ROS production was observed in HL-60/adr cells, which have shown 2-fold increase of resistance to LE compared to the parental cells.However, mitochondria seem to be involved only at tetherminal stages of LE-induced apoptosis (12h), indicating the extra-mitochondrial ROS production by LE at early timepoints.The mitochondrial respiratory chain inhibitors failed to inhibit the LEinduced ROS burst at both early and terminal stages of the LE-induced cell death, and only one of them (DPI) was shown to have synergistic effect with LE on the mitochondrial membrane depolarization at 6h of cell incubation with the drug.All these data indicate that there was no involvement of mitochondria at early stages of the LE action, which are accompanied by ROS burst, possibly produced by the drug itself due to its specific chemical structure.A low glutathione level in HL-60/adr cells, which is a well-known antioxidant, may explain a 2-fold higher ROS burst, observed under the cytotoxic action of LE towards these cells.
Fig. 1 .
Fig. 1.Comparison of anticancer activities of LE and Dx towards human leukemia cells of HL-60 line and its drug-resistant sublines HL-60/adr (MRP-1+) and HL-60/vinc (P-gp+).Cell viability was analyzed by MTT assay after 72 h exposure to indicated concentrations of LE and Dx, or by trypan blue exclusion assay after 24 h exposure to aforementioned drugs.Graphs represent the mean +/-SD of three independent experiments done in triplicates ** p<0.01, *** p<0.001 relative to HL-60/wt cell line, 2-way ANOVA, Bonferroni post-test.
Fig.
Fig.3.LE leads to significant increase of hydrogen peroxide level in human leukemia cells of HL-60 line already at 1h after addition to cell culture, which is partially inhibited by N-acetylsysteine, but not by mitochondrial respiratory chain inhibitors DPI and oligomycin A. The cells were pre-treated for 30 min with NAC, DPI and OM then treated with either vehicle, LE (2 µM and 4 µM) and then stained with DCFDA for analysis by flow cytometry.Bar graphs show an increase (over vehicle control) in the percentage of cells with positive DCF fluorescence in each sample.Baseline level of hydrogen peroxide in control cells is taken as 100%.DPI -diphenylene iodonium, NAC -N-acetylcyteine, OMoligomycin A * p<0.05, ** p<0.01 relative to control, unpaired t-test.p<0.05, relative to LE, unpaired t-test.
Fig. 5 .
Fig. 5.No major differences in ROS production under LE action between P-gp overexpressing HL-60/vinc cell line and HL-60 parental cells are observed.NADPH oxidase inhibitor DPI partially decreases LE-driven ROS at 24h cells incubation with the drug.Cells were pre-treated for 30 min with NAC, DPI and OM, then treated with either vehicle, LE (2 µM and 4 µM) and then stained with DCFDA for analysis by flow cytometry.Bar graphs show an increase (over vehicle control) in the percentage of cells with positive DCF fluorescence in each sample.Baseline level of hydrogen peroxide in control cells is taken as 100%.DPI -diphenylene iodonium, NAC -N-acetylcyteine, OMoligomycin A * p<0.05, ** p<0.01 relative to control, unpaired t-test.p<0.05, relative to LE, unpaired t-test.
Fig. 6 .
Fig.6.LE leads to late depolarization of mitochondrial membrane in HL-60 and HL-60/adr cells, which takes place mainly at 12h after incubation with the drug.DPI possesses strong synergistic effect with LE on depolarization of mitochondria, but this effect is also observed much later (6h) than massive ROS burst at 1h under LE treatment.Cells were pre-treated for 30 min with NAC, Oligomycin A or DPI, then treated with either vehicle, LE (2 µM and 4 µM) and then stained with JC-1.Intracellular JC-1 fluorescence was measured by FACS and analyzed by Cytomation Summit software. | 2018-12-05T04:34:08.162Z | 2016-06-30T00:00:00.000 | {
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271275246 | pes2o/s2orc | v3-fos-license | Thermal-Responsive Antibacterial Hydrogel with Photothermal Therapy and Improving Wound Microenvironment for Promote Healing
Skin damage is one of the most prevalent human injuries, which affects the health of human beings. However, skin damage is often accompanied by bacterial infection and wound microenvironment changes, causing damage to normal cells and inhibiting wound healing. Herein, we designed a thermal-responsive antibacterial hydrogel (GAG hydrogel) loaded with catalase (CAT)-like Au@Pt@MgSiO3 nanoparticles (APM NPs) and gentamicin (GM) to promote wound healing. The GAG hydrogel was used in a photothermal therapy (PTT)/antibiotic combination to kill bacteria, reduce the use of antibiotics, improve the wound microenvironment, promote cell proliferation, and accelerate wound healing. Under near-infrared laser irradiation, APM NPs in the hydrogel generated local hyperthermia to kill bacteria. Meanwhile, the generated heat led to a change in the hydrogel’s morphology, enabling it to release GM and APM NPs to prevent the overuse of antibiotics. Subsequently, the CAT-like ability of the APM NPs decreased the oxidative stress caused by hydrogen peroxide (H2O2), thus remodeling the wound microenvironment. Then, the weakly acidic microenvironment of the wound caused the decomposition of the APM NPs and the release of magnesium ions (Mg2+), promoting the growth and migration of cells for wound healing. Therefore, the studied thermal-responsive antibacterial (GAG) hydrogel has potential in the field of wound healing.
Introduction
The skin, as the largest organ in the human body, covers the entire body's surface and serves as a natural barrier.It plays a vital role in maintaining body fluids and preventing the invasion of harmful substances and pathogenic microorganisms [1].As a prevalent human injury, skin damage is often accompanied by bacterial infection, oxidative stress, inflammation, and cell proliferation disorders [2][3][4].Once bacteria accumulate, persistent chronic inflammation often occurs in the wound area, which hinders damaged tissues' transition and further delays wound healing [5].Furthermore, bacterial infection can cause changes in the wound microenvironment, including oxidative stress and a weakly acidic microenvironment [6].These changes may hinder cell proliferation and slow down wound healing [7][8][9][10][11].Thus, designing a new strategy that can enable the provision of antibacterial treatment and regulation of the wound microenvironment is of great significance for accelerating wound healing.In the past few decades, antibiotic therapy has been widely used in antibacterial therapy because of its high efficiency and universality, but the excessive use and abuse of antibiotics has led to the emergence of drug-resistant strains, which considerably affect the therapeutic effect of antibiotics [12,13].Methicillin-resistant Staphylococcus aureus (MRSA) is a common and highly toxic drug-resistant bacterium that is difficult to kill with antibiotics [14].When a wound is infected with MRSA, traditional doses of antibiotics are insufficient to effectively kill bacteria, impeding tissue regeneration and delaying wound healing.In addition, due to the absence of skin on the wound, the continued presence of bacteria further exacerbates the deterioration of the wound microenvironment and causes damage to normal cells, making it difficult for the wound to heal [15].Therefore, a multifunctional strategy must be designed that can effectively inhibit bacterial infection and optimize the microenvironment of wounds, which will play a crucial role in wound-healing treatment.
Photothermal therapy (PTT), as a novel non-invasive treatment strategy, has a unique advantage in inhibiting wound infection [16][17][18].It has been reported that local hyperthermia generated by photothermal agents (PTAs) under laser irradiation could destroy the structure of bacteria [16][17][18].With the development of nanotechnology, more and more PTAbased nano-antibacterial agents have been designed and used to promote wound healing, such as metal nanostructure-based nanoparticles, transition metal sulfide/oxide nanomaterials, and carbon-based PTAs [19].Under laser irradiation, this type of nano-antibacterial agent can generate local hyperthermia to effectively kill bacteria.The degree of hyperthermia can be adjusted by changing the laser parameters, achieving controllable regulation of the antibacterial effect and reducing the use of antibiotics and unnecessary thermal damage [20].Additionally, an appropriate heating temperature (below 45 • C) improves the permeability of bacterial cell membranes and accelerates the penetration of antibiotics, as well as rendering previously resistant bacteria sensitive to antibiotics, which has proven useful in the new antibacterial treatment strategy of PTT combined with antibiotics [21].Therefore, the combination of PTT and low-dose antibiotics can effectively prevent the overuse of antibiotics and has the potential to be applied in wound-healing treatments.
Wound microenvironment change is a complex process caused by bacterial infection and inflammation, and it is mainly characterized by oxidative stress and the inhibition of fibroblast proliferation [22].Oxidative stress caused by the overexpression of hydrogen peroxide (H 2 O 2 ) damages normal cells at the wound site and inhibits wound healing [23].Furthermore, as an important factor in tissue regeneration, fibroblast proliferation is inhibited by wound microenvironment changes, which is detrimental to wound healing [24].Therefore, alleviating oxidative stress by reducing H 2 O 2 and promoting fibroblast proliferation to modulate the wound microenvironment has a positive effect on wound healing.Some catalases (CAT) and their enzyme mimics (such as platinum nanoparticles) can catalyze the decomposition of H 2 O 2 to produce oxygen (O 2 ) and water, thereby reducing damage caused by oxidative stress [25,26].However, due to the loss of skin tissue, the wound is repeatedly infected by bacteria, leading to a reduction in the effectiveness of CATrelated oxidative stress alleviation and deteriorating the wound microenvironment [15].To ameliorate the wound microenvironment, wound shielding should be performed to reduce the continuous accumulation of external bacteria.Hydrogel-based wound dressings with good biocompatibility and adhesion are widely used for wound shielding and the treatment of various types of wounds [11,27].Hydrogels can not only fill the gap left by lost skin to isolate external bacteria, but also simulate the extracellular matrix (ECM) to provide a suitable environment for cell proliferation and wound healing [28].Gelatine methacrylate (GelMA), an ideal photo-crosslinked dressing, can be used to form hydrogels to simulate the ECM and fill in areas of missing skin [29,30].GelMA hydrogel also has a good load capacity and a thermal-responsive capacity for drug loads, making it suitable for wound healing [31].Moreover, upon doping with metal ions, hydrogels obtain additional functions [32].For example, magnesium ions (Mg 2+ ) in hydrogels can accelerate the migration and adhesion of cells, increasing the production of the ECM to promote wound healing [32,33].Therefore, designing a thermal-responsive antibacterial hydrogel which can kill bacteria, reduce the use of antibiotics, and mediate the wound microenvironment is of great significance in the field of wound healing.
Herein, we designed a thermal-responsive GelMA hydrogel (GAG hydrogel) loaded with Au@Pt@MgSiO 3 nanoparticles (APM NPs) as nano-antibacterial agents and gentamicin (GM) (Scheme 1).These consisted of gold-platinum bipyramid nanoparticles as the core and magnesium silicate (MgSiO 3 ) as a shell, which provided them with photothermal conversion performance, H 2 O 2 scavenging ability, and Mg 2+ release capability.Under near-infrared (NIR) laser irradiation, the GAG hydrogel induced local hyperthermia to kill bacteria by relying on the internal APM NPs, and achieved the thermal-responsive release of APM NPs and GM to the wound area.The APM NPs distributed in the wound area effectively killed bacteria through PTT and reduced the dosage of antibiotics, and were able to further optimize the wound microenvironment.The CAT-like properties of APM NPs enabled the catalysis of H 2 O 2 into O 2 and water, reducing damage to the cells caused by oxidative stress.The acidic wound microenvironment induced the decomposition of APM NPs to generate Mg 2+ , which accelerated the growth and migration of fibroblasts, increasing ECM production and promoting wound healing.Therefore, due to its ability to kill bacteria and improve the wound microenvironment, this thermal-responsive antibacterial GAG hydrogel represents a new wound-healing strategy.
Scheme 1. Illustration of thermal-responsive antibacterial GAG hydrogel for accelerating wound healing through a PTT/antibiotic combination to kill bacteria, reduce the use of antibiotics, remodel the wound microenvironment, and promote cell proliferation.
Preparation of Au Seeds
The Au seeds were synthesized using a simple method.CTAC (0.16 g) was dissolved in 10 mL of deionized water to prepare the CTAC solution [34].Next, 50 µL of HAuCl 4 •3H 2 O (20 mg/mL) and 500 µL of sodium citrate (0.1 mol) were added to the CTAC solution.Then, 0.25 mL of AA (25 mmol) was also added to the CTAC solution and heated at 80 • C for 90 min to obtain the gold seeds (Au seeds).
Preparation of Au BNPs
A total of 100 µL of AgNO 3 (0.1 mol) solution was dissolved in 100 mL of CTAB solution (100 mmol), and then, 2 mL of HAuCl 4 •3H 2 O (20 mg/mL) and 2 mL of hydrochloric acid (1 mol) were added [35].Next, 1.6 mL of AA (0.1 mol) was added dropwise to form the growth solution.Then, 250 µL of the Au seeds were introduced to the growth solution and kept at 30 • C for 120 min.Finally, Au bipyramid nanoparticles (Au BNPs) were generated via centrifugation (5000 RPM for 20 min) and dispersed in 50 mL water.
Preparation of Au@Pt BNPs
A total of 60 µL of H 2 PtCl 6 •6H 2 O (20 mg/mL) was added to the Au BNP (20 mL) solution.When the temperature reached 80 • C, 1.6 mL of AA (10 mmol) was added and the temperature was maintained for 30 min to obtain Au@Pt BNPs.
Photothermal Performance of APM NPs
To study the photothermal effect, temperature changes in the APM NP solution were measured under 1064 nm laser irradiation.A thermal imaging system was used to detect the real-time temperature and to obtain thermal images of the APM NP solution at different concentrations (0, 100, 200, 300, and 400 µg/mL) and intensities (0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 W/cm 2 ).In addition, the photostability of the APM NP solution was evaluated under 1064 nm laser irradiation over 5 cycles of an on/off process by monitoring the thermal imaging system.
Ability of APM NPs to Remove H 2 O 2
To study the effect of APM NPs on removing H 2 O 2 , a portable dissolved oxygen meter was used for detecting the concentration of oxygen to indirectly reflect the removal of H 2 O 2 .A total of 10 mL of H 2 O 2 solution (10 mmol) was prepared, and then, different concentrations of APM NPs (0, 2, 5, 15, and 20 µg/mL) were added.Finally, the change in oxygen concentration in the solution within 30 min was measured.
Ability to Release Magnesium Ions
To evaluate the Mg 2+ generation capacity of APM NPs, Mg 2+ detection kits were used for detecting the production of magnesium ions.The APM NPs were placed in PBS at different pH values, and we removed the PBS solution at different time points.Then, the absorption intensity of the PBS solution was measured to evaluate the release of magnesium ions by APM NPs at different pH values.
Degradation of GAG Hydrogels
To study the degradation of GAG hydrogels, the environment at the wound site was simulated by using collagenase type II.The GAG hydrogels were divided into three groups according to the proportion of GelMA and APM NPs: 100, 200, and 300%.Subsequently, 200 µL of hydrogel was placed in 0.5 mL of collagenase type II solution (2 U/mL).The solution was placed at 37 • C and treated at 80 rpm.The hydrogel was removed at different times and weighed and photographed.
The Photothermal Performance of GAG Hydrogels
We synthesized different hydrogels and investigated their photothermal ability.The hydrogels of the GelMA hydrogel (G hyd) and GelMA-GM hydrogel (GG hyd) groups were both synthesized without APM NPs.The hydrogels of the GelMA-APM hydrogel (GA hyd) and GAG hyd groups were synthesized with APM NPs.Consequently, the temperature change in the hydrogel under 1064 nm irradiation was measured.
The Release of APM NPs and GM
We used FITC small molecules instead of GM for the drug-releasing experiments.The hydrogel was placed in PBS, and then the PBS was removed after different durations of laser irradiation.Consequently, the changes in the APM NP absorption peak and FTIC fluorescence peak in the PBS solution were measured.
Bacterial Culture
All bacteria were first cultivated overnight on Luria-Bertani (LB) agar plates at 37 • C in a bacteriological incubator.After reaching the exponential growth phase, the MRSA cells were harvested via centrifugation and resuspended in a saline solution.The bacteria were adjusted to a 0.5 McFarland standard (1.5 × 10 8 colony-forming units [CFU]/mL) and then diluted to a concentration of 1.0 × 10 8 CFU/mL in Mueller-Hinton broth (MHB).
In Vitro Antibacterial Activity of GAG Hydrogels
Colony-counting analysis was performed by culturing MRSA strains on LB agar plates to determine the number of bacterial colonies.Briefly, the MRSA strains were grown on LB agar plates for 24 h at 37 • C. A 0.5 McFarland inoculum of MRSA was prepared in saline solution (0.9%) from the bacterial cultures.The bacterial suspension was diluted to a concentration of 1.0 × 10 8 CFU/mL, and then 1 mL of the diluted bacterial suspension was added to each group for further experiments.These treatments were divided into five groups, including the control, GA Hyd, GAG Hyd, GA Hyd + NIR, and GAG Hyd + NIR groups.After NIR (1064 nm, 1.5 W/cm 2 ) irradiation for 10 min, 100 mL of dilution of each group was spread on LB agar plates and incubated at 37 • C for 24 h.Finally, the number of bacterial colonies formed was counted to determine the effectiveness of the treatment.
Determination of Live/Dead Ratio
The MRSA suspension (1.0 × 10 8 CFU/mL) was seeded in hydrogel in preparation for the experimental treatments.Afterward, the treated bacterial suspension was removed and washed with saline solution.SYTO 9 was diluted in sterile water and used at a final concentration of 10 mmol, and propidium iodide (PI) was diluted in sterile water and used at a final concentration of 5 mmol.The mixture of SYTO 9/PI dye and the bacterial suspension was incubated under dark conditions for 10 min, and then live/dead cells were observed using a fluorescence microscope.
The Cytotoxicity of APM NPs
A CCK8 assay was conducted to evaluate the cytotoxicity of APM NPs on L929 cells.The L929 cells were seeded in 96-well microplates with 1 × 10 4 cells per well.After incubation for 24 h, the culture medium was replaced with fresh medium which contained different concentrations of APM NPs (0, 50, 100, 150, and 200 µg/mL).After incubation for 24 h, the activity of the L929 cells was detected using a CCK8 kit.
Cell Migration
In vitro, a scratch wound-healing assay was used to reveal the ability of Mg 2+ to induce cell migration and proliferation.L929 cells were seeded in 6-well microplates with 3 × 10 5 cells per well.After incubation for 24 h, a scratch was made on the bottom, and the culture medium was replaced with APM NPs.After incubation for 24 h, the cell migration before and after incubation for 24 h was studied with an inverted microscope.
In Vivo Wound Healing
All experiments involving animals complied with the Institutional Animal Care and Use Committee and the Administrative Committee of Laboratory Animals of Fujian Medical University (FJMU 2023-Y-1141).Balb/c mice (15-20 g) were provided by Shanghai SLAC Laboratory Animal Co., Ltd.The full-thickness circular wound models (d = 8 mm) were established on the backs of the mice.Each wound was infected with 30 µL of MRSA suspension with a concentration of 1.5 × 10 8 CFU/mL for 24 h.After grouping the wounds (control, GA Hyd, GAG Hyd, GA Hyd + NIR, and GAG Hyd + NIR), the hydrogel was placed on the surface of the wound and irradiated at 1064 nm (0.5 W/cm 2 , 10 min).On days 1, 4, 7, and 10, the wounds were photographed and analyzed.
Synthesis and Characterization of APM NPs
The APM NPs were prepared using a simple and convenient strategy (Figure 1A).First, ultra-small gold seeds (Au seeds) were successfully synthesized, which played an extremely important role in the formation of Au BNPs.Transmission electron microscopy (TEM) (Tecnai G2, FEI Company, Lausanne, Switzerland) data showed that the Au seeds had good dispersion, with a size of about 5 nm (Figure 1B).Then, Au BNPs were synthesized through seed-mediated growth under the action of AA and AgNO 3 .As shown in Figure 1C, the Au BNPs were characterized by long sides and a short middle.In order to decrease H 2 O 2 levels, CAT-like Pt NPs were introduced to the surface of Au BNPs to form Au@Pt BNPs.As shown in Figure 1D, the Pt NPs were mainly distributed around Au BNPs, with a size of about 5 nm.To further promote the release of Mg 2+ , a magnesium silicate shell was synthesized via a hydrothermal reaction [37].Through continuous agitation, a silicon shell with a thickness of about 20 nm was formed on the Au@Pt BNPs' surface (Figure 1E).Then, the silicon shell was transformed into a magnesium silicate shell via a hydrothermal reaction (Figure 1F).These TEM images prove that the APM NPs were successfully synthesized through a series of processes.The image obtained using the scanning electron microscope (SEM) (SU8100, HITACHI) further revealed that APM NPs had a uniform rough surface structure with a MgSiO 3 shell, validating the success of rough-surface APM NPs (Figure 1G).Then, the absorption spectrum of APM NPs was studied, which showed prominent absorption peaks at 1064 nm (Figure 1H).This suggests that it is possible for APM NPs to act as PTAs in PTT-based antibacterial therapy.Moreover, the size distribution data revealed that the diameter of APM NPs was about 123 nm (Figure 1I), and the polymer dispersity index (PDI) (Malvern Zetasizer Nano ZS, Malvern, UK) was about 0.303.Meanwhile, the Zeta potentials of Au BNPs was 40.16 mV, of Au@Pt BNPs was 15.24 mV, of APS NPs was −51.45 mV, and of APM NPs was −36.41 mV (Figure 1J).These results collectively indicate the successful synthesis and characteristics of APM NPs, and their potential for use in PTT-based antibacterial therapy and remodeling of the wound microenvironment.
The Photothermal Performance of APM NPs
In order to evaluate the photothermal effect, temperature changes in the APM NPs solution at different parameters of 1064 nm laser irradiation were tested (Figure 2A).After five on/off cycles with 1064 nm laser irradiation, the APM NPs still exhibited photothermal abilities, indicating their excellent photothermal stability (Figure 2B).In order to further study the effect of APM NP concentrations on photothermal performance, the temperature changes were tested under NIR laser irradiation with a power density of 1.0 W/cm 2 , and temperature rise curves and thermal images were obtained (Figure 2C,D).With 0 µg/mL of APM NPs under 1064 nm irradiation, the temperature increased by only 3.7 • C.Moreover, with 400 µg/mL of APM NPs, the temperature increment was about 27.4 • C, confirming the light-to-heat ability of AMP NPs.Moreover, photothermal durability is also an important characteristic of APM NPs in PTT-based antibacterial therapy.Figure S1 shows that the temperature rise was more obvious in the APM group under 1064 nm irradiation after 15 min.In addition, the photothermal ability of APM NPs was studied in relation to laser intensity (Figure S2).It was found that the APM NPs had a considerable photothermal effect under 1064 nm irradiation, and can therefore be used for PTT-based antibacterial therapy.
The CAT-like Performance of APM NPs
Due to the introduction of Pt NPs, we found that the APM NPs might have a CAT-like ability to catalyze the decomposition of H 2 O 2 to produce O 2 .The decrease in H 2 O 2 levels helped to decrease oxidative stress in the wound area.To study the CAT-like ability of APM NPs, we evaluated the oxygen concentration when mixed with different concentrations of APM NPs.As shown in Figure 2E, the O 2 concentration in the control group (without APM NPs) changed slightly over time.When 20 µg/mL of APM NPs was added, the O 2 concentration in the solution increased by about 6.7 mg/L, showing that the CAT-like APM NPs could decompose H 2 O 2 .Furthermore, Figure S3 shows that more and more oxygen bubbles could be observed on the pipe wall with an increase in APM NP concentration, verifying the CAT-like ability of APM NPs.
Mg 2+ Release in Acidic Environment
Due to the function of Mg 2+ in promoting fibroblast growth and migration, we expected the designed nanomedicine APM NPs to be able to carry and release Mg 2+ .To verify this mechanism, a blood magnesium content assay kit was used to detect Mg 2+ release.We compared the Mg 2+ content between PBS solutions with different pH levels.When the pH was 7.3, the content of Mg 2+ was slightly increased in the PBS solution, indicating that APM NPs may not produce many magnesium ions in a neutral environment (Figure 2F).However, when the pH dropped to 6.5, the level of Mg 2+ in the PBS solution was significantly increased, which explains the Mg 2+ release from APM NPs under acidic conditions.Therefore, APM NPs may release Mg 2+ in an acidic environment, suggesting their potential in promoting wound healing.
The Synthesis and Characterization of GAG Hydrogels
A thermal-responsive GAG hydrogel was prepared through LAP via a photo-crosslinking reaction (Figure 3A).The hydrogel appeared as a liquid and possessed fluidity before the photo-crosslinking reaction (Figure 3B).Then, after the photo-crosslinking reaction, transformation to a gel state occurred, indicating the successful synthesis of the GAG hydrogel.Subsequently, the GAG hydrogel was irradiated under a 1064 nm laser for 20 min, following which the hydrogel was partially dissolved.To explore the adhesion of the GAG hydrogel, it was placed on the surface of different materials and was found to adhere to them (Figure 3C).This showed that the GAG hydrogel had a certain adhesion ability, indicating its potential for filling in areas of skin loss.In addition, this soluble part returned to a gel state over time, which allowed the GAG hydrogel to more firmly adhere to the material (Figures S4-S6).These experimental results showed that the special thermal response capacity of GAG hydrogels might enable them to stick firmly to wounds, promoting wound healing.These experiments demonstrated the successful synthesis and the characteristics of thermal-responsive GAG hydrogels.
Biodegradation of GAG Hydrogels
In order to verify the biodegradation, the GAG hydrogels were evaluated in PBS with collagenase II at 37 • C. As shown in Figure S7, the degradation ability of GAG hydrogels in different proportions was studied.The results showed that the GAG hydrogel with a proportion of 100% was completely degraded after 3 h of incubation in the pathological microenvironment, confirming the self-degradation properties of GAG hydrogels.This could prevent secondary damage caused by GAG hydrogels to the wound area.In addition, upon comparing different proportions of GAG hydrogels, it was found that the degradation rate was related to the GelMA content in the hydrogels.These experimental results provide a basis for studying the biosafety of GAG hydrogels.
Photothermal Performance of GAG Hydrogels
Due to the inclusion of APM NPs, the GAG hydrogels were expected to have photothermal conversion properties.We evaluated the photothermal properties of GAG hydrogels under a 1064 nm laser irradiation.Figures 3D and S8 show that the temperature of the GelMA hydrogel (G Hyd) without the addition of APM NPs exhibited little change and only increased by 3.8 • C after 5 min.After adding APM NPs, the temperature of the GA hydrogel (GA Hyd) increased rapidly by nearly 43.9 • C, indicating that the GAG hydrogel with APM NPs had excellent photothermal conversion ability.Moreover, the temperature changes in the other groups (the GelMA-GM and GAG hydrogel groups) are shown in Figure S9, indicating the photothermal ability of GAG hydrogels.
APM NPs and GM Release of GAG Hydrogels
The GAG hydrogel exhibited a thermal-responsive performance, so it could be used for APM NPs and GM release.To verify the release of APM NPs, the UV-Vis-NIR absorption peak of the solution after laser irradiation was measured.The results in Figure 3E show that, with an increase in laser irradiation time, the absorption peak of the PBS solution increased significantly, indicating that APM NP release was constantly increasing.This showed that the APM NPs from the thermal-responsive GAG hydrogels could be released by adjusting the laser irradiation parameters.In addition, FITC fluorescent molecules were used instead of GM for hydrogel synthesis to explore the GM release property of GAG hydrogels.Figure 3F shows that the fluorescence signal of FITC increased with increasing laser irradiation time, indicating that GM in the GAG hydrogel could be released.Therefore, GAG hydrogels possess the capability of a thermal-responsive release of APM NPs and GM in the wound area.
Antibacterial Activity of GAG Hydrogels In Vitro
The GAG hydrogels were expected to induce hyperthermia and kill bacteria under 1064 nm irradiation (Figure 4A).To verify whether the GAG hydrogels possessed antibacterial abilities, bacterial experiments were performed, including a colony-forming assay, a bacterial morphology assay, and a live/dead staining assay.Based on the experimental results (Figure 4B,C), the average colony-forming units (CFU) of MRSA in different treatment groups were as follows: control group, 446 CFU; GA Hyd group, 428 CFU; GAG Hyd group, 362 CFU; GA Hyd + NIR group, 98 CFU; and GAG Hyd + US + NIR group, no bacterial growth.The colony-forming results showed that the control group and GA Hyd group without GM and NIR did not have an antibacterial effect.Upon adding GM (GAG Hyd group), the number of bacteria slightly decreased, while most bacteria still survived and the antibacterial effect was not obvious.In another group (GA Hyd + NIR group), the number of bacteria clearly decreased, indicating that PTT using the GA hydrogel had positive effects on antibacterial therapy.It is worth noting that the number of bacteria in the All group (GAG Hyd + NIR group) was 0 CFU, indicating that the GAG hydrogel had a strong antibacterial effect under 1064 nm irradiation.The SEM images were used to observe the morphological changes in bacteria induced by different treatments.Figure 4D shows that the MRSA in the control group (without any treatment) and GA hydrogel group exhibited a nearly spherical shape with smooth surfaces.However, MRSA treated with the GAG hydrogel and GA hydrogel + NIR showed varying degrees of damage.The damage was particularly evident in the All group, with the bacterial surface showing wrinkling, indentations, and numerous pores, indicating the loss of membrane integrity.These results showed that the bacterial membrane underwent reduced integrity and increased permeability, which may accelerate the penetration of GM and the killing of bacteria.
To further assess the antibacterial ability of GAG hydrogels, an experiment using a combination of SYTO 9/PI dye and a bacterial suspension was conducted.The permeant nucleic acid dye SYTO 9 could enter all cells, while only membrane-damaged cells were stained red by the impermeant nucleic acid dye PI.Fluorescence images and the results of the quantitative analysis of MRSA targeted with different treatments are shown in Figures 4E and S10.Only a few red spots were observed in the control group (without any treatment) and GA hydrogel group, while more red spots were observed for those treated with the GAG hydrogel and GA hydrogel + NIR.Additionally, the bacterial survival rates in the GAG hydrogel group and GA hydrogel + NIR group were 51.75% and 52.91%, respectively.Furthermore, the number of red spots increased significantly in the GAG hydrogel + NIR group, and the bacterial survival rate was only 16.5%, confirming the antibacterial effect of using GAG hydrogels in combination with PTT and GM release.These experimental results verified that the GAG hydrogel combining PTT and GM release possessed a good antibacterial ability.
Cytotoxicity and Cell Migration Ability
To investigate the safety of APM NPs at the wound site, L929 cells were used to detect cytotoxicity.As described in Figure 4F, cell viability was about 100% in the control group (without APM NPs).Upon adding different concentrations (0-200 µg/mL) of APM NPs, cell viability was still greater than 80%, indicating that the APM NPs promoted biosafety for the L929 cells.Furthermore, an easy and affordable scratch test was used to evaluate L929 cell migration with APM NPs.The scratch gaps were evaluated at 0 h and 24 h to learn about the APM NPs' promotion of cell migration ability (Figures 4G and S11).At 0 h, there were very obvious scratch gaps in both the control group and APM NP group.At 24 h, the scratch gap in the control group showed almost no significant change, while the gap in the APM group was narrowed and the cells gradually migrated to the middle region.These results indicated that the APM NPs had good cytocompatibility and could increase cell migration to promote wound repair.
In Vivo Wound Healing Effects of GAG Hydrogels
These results showed that the thermal-responsive GAG hydrogels had the ability to kill bacteria, reduce antibiotics use, remodel the microenvironment, and promote cell proliferation, which can promote wound repair.Therefore, a full-thickness MRSAinfected wound model was selected to evaluate the wound repair ability of GAG hydrogels (Figures 5A and S12).As described in Figure 5B,C, the wound in the control group without any treatment was still large after 10 days, and there was a scab on the surface.In the GA Hyd and GAG Hyd groups, the wound areas were similar to that in the control group, indicating that neither treatment had a therapeutic effect.The wound area of the GA Hyd + NIR group showed a significant reduction compared to the control group, which might be due to the killing of bacteria by PTT and the release of APM NPs to promote cell migration.In addition, the wound area was the smallest in the GAG Hyd + NIR group, and the wound healing effect was the most pronounced after 10 days.Furthermore, Figure 5D shows a comparison of wound area sizes over the 10 days with different treatment strategies, indicating that the thermal-responsive antibacterial GAG hydrogels combined with PTT and APM/GM release were highly effective in promoting wound healing.
To further analyze the wound-healing ability of the different hydrogels, hematoxylineosin (H&E) staining was used to assess the process of wound healing.In the early stage of wound healing, inflammatory cells would be recruited, there would be an acute inflammatory response at the wound site, and the degree of inflammation would decrease with the progress of healing [2].As shown in Figure 5E, in the control group, the epidermis was missing at the site of the skin tissue injury, and we noted an abscess (brown arrow), bleeding (yellow arrow), and a lot of necrotic cell debris.Additionally, the epidermis around the lesion was also thickened, accompanied by hyperkeratosis (black arrow) and stratum corneum thickening.Moreover, the dermis and subcutaneous structures were unclear, with no obvious skin appendages and a lot of connective tissue hyperplasia (red arrow).Furthermore, a large amount of lymphocyte and granulocyte infiltration (blue arrow) was shown.The results showed that there was an acute inflammatory response in the control group, indicating that the wounds healed slowly without treatment.After treatment with the GAG hydrogel under NIR irradiation, the epidermis was intact at the site of skin tissue injury (Figure 5F).We noted connective tissue proliferation (red arrow), neovascularization (green arrow), and reduced lymphocyte and granulocyte infiltration (blue arrow) at the wound site.These experimental results demonstrated that the thermal-responsive antibacterial GAG hydrogel possessed a good wound-healing capacity.Therefore, the thermal-responsive GAG hydrogel combining PTT and APM/GM release might represent a good therapeutic strategy for wound-healing therapy in the future.
Conclusions
In this study, we designed a thermal-responsive antibacterial GAG hydrogel for wound healing by loading CAT-like APM NPs and GM.The APM NPs were prepared using simple methods, including seed-mediated growth, a hydrothermal reaction, etc.The APM NPs were contained with Au BNPs, which exhibit good performance in photothermal conversion, reducing H 2 O 2 , and releasing Mg 2+ .Then, the GAG hydrogel was synthesized by mixing GelMA, APM NPs, and GM under a photo-crosslinking reaction.The GAG hydrogel possessed the ability to combine photothermal therapy and antibiotic therapy, which can kill bacteria, prevent antibiotics abuse, remodel the wound microenvironment, and promote cell proliferation.Under 1064 nm laser irradiation, the APM NPs in the GAG hydrogel generated local hyperthermia to kill MRSA, and the generated heat led to a change in GAG hydrogel morphology and the release of GM and APM NPs to prevent the overuse of antibiotics.Because of the enhanced bacterial cell membrane permeability caused by PTT, GM could more easily enter the bacteria and kill them.Subsequently, the CAT-like APM NPs decreased the H 2 O 2 levels to reduce oxidative stress and regulate the wound microenvironment.Then, the APM NPs were decomposed to release Mg 2+ under the weakly acidic wound microenvironment, promoting the growth and migration of cells for wound healing.Therefore, the proposed thermal-responsive antibacterial GAG hydrogel represents a promising solution in the field of wound healing.
Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox13070857/s1. Figure S1: (A) The temperature changes and (B) photo images in different solutions (water and APM NPs) during 15 min.Figure S2: The temperature changes in APM NPs solution at different laser intensities.Figure S3: The oxygen bubbles production in H 2 O 2 solution with different APM NPs concentrations (0, 2, 5, 15, and 20 µg/mL).Figure S4: The adhesion changes before and after 1064 nm laser irradiation.Figure S5: The healing of GAG hydrogels after 1064 nm irradiation.Figure S6: (A) The hydrogel synthesized using 5 % of GelMA was unable to form a stable shape.(B) The hydrogel synthesized using 10 % of GelMA.The hydrogel forms a stable shape and has good adhesion to the skin.(C) The hydrogel synthesized using 20 % of GelMA.Hydrogels can form a stable shape, but the skin adhesion is not good.Figure S7: (A) The degradation of GAG hydrogels in different ratios (1:1, 2:1, and 3:1), and (B) the weight changes in GAG hydrogels.
Figure 3 .
Figure 3. (A) Illustration of GAG hydrogel fabrication.(B) Photos of thermal-responsive GAG hydrogel in the process of synthesis and partial dissolution.(C) Photos of GAG hydrogels placed on the surface of different materials (rubber, skin, plastic, and PS).(D) Temperature photos of different hydrogels (G Hyd and GA Hyd).(E) The release of APM NPs at different durations of 1064 nm irradiation (0, 5, and 15 min).(F) The release of FITC (GM mimic) at different durations of 1064 nm irradiation (0, 5, and 15 min).
Figure S8: The temperature changes in different hydrogels under 1064 nm irradiation.Figure S9: Temperature photos of different hydrogels (GG Hyd and GAG Hyd).
Figure S10: The analysis of bacterial survival rates after different treatments (Control, GA Hyd, GAG Hyd, GA Hyd + NIR, and GAG Hyd + NIR).
Figure S11: The scratch test of L929 in different treatments (control, APM NPs, and MgCl2) after 1 day.Figure S12: (A) Changes in wound skin after NIR treatment in different groups (control and GAG hydrogel group).(B) Temperature change curves after different treatments (NIR and GAG hydrogel + NIR).(C) Temperature change after different treatments (NIR and GAG hydrogel + NIR). | 2024-07-19T15:21:58.763Z | 2024-07-01T00:00:00.000 | {
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19534384 | pes2o/s2orc | v3-fos-license | Evaluation Research of Traction Motor Performance for Mine Dump Truck Based on Rough Set Theory
This paper presents the traction motor evaluation method depending on the electric transmission energy transfer characteristics and different source of supply, including motor manufactures, diesel turbine manufacturers, wheel side reducer manufacturers and electric drive system integrated manufacturers. 9 evaluations are proposed in 3 levels from the motor body and control performance, electric drive system coordinate index, driving conditions and specific cycle. Motor performance evaluation system is published by the means of electric transmission tests and computer simulation platform, using rough set theory. Experimental results show that the model can accurate evaluation of state of the traction motor, Evaluation of the accuracy is better than the subjective weighting analysis, verifying the integrity and usefulness of this valuation method. At the same time, the comprehensive evaluations index of permanent magnet synchronous motors is high, it has important research value.
Introduction
Currently, large dump truck as the main means of transport for large open pit mine bears 40% of the world's coal, 90% of the iron ore mining traffic [1].Electric Drive is used in mining manufacturers of more than a hundred-ton mining dump truck except the Caterpillar Company.The electric drive system structure is simplified, easy to operate, maintain and energy efficient.Large mining trucks develop to electric drive is an inevitable trend.At the same time, Traction motor characteristics have an important impact on the vehicle dynamic parameter.The matching and evaluation of Motor vehicle driver and vehicle performance is now an issue that needs to be addressed as a matter of urgency.
In the process of choosing traction motor, its evaluation and assessment have been often out of the vehicle electric drive system; just relying on the drive motor bench test or basic parameters matching test.It's difficult to guarantee the accuracy and objectivity of the evaluation, more difficult to achieve efficient use of the system.In the electric drive system of large tonnage dump truck, optional range diesel turbine is limited in scope.Therefore, it is necessary to establish comprehensive evaluation system as the core of a traction motor to provide a theoretical basis for the determination of the overall design of dump truck.
Dump truck electric transmission evaluation at home and abroad is also less， and often for the match and the fuel consumption of the hybrid vehicle power system analysis and evaluation [2]- [9].Domestic Beijing Institute of Technology Wang Wei [10], finish the gray correlation and experimental simulation of the motor performance ;Foreign Livint Gheorghe [11] illustrated evaluation for hybrid electric vehicle control algorithm analysis ,Sung Chul [12] use hardware in the ring needle evaluation and analysis of electric car motors,These studies did not focus on the electric drive system.
According to a variety of vehicle traction motor and electric drive system features, different from the general hybrid vehicles.This paper presents the traction motor evaluation method depending on the electric transmission energy transfer characteristics and different source of supply ,including motor manufactures, diesel turbine manufacturers, wheel side reducer manufacturers and electric drive system integrated manufacturers.9 evaluations are proposed from 3 levels from the motor body and control performance, electric drive system coordinate index, driving conditions and specific cycle.Motor performance evaluation system is published by the means of electric transmission tests and computer simulation platform, using rough set theory.
Drive motor evaluation process
Depending on the type of traction motor basic performance and demand characteristics of mining dump truck motor, mining trucks traction motor evaluation system of "Two vertical and three horizontal" is proposed.As Figure 1 shows, three horizontal mainly refers to the traction motor manufacturers, manufacturers of electric drive systems and integrated vehicle manufacturers, two vertical mainly refers to drive motor bench testing and computer simulation platform which is the evaluation testing means of drive motor system.Bench test mainly tests the inherent performance of the motor system, the vehicle driving cycle simulation is to examine the run performance of drive motor in driving conditions.
Dump truck evaluation index
The main types of dump truck driving motor include [13]:AC induction motor (IM), permanent magnet synchronous motor (PMSM), brushless DC motor (BLDC), DC the brush motor (BDC)and switched reluctance motor (SRM).Each type of drive motor has a unique structure design and control algorithms, while the selected drive motor type the hybrid vehicle of different structural forms is not the same.Therefore, combined with the basic characteristics of different types of vehicles and the vehicle condition, nine types of evaluation is presented comprehensively.Among these motors, AC asynchronous motor is the mostly widely used.In order to evaluate more intuitive and full use of existing data, multiple sets of ACIM is used for comparison.
Motor body and Control indicators
Electric dump truck wheel motor is placed directly in the wheel hub, and combined with the drive system, brakes and wheel side reducer as a whole; this installation and transmission form has a high demand for the volume of the motor.Consider the mine dump truck tonnage load characteristics, to stress overload and constant power range, and to meet the basic requirements of the rated condition, the following six indicators is comprehensively proposed: Efficient area overload capacity, constant power range, response time, cost and volume manipulation.Among them, overload capacity and control response time refers to the data of the rated conditions.
Electric drive system combining index
The so-called electric drive system is that: fuel energy pass through the engine, generator rectifier inverter, and finally export energy after the mechanical part of the wheel side reducer tire.To make the system fully efficient operation, the entire system should run on efficient district.On the base of definition of motor itself efficient district，motor development Where, N mo-i is amount of motor operating points drop in the efficiency zone, N mo is all amount of motor operating points According to the corresponding speed relationship between the motor and diesel motor group, efficient utilizing index of diesel electric group can be obtained as following: Where, N mo-de-i is amount of motor and diesel electric group operating points drop in the efficiency zone at the same time, N de-i is all amount of diesel electric group efficient operating points The efficient utilizing formula is similar for wheel side reducer and diesel electric group, the range which motor accounts for should be concluded in the wheel side reducer efficient range.
Driving cycle combining index
Mining dump truck driving conditions are complex and changeable.With regard to such working condition, we must also consider the basic speed of the vehicle and motor to match the design of the entire system.In this propose, the vehicle index in the efficient use of interval is presented as following : Where, N mc-i is amount of motor common operating points drop in the efficiency zone.Nmc is all amount of motor efficient operating points.
Rough set evaluation method
Main idea of rough set theory is to approximate portrayed imprecise or uncertain knowledge by using the knowledge in the knowledge which we have known.That is, when the object information is uncertain, inexact approximate, classifying the data and inferring the relationship between the reasoning data in order to identify implicit knowledge to reveal potential law, and then complete the judgmental forecasting and decision-making of things [14].
Determination of the functional properties
Relational data modeling: evaluation index is used as the condition attribute, and then the condition attribute collection C=﹛c 1 ,c 2 ,…,c n ﹜; Regarding expert evaluation of the results as the decision attribute, then the decision attribute set D=﹛ ﹜ y .Regarding the index value of the kobjects to be evaluated and the final composite score as a system of knowledge, then we can define uk=( c 1k ，c 2k ，…c nk ,y k ) ， ﹛ thus U= u 1 ,u 2 ,…,u m ﹜, Two-dimensional information table constituted by uk is relational data model about the evaluation object.
The function attribute data processing
First, some attribute indicators can be quantified in the evaluation system, some attribute index can not be quantified.The indicators which can not be quantified may take the form of scoring by experts to determine the possibility of good or bad, Quantifiable indicators ISSN: 1693-6930 TELKOMNIKA Vol. 12, No. 2, June 2014: 333 -342 336 data can be rated according to the measure values.Because different indicators is different on the magnitude and the dimensionless, even the impact direction on evaluation objectives is also inconsistent.Therefore, the the rating rules should be in unified magnitude and eliminate dimensionless, the scoring method is as follows [15]: When indicators are bigger the better When indicators are smaller the better Continuous data must be discretized, there are many discretization methods.According to the actual situation someone can be selected.In this paper, the the equidistant attributes discretization is choosen.a.calculatin of the attribute interval length where, zimax is the maximum of the i-th attribute; zimin is the the minium of the i-th attribute; ni the number of intervals.b.Determine the properties of the interval range.The range of each interval of the i th attribute is c.Calculate the quantized value of the property.
Determination of functional attribute weights
In the multi-index evaluation, different functional attributes may have different importance.By rough set theory, when some properties added to the classification,the system will directly be affected.In order to find out the importance of certain functional properties,the method is to remove some functional properties, and then examine the classification changes after the attribute.If the attribute is removed, the corresponding changes in the classification is great, the strength of the properties is high, that is of high importance.Conversely, the strength of the properties.That is of low importance .Weight multi-index evaluation can be determined by importance principle in rough set attribute Dependence of the knowledge D (decision attribute index) to the knowledge of C (evaluation set): Calculation of the evaluation importance Weight coefficient of evaluation:
Data processing
Motor evaluation shows that they are all quantifiable indicators, except C 4 , C 5 is the smaller the better,the rest are the bigger the better indicators.According to the national institute standards and the actual use requirements,evaluation of the upper and lower limits are presented as equation (11).
Weight determination
Using Eq.11 to calculate the score of each index, such as shown in Table 2; Using Equations ( 6) and ( 7) to determine discrete each attribute interval, according to the size of each attribute range.Discrete data should be finished.The results are shown in Table 3.
For example, take the original average the 7th highest permanent magnet synchronous motor analysis, we can see than E7 = 74.99%,further highlighting the advantages in the original basis.Overall evaluation of permanent magnet synchronous motor in the cost and control index is not very good, but the good comprehensive utilization rate makes the comprehensive evaluation index is high; on the other hand, induction motor although cost is low, but the comprehensive utilization rate is low, unable to play a maximum efficiency of the system.Switched reluctance motor and brushless DC motor control system based on the mature insufficiently, the comprehensive evaluation index in this field is not high, but has broad popularization prospect.This analysis shows that this system can be convenient for comprehensive evaluation of multi-motor.
The actual road conditions efficient use and the efficient using of diesel electric group must be combined with the real vehicle and platform experiment and analysis, In the platform experiment,diesel electric group and generators are as shown in Figure 2, Motor and dynamometer section is shown in Figure 3, the data in Table 3 is for manufacturers to provide a basic design data.The system we built ensures that energy transport from the diesel-electric group to motor after the rectifier inverter, by way of rectifier and inverter.Then the dynamometer turbine consumes the power.We selected the Cummins Engine (Cummins QSL9-C325) as shown in Figure 2. As shown in Figure 4, it's the engine speed-torque curve we measured in the laboratory.The engine common interval is from 1200 to 1800 rpm.This is enough to meet the demand for generators.Table 4 for the actual vehicle measurement data: For efficient use of high efficient utilization of the actual road conditions and diesel generator, the wheel reducer must be combined with the real vehicle and platform experiment were measured and analyzed, the original data in Table 3 provide basic design data for the factory.Table 4 shows the real measurement data.Comparison of the measured data, we found that the difference is not big for road use efficiency, the weight should be appropriately reduced, relative to the differences in the efficiency of the use of firewood in the electric drive is big different from hybrid cars drive turbine wheel side reducer, should be given full attention, adjust the weights, the advantage of permanent magnet synchronous motor is more apparent, which also bears out our rough sets to the accuracy of the theoretical system evaluation.
Conclusion
In accordance with the main source of supply of the dump truck electric drive system and electric drive energy transfer characteristics evaluation index system, a series of evaluation indicators proposed to make the motor evaluation more objective.The method of comprehensive evaluation based on rough sets mining dump truck traction motor, the evaluation index to make effective use of the rough set evaluation system.According to the importance of performance indicators index, based on rough sets empowering way, provides a theoretical basis for the selection of parameters of transmission system and electric motor matching, shorten the development cycle.The combination of platform experiment data, simulation results and real car long-term measurement data verify the accuracy and usefulness of the overall performance evaluation method based on the rough set method motor.
Figure 1 .
Figure 1.Traction motor evaluation system diagram
Figure 2 .
Figure 2. Diesel electric group and generators
Table 1 .
: motor evaluation parameters of various types | 2017-11-28T22:52:38.052Z | 2014-06-01T00:00:00.000 | {
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234266450 | pes2o/s2orc | v3-fos-license | Identification of connected arguments based on reasoning schemes “from expert opinion”
The work presented describes a combined approach to the partial extraction of the argumentative structure of a text that can be employed in the absence of sufficient annotated data to apply efficiently the machine learning methods for the direct detection of arguments and their relations. In this case, argument identification is performed by using the patterns of argumentation indicators created by a linguist and automatically expanded. These patterns enable the recognition of specific argument types with fine precision. In this study, arguments “from expert opinion” serve as such a pivot type. Besides, potential relations between recognized arguments are analyzed by dividing the text into superphrasal units (fragments united by one topic). The criterion for connecting arguments in an argumentative structure is their inclusion in the same superphrasal unit. An experiment for identifying potentially related arguments is conducted on a set of popular science texts with a minimum size of 1000 words.
Introduction
The field of research related to the automatic extraction of arguments has recently become quite independent (with the first specialized workshop held in 2014 [1]), stemming from the areas of opinion mining and sentiment analysis, since the tasks of recognizing emotional attitudes (positive, negative or neutral) towards entities or phenomena and recognizing opinions on them have been expanded with the problem of distinguishing the reasons and justification of specific opinions. At the moment, automatic identification of arguments and argumentative strategies across the texts of different genres is useful, for instance, for understanding and organizing debates, making decisions (for / against) in decision support systems, recognizing misguiding texts or radical opinions. Analysis of this type requires an automatic recognition of argumentative structure and argumentation units in applications performing it.
The employment of machine learning technique for extracting arguments and argumentative structures demands the corpora of texts annotated in a special way. We do not know whether such corpora exist for the Russian language. Deep annotation, an example of which is argumentation annotation, is labor-intensive. This is why the identification of argumentative structures can be performed with the use of combined methods which include, in particular, a pattern-based search with patterns created by a human expert.
Arguments in a text are often explicitly signaled by indicators called discourse connectives (because, therefore, consequently, etc.) or indicator patterns. Several types of these patterns indicate reasoning that refers to the opinion of experts (acclaimed in a given research field), such as the pattern Light verb + Opinion noun with the expert denoted in the subject of the phrase: Indicators of expert opinion reasoning schemes are notable for their high precision in the identification task. These easily identified anchor elements can be used to recognize text fragments that contain the argument and to predict the reasoning structure that reflects the process of opinion formation. One of the formal criteria for the identification of argumentative claim as a structure fragment can be its occurrence in a group of sentences united by the same topic.
The objective of the research is to develop a method for constructing a fragment of the argumentative structure based on the textual presence of arguments of a specific type and their role in the text topical structure and to apply this method to a collection of popular science texts.
Related works
The sequence of steps constituting the automatic identification of arguments is similar to the one for the manual annotation of text corpora [2]. This sequence includes 1) search for sentences and/or clauses containing argumentation, 2) identification of components and their roles in argument composition (premise, conclusion), 3) extraction of relations between arguments and their types (attack, support), and creation of the argumentative structure of the whole text.
Each step of the identification procedure can be performed by employing either machine learning methods or a set of expertly-created rules, or through the combined use of these approaches. If appropriately annotated text corpora are available, the use of machine learning methods is preferable as they do not require a time-consuming detailed study of the object. The exact machine learning method to be employed is chosen empirically as there are no obvious reasons for preferring one or another [3].
The quality of the identification procedure has been shown to depend significantly on the choice of features and combination of machine learning technique with rule-based methods. For instance, employing patterns with the supervised probabilistic sequence model for recognizing argumentcontaining sentences yields a 17% increase in the F-measure [4]. The advantages of methods employing linguistic knowledge are demonstrated in the study [5], where using lexical-syntactic trees for recognizing discourse boundaries results in the F-measure reaching the value of 0.83.
As shown in [6], capturing contexts of words when training a neural network along with using structural, syntactic, lexical and pragmatic features allows identifying discourse boundaries with the Fmeasure = 0.89. It is important to note that the comparison of different methods employed in Argumentation Mining tasks is difficult because different data sets are used, and their complexity influences the quality of the result. The components of an argument are also identified, as a rule, through the use of annotated texts and machine learning methods; for example, in [7] premises and conclusions are extracted with the SVM method (F = 0.74) while the preliminary classification of sentences as argumentative or non-argumentative is performed with the maximum entropy method. Along with structural, lexical and syntactic features, classification features include a model of the local context, discourse and rhetorical relations.
In [8], four classification methods (SVM, Naïve Bayes, Decision Tree and Random Forest) are used to identify the clauses of four types (major claim, claim, premise, non-argumentative) with the SVM recognized as the best. Classifiers are trained on structural (the location and punctuation of the argument component), lexical (n-grams (n = 1, 2, 3), verbs, adverbs and modals), syntactic and contextual features along with discourse markers (55 units in the dictionary used).
Knowledge-based approaches can be used if there are no annotated training data. Structural components of an argument are then identified through the use of expertly-created patterns similar to those described in [4]. For example, an author's attitude towards an opponent's stance is recognized by creating and using the patterns of the form SHELL I [MODAL] [ADVERB] AGREEVERB with the AUTHORNOUN, which include the lexemes of certain semantic classes: ADVERB, AGREEVERB etc. In this paper, linguistic data for identifying argumentative sentences are modeled with the created patterns of argumentation indicators [9], which in certain cases provide an identification of arguments' boundaries and recognition of their components.
The tasks of identifying relations between textual fragments, argument components, and argumentative textual segments are also primarily approached with machine learning methods if annotated corpora are available. The group of these tasks can be represented as follows.
1) Textual entailment recognition. The solution to the relations identification problem occasionally implies the possibility of inferring the content of one sentence from another, as in [10], performed by several research groups on the pairs of fragments (or sentences). It is shown that the best way to recognize the entailment of this type is to use lexical and syntactic features, labels of semantic roles and statistics of n-gram coverage, with the average precision reaching 0.8. In [11], topically related sentences are identified through the use of the LDA topic model. If argumentative relations connect a sentence and its predecessor from the same topic, then the entailment between the argument and preceding and succeeding sentences is checked. The precision of correspondence to the manual annotation is 0.72. The direction of the connection is not returned by the identification system.
2) Conflict relations detection. Identification of segments in microtexts (newspaper commentaries) is performed in [12] at the level of proponent \ opponent (pro and contra). Their recognition is based on the linear log-loss model; the features employed include the statistics of relative segment positions and their lengths, context windows, bigram coverage of segments and discourse connectives from a predefined list.
3) Identification of relations of a certain type. For example, argumentative relations, such as support and non-support, are identified in the already mentioned study [8] with the SVM classifier (F = 0.72), where the employed features are identical to those noted above but are used to characterize pairs of clauses. 4) Identification of attack \ support relations. A Naïve Bayes classifier which is trained on segments of premises and conclusions is used in [1] to detect distinct components of an argumentation scheme (two schemes are examined, "Expert Opinion" and "Positive Consequences"). After that, discourse markers are used to identify attack \ support relations between statements. The components of a scheme remaining unconnected are integrated into the structure in accordance with the results of the topical analysis of the text (using the LDA model). Similarly, the authors of [8] adapt models from [13] by introducing several alterations: changing the set of structural features, extending the list of discourse and argumentative markers. Additionally, they use the LDA method to identify topics and to create a topic list. Subsequently, a window-context model (in which a window covers the structure of the premise + conclusion) is employed with the result of a 10% F-measure increase in identifying attack relations, 4% and 1% in identifying support and non-support relations correspondingly.
Importantly, topical analysis is becoming a popular technique for detecting the relations of different types. In this work, topically related textual fragments are identified using the clustering profile for words and phrases [14], which for a given argument allows detecting potentially related fragments.
3. An approach to constructing argumentative structures An argument is a set of related statements used to prove the final statement (thesis, or conclusion). Statements that constitute an argument can be divided into premises and conclusion (claim) connected by certain relations. The relation type indicates whether a given argument is a proof (support) or refutation (attack) of the concluding thesis. A statement is a proposition formulated in a natural language (assertion). Relations between arguments unite them in the argumentative structure. In the proposed approach, external connections of the argument with other arguments are naturally represented by using its conclusion as a premise (assumption, exception) in the structure of another argument or as a shared claim. Alternatively the whole argument may serve as the conclusion of some inference.
Standards for describing arguments and argumentative structures are established in the Argument Interchange Format (AIF) [15]. As postulated in the AIF, arguments are represented by directed graphs with two different types of nodes: information nodes (statement nodes) and scheme nodes (argument nodes). Information nodes correspond to claims and premises while scheme nodes are used to represent typical reasoning models. Several dozens of argumentative schemes from Walton's Compendium are considered [16].
At the current stage, our approach to argumentation mining allows determining whether information nodes can be connected into a joint structure. However, the type of a reasoning model (except for the reasoning "from expert opinion") is not identified automatically. Expanding the patterns of reasoning "from expert opinion" with perception verbs (see, hear) enables partial recognition of the reasoning scheme "from position to know".
Constructing indicator patterns of expert opinion
At the first stage of constructing indicator patterns, an expert analyzes the content of argumentative textual fragments. He/she identifies the structure of the indicator and creates its description in the form of a lexical-grammatical pattern for the automatic search of corresponding text occurrences. Several components are defined in the structure of the pattern: constants (punctuation marks, designations etc.) and words and phrases of low variance that are further divided into discourse connectives (prepositions, conjunctions, parenthetical words: poetomu 'that is why', sledovatel'no, 'consequently', tak kak 'as', znachit 'hence', tem bolee chto 'all the more so that', naprimer 'for example', v chastnosti 'in particular' etc.) and content words (verbs and nouns) along with phrases expanding them (their relevant grammatical features and compatibility are also noted if necessary).
Here are some examples of the verbal constructions of a direct or indirect speech or opinion, with a core speech or mental verb as the predicate and a lexical unit for authoritative expert (class "expert") as the subject: professor… utverzhdaet: "…"; uchenye…zayavlyayut o tom, chto. In addition, indicator patterns have been constructed for parenthetical opinion phrases («po …mneniyu …lingvistov», «v sootvetstvii so slovami rukovoditelya proekta») and the Light verbs' constructions with opinion nouns: «uchenye vydvinuli gipotezu o…», «lingvisty prishli k vyvodu o tom, chto».
These constructions are generalized to the class "knower" by expanding the predicates of speech and opinion with the predicates of perception while the subjects are considered as possible witnesses of the situation in question, for instance: «Vo-pervyh, u kochevnikov ne bylo obychaya skvernoslovit'» or «V podtverzhdenie etomu -zapisi ital'yanskogo puteshestvennika Plano Karpini, posetivshego Central'nuyu Aziyu. On otmechal, chto u nih brannye slova voobshche otsutstvuyut v slovare».
Contextual analysis of indicators and representation of indicator structures in the form of patterns involves using the KLAN software [17], which performs preprocessing of a text (graphematic analysis, lexical-morphological analysis, extraction of phrases by grammatical models). Examining contexts provides an opportunity to generalize lexical components of the pattern on the lexicalsemantic level.
The analysis results in the identification of indicators with a simple structure and compound constructs. The former denote discourse connectives (single-or multi-word units) and content lexemes (inference predicates, speech and mental predicates etc.), while the latter stand for simple sequences of lexemes and punctuation marks, sequences with grammatical constraints and split constructions.
The following properties of compound indicators are worth mentioning: 1) constructions with constituents defined by grammatical characteristics (for example, predicates and governed objects); 2) constructions with constituents defined by semantic classes of lexical units (for example, predicates of specified semantic classes); 3) constructions with multiple splits (distant contexts).
A textual fragment with a formal description of the construction of an indicator that marks reference to an opinion / indirect speech is presented below (the construction is highlighted bold).
The verbal construction «lingvisty … schitayut, chto» is a compound indicator which contains a lexeme of the expert class in the subject position.
The pattern of the indicator can be formally represented as follows: Extension of indicator lists with words and phrases of low variance increases the recall measure in identifying arguments. It can be achieved automatically with the use of complementary methods of forming samples with variables and iteratively filling patterns on the sets of texts of a similar genre [9]. This type of extension enables distinguishing such types of variation as conditional synonymic variation at the level of morphological derivation, usage of the simplest negation forms, transposition of elements, and variation of indicator sequences due to inserted constructions limited in length.
Identification of superphrasal units, construction of clustering profiles
In this study, we assume that words unevenly distributed in the text are topically significant. The task of identifying non-uniformities of positional distributions of distinct words can be reduced to the examination of different schemes of arrangement of points on a line (each point can be interpreted as an occurrence of the analyzed word form in the text). Statistically significant topical clusters are effectively identified with the use of scan statistics [18].
Let x1, x2, …, xM be an arbitrary set of points from the unit interval (0, 1]. A uniformity hypothesis (H0) is tested against the alternative (H1) which represents a certain type of a deviation from uniformity. In the clustering case, the effective solution is based on the usage of a scan statistic n(d) which traces the maximum number of points n captured by an interval of the length d for all possible positions of this interval inside the unit interval. n(d) is calculated by counting the number of points captured by a window of length d which is sliding along the unit interval.
In this study, the statistic n(d) is replaced by a related statistic d(n) tracing the length of the minimal interval to capture exactly n points (2 ≤ n ≤ M). As the tabulation of the distribution of this statistic over a wide range of values for n and M is algorithmically difficult, simulation modeling can be used to evaluate how significant is the deviation of a statistic d(n), calculated for a specific text, from the value postulated by hypothesis H0 (uniformity).
The scheme of identifying positional anomalies (of the clustering type) in the distribution of lexical units appears as follows [14].
1. Frequency of each lemma is calculated in the normalized text. 2. Let x be an arbitrary lemma (lemmatized phrases can also be considered), F(x) is the number of its occurrences in the text, n is the fixed number of its subsequent occurrences in the text (2 ≤ n ≤ F(x)), d(n) is the length of the minimal textual fragment that captures n occurrences of x. Further analysis is conducted for word forms with the value F(x) ≥ Fb, where Fb is the threshold frequency value that depends on the length of the text N in word forms.
3. For each lemma, a cycle is organized over all possible values of n (Fb ≤ n ≤ F(x)). For the currently fixed n: a) the value of d(n) is calculated in the analyzed text; b) simulation modeling is used to evaluate the distribution of this statistic in accordance with the hypothesis H0. This is achieved by repeatedly shuffling words of the original text and forming its m 4. The value So, which is observed for the statistic d(n) in the original text, is compared to values calculated in the simulation experiment. Abnormal deviation from the uniformity of the clustering type is assumed to take place if the following condition holds true: (So ≤ Smin)&(So ≤ Savr -3s).
The significance of the identified cluster can be characterized with a dimensionless quantity δ(x) equal to the ratio of the average distance between the occurrences of x to the average inter-cluster distance between the occurrences of x.
The notion of a clustering profile is introduced to accumulate data about all the fragments where different lemmas cluster on the same figure. Formally, a clustering profile is a step function whose argument is the index number of a sentence in the text, while its value is the number of different clusters containing this sentence. At the same time, a given separate sentence cannot simultaneously include all lemmas clustered in the text fragment examined. The peaks of the clustering profile usually correspond to distinct micro-topics in the text, while gaps between them denote transitions from one micro-topic to another. The clustering profile reflects different relations (part\whole, general\specific, associations) between the lemmas clustered in the same text fragment.
An example of the clustering profile for lemmas in a popular science text is provided below, in Here the abscissa with the index numbers of sentences is directed downward while the ordinate (which represents the numbers of clusters) is placed horizontally, flowing from left to right. It indicates only the numbers of the sentences (ns) in which the value of the clustering profile changes (either new clusters are added or some of the previous disappear; nc is the number of clustered lemmas). The profile in Fig. 1 demonstrates the presence of two explicitly expressed micro-topics in the text in the ranges (1-31) and (32-92). One micro-topic corresponds to the textual content, while another is related to its discussion.
Different variants of cluster allocation in relation to each other are examined. Clusters can be positioned differently in relation to each other: one might be nested in another, they might intersect or appear disjoint. Their mutual position is considered in the following hypothesis: connected argumentative statements can belong to those text fragments that correspond either to the same cluster or nested \ intersecting clusters in the same micro-topic \ adjacent intersecting micro-topics.
The algorithm for identifying potentially connected textual fragments consists of the following steps (steps 2 and 3 can be performed independently): S1. Text preprocessing (lemmatization, morphological analysis). S2. Search for argument indicators in the text. S3. Construction of the clustering profile. S4. For every argument recognized at S1, the arguments or textual fragments potentially connected with it are identified in accordance with the clustering profile.
Results of the experiment
The collection of 68 popular science texts about linguistics (С1) is used to construct a system of 65 patterns (which are described in terms of lexical-semantic classes, individual lexical units and symbol sequences and which are characterized by the nesting property) representing 4 classes of the constructions of "expert opinion" indicators. An example of one such class is provided in section 4. The list of indicator constants (markers) is automatically extended using the proceedings of the international conference Dialogue-2005 "Computational linguistics and intellectual technologies". This list of markers is further increased by a factor of 1.5 and currently contains about 300 elements.
The test collection (С2) consists of 950 texts, with the average size about a thousand word forms. These texts belong to the genre of popular science online publications and are presented on the websites of different online publishers. Collected publications are not limited to any specific subject, structure or narrative type. The precision of the expertly created patterns reaches 74.18% on the training collection and 65.73% on the test collection. The precision measure on the training set demonstrates the frequency of an indicator construction in argumentation (as opposed to nonargumentative narration). False positive results often result from structural ambiguity, for example, the usage of quotation marks and dashes, the absence of annotated coreference relations for the experts described, mentioned with a proper noun or a pronoun; less frequent causes are the polysemy of lexical constants (pokazyvat' -dokazyvat' vs. delat' vidimym), homonymy of the "science-scientist" type (logika-logik), an object or an incomplete proposition in the actant position of a speech predicate. The increase of the number of test fragments by a third has decreased the precision of enriched patterns by 2−3%.
The size (n) and the parameter δ(x) of significant clusters are established experimentally using the С2 set. Dependence of these values on the textual length is apparent: short texts (around 1000 word forms) are characterized with n ≥ 3 and δ(x) > 2 or 3, while longer texts require n ≥ 5 and δ(x) >4.
The set С1 is employed to assess the possibility of identifying potential relations between arguments through the use of a clustering profile (an example of the profile is given in Section 5). An expert manually checks the presence of arguments related to the automatically identified argument "from expert opinion" in the textual fragments corresponding to the same cluster (I) or nested \ intersecting clusters (II) in the same micro-topic; then he/she examines the presence of connected arguments from different micro-topics (III).
I. An example below demonstrates textual fragments in which all arguments belong to the same "Language" cluster. The bold font is used to highlight statements numbered in brackets in accordance to the sequential order. Reformulations of a previous statement are connected to it by an equivalence relation (indicated by the sign "=" before the number of the sentence). Indicators of "expert opinion" are underlined by a single line, while the double line marks indicators of other arguments and the dashed line underlines structural indicators. | 2021-01-07T09:07:23.920Z | 2021-01-01T00:00:00.000 | {
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235084805 | pes2o/s2orc | v3-fos-license | FORMATION OF PROFESSIONAL COMPETENCIES IN FUTURE TEACHERS OF PHYSICAL EDUCATION IN THE CONDITIONS OF INTERACTIVE INTERACTION Formulation
The dynamics of development of the modern world causes progressive changes in education, reflecting the creative search for the most effective forms of organization and technology of learning. Recent studies outline the strategy of practical constructions, involving the education system in the development of the value-semantic sphere of personality on a pedagogical basis. In the offered article the authors consider actual problems of formation of professional competence which is considered as the integrated property of the person possessing a complex of professionally significant for the future teacher of physical culture qualities. The study identified the concept of professional competence of the future teacher, value orientations. The classification of value orientations of the future teacher of physical education which allows to adapt more successfully masters to modern educational processes is proved. The importance of the teacher's value orientations in the modern process of forming the moral beliefs of future teachers is determined. One of the possible ways of forming professional competence in the process of training future physical education teachers through interactive interaction is presented. It is this system of training allows by determining the general laws of psychology and pedagogy, specifying their basic provisions to determine the specifics of coaching.
An analysis of the latest research and publications in which the problem is solved and which the author relies on. Analysis of recent research and publications indicates that the problem of forming the value orientations of young people is in the spotlight of many scientists and educators. We can note that the very term "Orientation" has acquired scientific significance due to the work of T. Parsons -the founder of the theory of action and system-functional school in sociology. Orientation comes from the French orientation and literally means "eastward". Further, in particular, such scientists as L. Kondrashova studied in her work the management of students' personal self-improvement as a tool for personal and professional growth of young professionals, A. Lazaruk introduced the scientific substantiation of human values, J. Omelchenko studied the process of formation of universal human values, G. Pyatakov and N. Zayachkivska in their works dealt with the application of interactive teaching methods in higher education. L. Galitsyna also offered a number of trainings to improve learning outcomes.
The purpose of writing the article is to reveal the possibilities of using training in the educational process of higher education with the formation of value-semantic norms in the minds of students of the Faculty of Physical Education on the one hand, and on the other -the formation of cost standards that would ensure personal development in socio-cultural conditions. The main task of our article is to substantiate the methods of applying training as a tool for forming the value orientations of students.
Definition of research methodology. To determine the level of philosophy of competence of future teachers of physical education use diagnostic methods: the technique of self-assessment of Dembo-Rubinstein's personality, diagnostics of the cognitive component of bilingual culture, diagnostics of the communicative component of bilingual culture.
Methods of generalization, comparison, and systematization were used to justify the conditions of competence formation for the professional coaching activities of future physical education teachers. In order to determine the level of professional competence, we conducted a survey of 52 students of SHEI of Donbass State Pedagogical University of the 2nd and 3rd courses of the Faculty of Physical Education (Slovyansk).
The survey found that almost all students are focused on professional development, 78% understand the need to study specialized literature aimed at improving the professional skills of the future specialist, 56% believe that professional qualities are formed only in the course of production or practice, but the vast majority understand the need formation of professional competence through psychological and pedagogical methods.
Presenting main material. Every young person is aware of the conditions of their own existence, the purpose and means of its implementation, the regulatory social framework, forms of control, their own efforts and the extent of their implementation. The peculiarity of the individual is the ability to give things and phenomena of reality certain value traits, to make a certain assessment. In this case, values to some extent control human actions, determine their content, sequence, form a set of signs and phenomena that guide the individual in their behavior.
Value orientations are closely correlated with the life goals of the individual, needs, lifestyle, psychological attitudes, interests and are fundamental to the worldview of man, his moral, ethical, political and cultural preferences and beliefs. Studying the term "Value Orientation", researchers note a complex socio-psychological phenomenon that determines the direction and content of personal activity, is a key factor in the worldview of oneself and the world as a whole. The analysis of the classification of value orientations given by A. Lazoruk (Lazoruk, 2012, p.175) deserves attention. He groups them according to the following categories: -By level of development (higher or lower); -By object of appropriation (material, or moral and spiritual); -For the purpose of use (selfish or altruistic); -By method of detection (situational or clear); -By level of generalization (concrete or abstract); -By role in human activity (terminal or instrumental); -The content of activities (cognitive, creative, aesthetic, scientific); -By affiliation (individual, group, collective, social, national, universal); -By opposite meaning (positive-negative, primary-secondary, realpotential, direct-mediated, absolute-relative).
Thus, we conclude that value orientations have a significant role in selfrealization and the formation of a young person in the process of forming professional orientations. The task of high school teachers is to properly organize the learning process, which contributes to the formation of independent thinking of masters of physical education, to encourage them to be able to solve various problems that they face during education. It is with the help of training, as a form of interactive interaction, you can organize the learning process most effectively and creatively. In the training group, masters gain some social experience, offer examples of solving artificial learning situations that can be the basis for solving real-life situations and the formation of clearer professional orientations. Regular use of interactive trainings during the educational process allows to deepen such behavioral reactions as awareness of one's own motives, deepening responsibility for oneself, accepting oneself as one is at the moment. By including interactive training in the educational process, the teacher fundamentally changes his role, he becomes a facilitator, a student's assistant and accompanies him on the path of personal growth. (Domina 2019, p.105). The teacher has a task to organize the activities of masters so that the most important goals of personal growth are achieved during the training. In a game situation, masters can show their creative, professional and intellectual abilities, look at themselves from another angle, feel the collective spirit and learn to solve various problems together with a team of their peers.
The modern period of development of society, renewal of all spheres of social and spiritual life requires a qualitatively new level of education that would meet international standards. We can say that the interactivity of education in the process of physical fiction contributes to the formation of both subject skills and general education, the development of life values, creating an atmosphere of cooperation, interaction, development of communicative qualities of the individual. "Interactive learning (from the English. Inter -mutual, act -to act), according to O. Pirozhenko, is a special form of organization of cognitive activity, which aims to create comfortable learning conditions in which each student feels his success and intellectual ability" (Pometun, 2014, p.192).
Interactive learning changes the usual illustrative forms into dialogical ones, which are based on interaction and mutual understanding. Given the above, we can note the following methodological features of interactive learning: the use of problem situations and formulations, appropriate organization of learning space that promotes dialogue, motivational support for joint activities, compliance with the rules of educational cooperation, use of communicative methods and techniques, optimization of evaluation process and results activities, development of skills of self-analysis and self-control of individual and group activities.
The main feature of interactive learning is the use of their own experience by masters in solving problems. They are given maximum freedom of mental activity in the construction of logical circuits. The use of modern innovative technologies, in particular interactive learning technologies, significantly increases the efficiency of the educational process, promotes high intellectual development of masters, provides mastery of personal selfdevelopment skills, the ability to think and create. A feature of interactive learning is the preparation of future teachers for life and civic activity in civil society and the rule of law.
In order to identify the professional orientations of future physical education teachers, an interactive training "Auction of life values" was conducted. Each of the masters was asked to indicate the statements of life values on the cards. They had to choose the values that are closest and most relevant to them, then the masters were asked to justify their choice. The analysis conducted after the training gave the following results: in the group of masters of 20-25 people, only 4 participants spent all 10 cards, the latter were somewhat confused and could not fully decide on their choice. The special benefit of this training is that masters not only participate in the game situation, but also can analyze their choices, make a self-assessment of their own actions and decisions. After the training, the teacher should ask the students a few questions to determine if they are satisfied with their choice and if they would like to change that choice if given the opportunity. Masters must conclude that it is impossible to cancel the consequences of a decision already made, it is impossible to achieve all goals and take advantage of all opportunities, there are always certain limitations in time, strength, choice of opportunities, money, interests, etc., and for every choice made in life. a person pays a certain price. By modeling life situations, training interactive interaction provides invaluable experience and can teach you to be proud of yourself, your life, your choices. It allows you to experience the difficulties that can be encountered in real life, helps to feel inner strength, to form a focus and desire to implement their personal plans.
Therefore, the professional competence of the teacher is an integrated personality trait, possessing a complex of professionally important qualities for the teacher, has a high level of scientific-theoretical and practical preparation for creative pedagogical activity and effective interaction with students in the process of pedagogical cooperation based on the implementation of modern technologies results.
That is why the development of professional competence is a matter for the future physical education teacher himself, since he consciously regulates the standards of his behavior on the basis of acquired pedagogical experience. The professional growth of the teacher and the improvement of his professional competence are facilitated by: high spiritual and moral image of ZVO, which provide favorable creative working conditions; systematic selfeducational activity of the teacher; participation in projects, internships, competitions, conferences, seminars, etc .; emphasis on innovative, creative search component of scientific activity; objective evaluation of work; moral and material stimulation (Domina, 2019, p.105).
With the growing importance of interactive teaching methods, the number of interactive methods, forms of learning, a variety of trainings is increasing. Methods of cooperative learning, work in small groups, in pairs. Next, we will consider in more detail some types of exercises that a teacher can use during their classes. "Aquarium" -a variant of cooperative learning is effective for the development of masters of communication skills in a small group, the formation of the ability to discuss and argue their views, "Circle of ideas" -the purpose of this method is to resolve controversial issues, create a list of ideas and involve all students question, the technology is used when all participants have to perform the same task, consisting of several questions (positions). The "Press" method is used in cases when conflicting issues arise and you need to take and argue a clearly defined position on the social issue under discussion. The purpose of applying this method is to give garlic the opportunity during the lesson to learn to formulate and express their views on the discussion question in a clear and concise manner. "Carousel" version of cooperative learning is most effective for the simultaneous involvement of all participants in active work with different partners to discuss issues of discussion, "Discussion" is a broad public discussion of a controversial issue. It is an important means of cognitive activity that promotes the development of critical thinking, gives the opportunity to determine their own position, develops the skills of argumentation and defense of personal opinion, deepens knowledge of the problem. Rotational triplets students' activities in this case are similar to working in pairs. This version of cooperative learning promotes active, thorough analysis and discussion of new material in order to comprehend, consolidate and assimilate., Aimed at developing communication skills in the group, the ability to think critically, persuade, discuss, facilitate the analysis and discussion of new material (Garna, 2020, p. 60).
The methods of situational modeling include role-playing, staging, dramatization. All these forms of interactive learning form students' skills of cooperation, provide maximum freedom of intellectual activity, teach free discussion and confident defense of one's own opinion.
Conclusions and prospects for further research. The use of training technologies gives future teachers the opportunity to get rid of internal stress. In the artificially created conditions of interactive interaction the reassessment of vital values is carried out. In a safe game situation, which allows training participants to make sure of their own beliefs about the choice of profession, to determine their own moral principles and material beliefs, gives the opportunity to change their life positions. Prospects for further development of the use of methods of interactive training interaction, we see in the analysis of the problem "teacher-master" and the development of innovative training programs, as well as a reasonable establishment of order between different objects that fall into the value orientations of the future teacher. their factors that determine their behavior.
Domina V., Gutareva N., Sedova J. Formation of Professional Competencies in Future Teachers of Physical Education in the Conditions of Interactive Interaction
The dynamics of development of the modern world causes progressive changes in education, reflecting the creative search for the most effective forms of organization and technology of learning. Recent studies outline the strategy of practical constructions, involving the education system in the development of the value-semantic sphere of personality on a pedagogical basis.
In the offered article the authors consider actual problems of formation of professional competence which is considered as the integrated property of the person possessing a complex of professionally significant for the future teacher of physical culture qualities. The study identified the concept of professional competence of the future teacher, value orientations.
The classification of value orientations of the future teacher of physical education which allows to adapt more successfully masters to modern educational processes is proved. The importance of the teacher's value orientations in the modern process of forming the moral beliefs of future teachers is determined.
One of the possible ways of forming professional competence in the process of training future physical education teachers through interactive interaction is presented. It is this system of training allows by determining the general laws of psychology and pedagogy, specifying their basic provisions to determine the specifics of coaching.
Key words: value orientations, professional competencies, modernization of the educational process, interactive interaction, physical education, behavioral reactions. | 2021-05-22T00:04:39.449Z | 2020-01-01T00:00:00.000 | {
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260514938 | pes2o/s2orc | v3-fos-license | Tinea pedis: a clinico mycological study
Dermatophytosis is the superficial fungal infection of keratinized tissues caused by dermatophytes. They are a group of taxonomically related fungi that utilize keratin as a source of nutrients. Tinea pedis is a common superficial dermatophyte infection of the feet. It may present in several clinical varieties such as intertriginous, hyperkeratotic, vesiculobullous, ulcerative or a combination of these. It is often referred to as “Athlete’s Foot”. Tinea pedis may be accompanied by dermatophyte infection of other parts of the body including groin, hands or nails. It is estimated to affect about 15% of the population at large, being more common in closed communities such as army barracks and boarding schools, in warm weather, among those frequently using swimming pools, and when the feet are occluded with nonporous tight fitting shoes.
INTRODUCTION
Dermatophytosis is the superficial fungal infection of keratinized tissues caused by dermatophytes. They are a group of taxonomically related fungi that utilize keratin as a source of nutrients. Tinea pedis is a common superficial dermatophyte infection of the feet. It may present in several clinical varieties such as intertriginous, hyperkeratotic, vesiculobullous, ulcerative or a combination of these. It is often referred to as "Athlete's Foot".
Tinea pedis may be accompanied by dermatophyte infection of other parts of the body including groin, hands or nails. It is estimated to affect about 15% of the population at large, being more common in closed communities such as army barracks and boarding schools, in warm weather, among those frequently using swimming pools, and when the feet are occluded with nonporous tight fitting shoes.
In the west, tinea pedis is estimated to be present in about 40% of all patients who attend clinics for any medical concern. 1 Those patients with more severe symptoms seek medical help and often have concomitant fungal infection of the toenails. 2,3 There are many undiagnosed cases, many of which may be asymptomatic and unsuspecting of having tinea pedis and be a possible source of infection for others. 2 Chronic infection is common in patients with concomitant diabetes, atopy, and immunosuppression. In an increasingly ageing population and with the increasing numbers of immune compromised patients, tinea pedis is emerging as an important and a significantly prevalent infection.
METHODS
Seventy five randomly selected patients with tinea pedis, during the period August 1 st 2007 to September 3o th 2009, were enrolled to the study. The study was conducted in Mycology department of Department of Dermatology, Madras Medical College. A clinical diagnosis of tinea pedis was made by the presence of scales, fissuring and morphology of the lesions and all cases of tinea pedis were first confirmed by KOH examination.
Age, sex and duration of the disease were recorded. A detailed history was obtained in all the patients with regard to socioeconomic status, occupation, about the habit of shoe wearing, any similar episodes in the past with exacerbation during summer months or associated with hyperhidrosis. Any contact with pet animals and living in institutions with habit of sharing of shoes or socks, similar illness among the residents or the family members were especially enquired into. History of any systemic illness and treatment were also recorded.
The dermatological examinations included the clinical type of infection with areas of involvement. Particular importance was given to the presence of any anatomical deformities of the foot, spacing between the toes and the nature of sweating. Any other associated dermatological condition of the foot was also noted. Screening for dermatophyte infection over other areas of skin and nails especially the palms for ide eruptions were done. Systemic examination and screening for other dermatological disorders were done in all the patients.
In all the 75 patients, mycological examination of the skin scrapings from the affected site or blister top specimens from the vesiculobullous lesions were carried out in wet mount in 10% potassium hydroxide (KOH). Isolation of the agent was done through inoculating the specimen (scales or crusts) in Modified Sabouraud's Dextrose Agar medium with cycloheximide. The isolates were studied with regard to macroscopic, microscopic colony morphology and pigment production. Differentiation of the species was done by culturing on corn meal agar and looking for the persistence of pigmentation.
Inclusion criteria
All cases of tinea pedis who are comfirmed by KOH positivity
Exclusion criteria
KOH Negative tinea pedis patients and patients with pompholyx lesions
Sex distribution
Out of the 75 patients enrolled into the study 52 (69.3%) were males and 23 (30.7%) were females.
Age distribution
The youngest patient in the study was a 5 year old male child and the oldest a 60 year old man. Majority of patients were in the age group 21 -30 years.
Association with shoe wearing
Among the patients studied shoe wearing habit has been noted in only 23 cases. Most of these patients are students and others were policemen, drivers and businessmen who must wear shoes as routine as their occupation demands.
Episode of tinea pedis
In the study 67 cases presented to us with tinea pedis for the first time and 8 patients had recurrent episode. 7 patients with recurrent episode were having regular shoe wearing habit. Bilateral affection of foot by tinea pedis was seen in 7 patients (9.3%).
Systemic associations
Among the 75 patients studied 12 patients were diabetics (16%). Hyperhidrosis as associated in 11 patients (14.6%). 7 patients gave history of atopy and 2 patients were known HIV positives.
Blood group distribution
The blood group distribution in dermatophytosis patients demonstrated that 37.3% of the patients belonged to group O+, 32% to group B+ and 21.3% to group A+. Other blood groups found were AB +ve 4 cases, AB-ve 2 cases and 1 patient O -ve.
Association with other types of dermatophytosis
Tinea pedis was an isolated entity in 46 patients (61.3%). Among the remaining, 29 patients (38.7%) had other types of dermatophytosis and 11 patients studied had associated tinea unguium infections. Ide eruption was seen among 2 patients with vesiculobullous tinea pedis.
Association of other foot dermatoses
The foot was examined for the presence of other dermatosis and the other dermatoses associated are given in the following table. Traumatic fissure was the most common finding seen among 15 patients. Crowding of toes was noted in 8 patients.
The various types of tinea pedis observed
Of the 75 patients, 65 patients (86.7%) had a single clinical type fitting into the 4 major forms. Other 10 cases (13.3%) presented with combination of two types of the clinical varieties. The type of tinea pedis and the organism isolated with each are recorded in the table given below.
Web space affected
Total number of interdigital type of tinea pedis= 36. The predominant web space involved was the 3 rd space with a total of 15 patients. Next commonly involved was both the 3 rd and 4 th web spaces together with 10 cases.
Culture positivity
Positive isolates were obtained in culture among 61 patients from the total 75 patients, giving the culture positivity rate of 81.3%. Table 13 below gives the various organisms isolated and the morphological type with which they were associated. The macroscopic morphology of the colony of Trichophyton rubrum was downy with the diffusible red pigment on the reverse. On lactophenol cotton blue staining, microscopically the hyphae were thin with few to plenty of microconidia both in enthyrse and engreppe distribution. Pencil shaped macroconidia were also seen. Subcultures in corn meal agar produced diffusible red pigment characteristic of Trichophyton rubrum.
Type of tinea pedis and the organism isolated
The macroscopic colony of Trichophyton mentagrophytes appeared as white powdery colony on the surface which in some cases developed a cream centre. The reverse appeared tan to golden in colour with no pigment production. On microscopic examination, spherical microconidia arranged in bunches and linear distribution around the hyphae were seen. Spiral hyphae and macroconidia were present in some isolates. Subcultures in corn meal agar did not produce any diffusible red pigment into the medium.
DISCUSSION
Out of the 75 cases taken in our study 52 were males and 23 were females. This gave a male: female ratio of 2 shoe wearing in our study the maximum were students (34.7%) followed by policemen, businessmen and car drivers whose occupation compels them into the habit of shoe wearing. Tight fitting occlusive footwear, and usage of non-absorbable socks were the most important predisposing factors which caused fungal infection in these patients. In the West it has been called penalty of civilization as tinea pedis has been the predominant type of dermatophyte infection among the most active younger age group people.
Figure 2: (A) Intertriginous type of tinea pedis, (B) Vesiculobullous type of tinea pedis-blister top, (C) Hyperkeratotic type of tinea pedis: Moccasin type, (D) Ulcerative type of tinea pedis, (E) Hyperkeratotic type of tinea pedis with tinea manuum, (F) Associated with tinea unguium, (G) Associated with traumatic fissure, (H) Associated with keratolysis punctate, (I) Vesiculobullous type of tinea pedis with ide eruption.
Hyperhidrosis was associated with tinea pedia is 11 cases (14.6%). Bilateral affection of foot by tinea pedis was seen in 7 patients (9.3%). Such bilateral involvement was observed in the regular shoe wearing population especially school and college students. Among the patients 8 of them had recurrent episode of tinea pedis at presentation and others in the study presented with their first episode. Like the bilateral involvement of the foot, recurrent episodes were also found in the majority of regular shoe wearing population.
Among the 75 patients 12 patients are diabetics (16%). Among the 12 cases 5 were newly diagnosed as diabetics on investigation and 7 were already known diabetics. The prevalence rate of diabetes among the patients in our study is less when compared to study by Abbas Ali et al which recorded 24.8%. In our study 6.6% of patients turned out to be diabetics on screening. 9 Diabetes is one of the most important predisposing factor for foot diseases including tinea pedis among the elderly age group patients. The compounding factors of immune suppression, local trauma and vascular problems that are associated with diabetes make the person more susceptible to fungal foot diseases. 10 Any patient diagnosed with tinea pedis should be screened for diabetes as routine.
Figure 4: (A) Sabouraud's dextrose agar-Trichophyton rubrum, (B) Sabouraud's dextrose agar-Trichophyton rubrum-reverse pigmentation, (C, D) Lactophenol cotton blue mount Trichophyton rubrum-enthyrse/engreppe arrangement of microconidia, (E) Trichophyton rubrum-persistence of pigmentation.
Atopic diathesis history was obtained in 7 cases (9.3%). 11 Similarly, Balajee et al suggested that individual from group A are more susceptible to dermatophyte fungi infections, once they found an increased percentage of patients belonging to this blood group when compared to the control group. 12 But the study results of Neering et al and from the study done in our hospital by Selvi et al, no significant association with specific blood group and increased incidence of fungal infection has been proved. 13,14 The blood group distribution in our study showed 37.3% of patients belonging to blood group O +ve, 32% patients belonging to B+ve group, 21.3% patients belonging to A +ve and rest 9.4% to other groups. This distribution reflects the blood group prevalence that is noted in the general population.
Figure 5: (A) Sabouraud's dextrose agar Trichophyton mentagrophytes var interdigitale, (B) Lactophenol cotton blue mount Trichophyton mentagrophytes-spiral hyphae, (C) Corn meal agar Trichophyton mentagrophytesno pigmentation.
Intertriginous scaling type with slight extension to the adjacent plantar and dorsal surface was the commonest presentation observed in these series (36 cases, 48%) followed by scaling hyperkeratotic type in 20 cases (26.6%). The vesiculobullous type of tinea pedis came next with 7 cases and the least common was the ulcerative type with 2 cases. Mixed types were observed in 10 patients. 7 patients with interdigital and hyperkeratotic variety and 3 patients with interdigital and vesiculobullous variety.
As per the literature and the study by Abbas ali et al, 4 th web space and 3 rd web space were the most commonly involved sites. In our study, among 36 patients with the interdigital type of tinea pedis, the web space most commonly involved was the third with 15 patients presenting with such involvement which is in coherence with above study. Both 3 rd and 4 th web spaces were involved in 10 patients. 9 Fourth web space was involved in 5 patients, second web space in 4 patients and first web space in 2 patients.
On examination of foot anatomical problems like crowding of toes was found in 8 of the patients studied. 5 patients had associated keratolysis punctata. Most were women who had history of frequent immersion in water for long periods during the house hold works. The maceration and trauma associated with such predisposition are the major factor responsible for the occurrence of tinea pedis among the house wives who does not have the habit of shoe wearing or other factors that usually predispose men for fungal infection. Other foot problems noted were traumatic fissure in 15 patients, plantar wart among 6 patients and corn foot in 4 patients.
As per study by Jacek et al one third of patients with toe nail onychomycosis had tinea pedis. Infected toe nails may be the site of primary infection and fungal disease can spread to other body areas from them. 10 In a study by Zais et al one third of patients with toe nail onychomycosis had tinea pedis. 15 Other dermatophyte infections noted among the patients with tinea pedis, in our study, tinea unguium was the most commonly associated problem among 11 patients (14.6%). The toe nail involvement was seen in 9 cases.
Tinea cruris was the next common dermatophyte infection among the tinea pedis patients with 6 patients, followed by tinea corporis 5 cases and tinea manuum 2 cases. A 13 year old school student who presented with vesiculobullous type of tinea pedis had associated tinea capitis.
CONCLUSION
Intertriginous type of tinea pedis was the most common type observed in this study and 3 rd web space was the most commonly affected. Trichophyton rubrum was the chief isolate among the patients of Tinea pedis in our study which affected young males more commonly. Epidermophyton floccosum was not isolated from any of the culture specimens. Most common associated factor and the cause of recurrence is the presence of tinea unguium lesions in toes. No common blood group associations as suggested by other studies were not noted in our study. Any patients diagnosed with tinea pedis should be screened for diabetes as implied by the outcome of the study. Among the 12 patients who were associated with diabetes 5 were newly diagnosed patients after our investigation. In all patients diagnosed with chronic recurrent dermatophytosis screening for foot lesions and treating the tinea pedis lesions completely helps in preventing the relapses and recurrences.
Points:
Prevalence of tinea pedis infection was more common among males with male: female ratio of 2.26:1.
The 21-30 years age group was commonly affected by tinea pedis. The mean age was 28.8 years. Shoe wearing habit has been encountered among 30.6% of patients. Most of them were school or college students and policemen who have to wear shoes compulsorily. More incidence of bilateral involvement and recurrent episodes were noted among the shoe wearing population than in the non shoe wearing population Bilateral involvement was seen in 9.3% and hyperhidrosis was associated among 14.6% patients. Any patients diagnosed with tinea pedis should be screened for diabetes as implied by the outcome of the study. Among the 12 patients who were associated with diabetes 5 were newly diagnosed patients after our investigation. The blood group distribution in our study showed 37.3% of patients belonging to blood group O +ve. The distribution of blood groups reflected the general distribution pattern of the population. Intertriginous type of tinea pedis was the most common type observed in this study in 36 patients. 3 rd web space was the most commonly affected with involvement seen in 15 patients. 4 patients had extensive dermatophytosis and ide eruption was noted in 2 patients. Among other dermatophyte infections noted in the patients with tinea pedis, tinea unguium was the most commonly associated problem in our study with 11 patients (14.6%). Infected toe nails may be the site of primary infection and fungal disease can spread to other body areas from these primary sites. Other foot abnormalities noted were crowding of toes, traumatic fissures, keratolysis punctata, corn foot and plantar wart. Culture positivity was recorded in 81.3% (61 cases) of cases studied. Trichophyton rubrum happened to be the chief isolate. Among the 61 culture positive specimens Trichophyton rubrum was isolated in 47 cases. Trichophyton mentagrophytes was isolated from the rest of the tinea pedis patients. Trichophyton rubrum was isolated more commonly from the dry squamous or hyperkeratotic lesions of tinea pedis and Trichophyton mentagrophytes was isolated from wet vesicular lesions. | 2019-03-17T13:06:51.096Z | 2017-08-24T00:00:00.000 | {
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244700166 | pes2o/s2orc | v3-fos-license | Nogo receptor impairs the clearance of fibril amyloid‐β by microglia and accelerates Alzheimer’s‐like disease progression
Abstract Alzheimer's disease (AD) is characterized by the progressive accumulation of β‐amyloid (Aβ)‐containing amyloid plaques, and microglia play a critical role in mediating Aβ clearance. Mounting evidence has confirmed that the ability of microglia in clearing Aβ decreased with aging and AD progress, but the underlying mechanisms are unclear. Previously, we have demonstrated that Nogo receptor (NgR), a receptor for three axon growth inhibitors associated with myelin, can decrease adhesion and migration of microglia to fibrils Aβ with aging. However, whether NgR expressed on microglia affect microglia phagocytosis of fibrils Aβ with aging remains unclear. Here, we found that aged but not young microglia showed increased NgR expression and decreased Aβ phagocytosis in APP/PS1 transgenic mice. NgR knockdown APP/PS1 mice showed simultaneous reduced amyloid burden and improved spatial learning and memory, which were associated with increased Aβ clearance. Importantly, Nogo‐P4, an agonist of NgR, enhanced the protein level of p‐Smad2/3, leading to a significant transcriptional inhibition of CD36 gene expression, which in turn decreased the microglial phagocytosis of Aβ. Moreover, ROCK accounted for Nogo‐P4‐induced activation of Smad2/3 signaling. Finally, the decreasing effect of NgR on microglial Aβ uptake was confirmed in a mouse model of intra‐hippocampal fAβ injection. Our findings suggest that NgR may play an important role in the regulation of Aβ homeostasis, and has potential as a therapeutic target for AD.
age-dependent decrease in the ability of microglia to phagocytose Aβ fibrils, as well as a decrease in the expression of Aβ-interacting protein in vitro (Floden & Combs, 2011). Thus, microglia are emerging as critical regulators of innate immune responses in AD, and understanding the molecular and cellular mechanisms that cause microglial dysfunction may lead to the identification of strategies to restore healthy microglial function and prevent the development of AD.
The Nogo receptor (NgR), as a receptor for three axon growth inhibitors associated with myelin, has been extensively studied due to its role in triggering growth cone collapse and arresting neurite/axon growth (Fournier & GrandPre, 2001). In addition, recent studies have provided direct evidence of a role of the NgR in AD. For example, in patients with AD and elderly rats with spatial cognitive deficits, Nogo-A and NgR expression are increased (Gil & Nicolas, 2006;VanGuilder & Sonntag, 2013;Zhu & Guo, 2007), suggesting their potential roles in AD pathogenesis and progression. Furthermore, NgR physically interacts with amyloid precursor protein (APP) and Aβ in a pocket on the concave surface, which can be distinguished from the surface required for Nogo-66 binding (Park & Gimbel, 2006). Subcutaneous NgR (310) ecto-Fc treatment reduces the brain Aβ plaque load and improves spatial memory in APPswe/PSEN-1ΔE9 transgenic mice (Park & Widi, 2006). Under normal circumstances, NgR is reported to be detected mainly in neurons (Zhang et al., 2014), but in some pathologic state including AD, NgR is also expressed on the surface of microglia and has indispensable effects (Fang & Yao, 2016;Satoh et al., 2005).
Our previous study demonstrated that the Nogo/NgR pathway plays a role in the Aβ pathology that characterizes AD by modulating microglial adhesion and migration to Aβ (Fang & Wang, 2018). However, the effects of NgR expressed on microglia in Aβ phagocytosis and the underlying molecular mechanism are not entirely understood.
Cluster of differentiation 36 (CD36) is a member of the class B scavenger receptor family, which could transport various molecules into cells such as fatty acids, collagen, and oxidized low-density lipoproteins (Endemann & Stanton, 1993). CD36 as a major pattern recognition receptor expressed on microglia had been show to bind to Aβ (El & Moore, 2003), and impaired recycling of CD36 reduces the phagocytic efficiency of microglia toward Aβ (Lucin & O'Brien, 2013). Moreover, CD36 is increased by blockade of the Nogo/ NgR pathway in the microglia of double transgenic mice expressing a chimeric mouse/human APP and mutant human presenilin 1 protein (APP/PS1) mouse (Fang & Wang, 2018). In addition, Nogo-66 binding to NgR inhibits the adhesion and migration of microglia through the RhoA/Rho-associated kinase (ROCK) pathway in vitro (Yan & Zhou, 2012). However, the precise mechanisms involved remain unclear. Blockade of transforming growth factor beta (TGFβ)activated Smad2/3 reportedly increases Aβ phagocytosis (Town & Laouar, 2008), and TGFβ signals through Smad2/3 to downregulate the scavenger receptor CD36 (Rustenhoven & Aalderink, 2016).
Here, we examined the role of NgR in microglial phagocytosis that occurs in AD using microglia of different ages, and found that aging microglia exhibited decreased Aβ clearance in APP/PS1 transgenic mice. Conditional knockdown of NgR reduces amyloid burden and improves learning and memory by enhancing Aβ phagocytosis in the brain. In addition, we elucidated that NgR knockdown promoted Aβ phagocytosis by regulating ROCK-Smad2/3-CD36 signaling in primary microglia. Furthermore, NgR decreased microglial Aβ uptake in a mouse model of intra-hippocampal fAβ injection. Together, NgR signaling mediates an age-dependent decrease microglial Aβ phagocytosis in the development of AD-like pathology, suggesting that NgR is a potential therapeutic target for AD.
| Aging decreases Aβ phagocytosis and upregulates NgR levels in APP/PS1 mice microglia
Microglia play a critical role in AD by contributing the clearance of Aβ, but the ability of microglia to clear Aβ may decrease with age in the APP/PS1 mouse (Hickman & Allison, 2008). To further confirm whether aging affects Aβ phagocytosis, primary microglia isolated from APP/PS1 mice and their age-matched wild-type (WT) littermates, in both young (3 months) and aged (8 months) mice, were incubated in the presence of green fluorescein amidite (FAM)-Aβ (0.5 μM). After 4 h exposure to Aβ, intracellular levels of Aβ were determined. The uptake of Aβ was significantly decreased in aged, but not young microglia in APP/PS1 mice and slightly dampened in WT mice (no significant difference) (Figure 1a-b). Consistent with this, at 8 months of age microglia from APP/PS1 transgenic mice showed significantly reduced Aβ phagocytosis compared to their age-matched WT littermates, but there was no significant difference at 3 months of age ( Figure 1b). Western blotting and quantitative PCR (qPCR) were used to investigate whether aging or AD transgene also induced NgR expression changes in microglia, respectively. Aged microglia expressed a higher NgR expression compared to young microglia from APP/PS1 transgenic mice, and it also can be seen in WT mice that the expression of NgR slightly increased over time (no significant difference) . Interestingly, at the 8 months, microglial NgR expression was much higher in APP/ PS1 transgenic mice than WT mice instead of 3 months, which indicated that AD transgene and aging together induced the increase of NgR expression in microglia. Taken together, these findings suggest that microglia show age-dependent upregulation of NgR in the APP/ PS1 mouse, which may be associated with the decline in Aβ phagocytosis levels.
| Knockdown of NgR in microglia decreases amyloid deposition, rescues cognitive deficits, and increases microglial clearance of Aβ in the APP/PS1 transgenic mice
The APP/PS1 transgenic model exhibits an increase in plaque burden between the ages of 4 and 14 months (Jankowsky & Slunt, 2001).
Therefore, in this study, the APP/PS1+LV-control and APP/PS1+LV-shNgR animals were examined at the stage of compact and diffuse plaque burden (14 months). A widespread distribution of Aβ plaques stained with 6E10 was found throughout the hippocampus and cerebral cortex of 14-month-old APP/PS1+LV-control mice. By contrast, APP/PS1+LV-shNgR mice showed a marked reduction in Aβ plaque To investigate whether the reduction of amyloid plaque deposition and Aβ levels induced by NgR knockdown is associated with improved memory performance, we assessed spatial memory in 14-month-old WT+LV-control, WT+LV-shNgR, APP/PS1+LVcontrol, and APP/PS1+LV-shNgR mice using the novel object recognition (NOR) test, which is a memory task that relies on the innate preference of mice for a novel rather than familiar object.
Mice were trained as illustrated in Figure 2c. APP/PS1+LV-shNgR mice performed normally, in contrast to APP/PS1+LV-control mice, which were not able to distinguish the old from the new object at 24 h ( Figure 2d). Next, we used the Morris water-maze (MWM) test to further confirm these results. Spatial learning and memory in WT+LV-control and WT+LV-shNgR mice were unaltered (Figure 2e-h). Compared to WT+LV-control mice, APP/ PS1+LV-control mice displayed impaired spatial learning, reflected in the time traveled to the platform at Day 6 ( Figure 2f, h). NgR F I G U R E 1 Microglia shows age-dependent decline in Aβ phagocytosis and NgR upregulation from APP/PS1 mice. (a) Representative images of phagocytosed by primary microglia (Iba-1, red) isolated from 3-month-old or 8-month-old WT mice and APP/PS1 mice after incubated with 0.5 μM FAM-Aβ (Anaspec, green) for 4 h. Scale bar: 20 μm. (b) The quantification of the phagocytosis assay. n = 3wells/ group, and N = 3 independent experiments (data presented are the average of all experiments). (c-d) NgR protein (c) and mRNA (d) levels was measured in microglia isolated from 3-month-old or 8-month-old WT mice and APP/PS1 mice. n = 3-5 samples/group in every experiment, and N = 3 independent experiments. Values were reported as the mean ±SEM. Statistical analyses were performed using a two-way ANOVA with Bonferroni's multiple-comparison post hoc test, *p < 0.05, **p < 0.01 for microglia from 8-month-old APP/PS1 mice vs. 8-month-old WT mice; and #p < 0.05, ##p < 0.01 for microglia from 8-month-old APP/PS1 mice vs. 3-month-old APP/PS1 mice
| Inhibition of NgR promotes Aβ phagocytosis by upregulating CD36 in primary microglia
Although NgR physically interacts with APP and Aβ (Park & Gimbel, 2006), and inhibits microglial adhesion and migration to Aβ (Fang & Wang, 2018), little is known regarding the molecular mechanism underlying the direct regulation of Aβ phagocytosis by microglial NgR. To determine how and whether Aβ phagocytosis is directly regulated by microglial NgR, phagocytic efficiency was evaluated by measuring the intracellular levels of FAM-Aβ in primary microglia purified after treatment with control siRNA or NgR siRNA for 24 h.
Troy as a partner of NgR, found to be expressed on the surface of microglial cells (Park & Yiu, 2005), was also silenced by siRNA interference at the same condition. Nogo-p4 is an agonist of NgR, and Rtn-p4 (Reticulon 1(Rtn1) non-inhibitory control/blocking peptide) was used as the negative control. Our data showed that Nogo-p4 decreased phagocytosed Aβ compared to Rtn-p4 (Figure 3a-b).
Conversely, both NgR siRNA and Troy siRNA reversed this effect.
These data indicate that NgR on microglia can mediate the clearance of Aβ.
CD36, as a pattern recognition receptor on macrophages, mediates the clearance of Aβ, as well as of microbial pathogens (El & Moore, 2003). APP/PS1+LV-shNgR mice showed greater expression of CD36 compared to APP/PS1+LV-control mice ( Figure 3c). Given that the expression of CD36 was up- However, the protein levels of RAGE and Trem2, as the microglial surface receptors genetically linked to Aβ phagocytosis, were not affected by NgR siRNA (Figure S1a-b). Aβ internalization in microglia is increased by blockade of TGFβ-Smad2/3 signaling (Liu & Liu, 2014;Town & Laouar, 2008), and TGFβ downregulated the scavenger receptor CD36 through Smad2/3 transcription F I G U R E 2 NgR knockdown leads to a strong reduction of amyloid deposition, rescues memory impairment and enhances microglial Aβ phagocytosis in APP/PS1 mice. (a) Brain sections of 14-month-old APP/PS1+LV-control or APP/PS1+LV-shNgR mice were stained with Hoechst (blue) and 6E10 (red) to detect Aβ deposition. Scale bar: 500 μm. (b) The brain was homogenized and separated Aβ into insoluble and soluble fractions. Amounts of Aβ42 and Aβ40 were measured by ELISA and normalized to the homogenate protein concentration in hippocampal and cortical regions. Statistical analyses were performed using 2-tailed Student's t-test, n = 5-8 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. (c) Overview of the NOR memory test, with the novel object (green triangle). (d) Recognition memory (recognition session, 24 h) of the old versus new object was assessed as the discrimination index (DI), the ratio of time spent exploring the old object to time spent exploring both objects. Statistical analyses were performed using 2-tailed Student's t-test, n = 9-12 mice per group. *p < 0.05; **p < 0.01. (e-h) Morris water-maze analysis of 14-month-old mice on 6 consecutive days, including 5 days training test (Days 1-5) and 1 day probe trial test (Day 6). Data were shown as mean ±SEM. Statistical analyses were performed using a two-way ANOVA with Bonferroni's multiple-comparison post hoc test, n = 9-12 mice per group. *p < 0.05, **p < 0.01 for APP/PS1+LV-control vs. WT+LV-control on Day 6; #p < 0.05, ##p < 0.01 for APP/PS1+LV-shNgR vs. APP/PS1+LV-control on Day 6. (e) Representative runs from the probe trials on Day 6. (f) Mean escape latency from Day 1 to Day 6. (g) Percentage of finding the platform from Day 1 to Day 6. (h) Left panel: Mean escape latency during the probe trial on Day 6; Right panel: Percentage of time spent in the target quadrant during the probe trial on Day 6. (i) Coronal sections from 14-month-old APP/PS1+LV-control or APP/PS1+LV-shNgR mice were stained with Hoechst (blue) for nuclei, 6E10 (red) for Aβ, and Iba-1 (green) for microglia. Representative images of the cerebral cortex and hippocampus regions were shown. Scale bar, 25 μm. (j) Left panel: Quantification of the Aβ plaque area in brain regions (cerebral cortex and hippocampus); Right panel: Quantification of the Iba-1+ microglia area in brain regions. (k) The ratio of Iba-1+ microglia area to 6E10+ Aβ plaque area in brain regions. (l) CD68+ lysosomes (green) containing Aβ immunoreactive content (red) were detected in mice (arrowheads). Scale bars: 20 μm. (m) CD68+ lysosomal content associated with Aβ plaques were quantitatively analyzed. (n) The lysosomal perimeter of microglia. (i-n) Data were shown as mean ±SEM. Statistical analyses were performed using 2-tailed Student's t-test. n = 5-8 mice per group, and 2 brain sections per animal were used for the analysis. *p < 0.05, **p < 0.01 (Rustenhoven & Aalderink, 2016). However, whether NgR modulates CD36 by mediating Smad2/3 signaling has not been investigated. Immunofluorescence staining showed that NgR siRNA decreased Smad2/3 nuclear translocation to microglia (Figure 3f-g). Furthermore, NgR siRNA or Troy siRNA significantly attenuated the phosphorylation of Smad2/3 in microglia that was increased by Nogo-p4 (Figure 3h-i). The results indicated that Nogo-P4 bound to the NgR and triggered the activation of the Smad2/3 signaling. Collectively, these data showed that Smad2/3 and CD36 are essential for Aβ phagocytosis in primary microglia regulating by NgR.
Of note, these data suggest that NgR decreases Aβ phagocytosis through ROCK-Smad2/3-CD36 signaling, but not the NF-кB or PPARг pathway.
| NgR decreases microglial Aβ uptake in the AD model after intra-hippocampal fAβ injection
To better understand the role of NgR in microglial Aβ uptake in vivo, WT and Cx3cr1CreER mouse were administered adeno-associated virus (AAV)-control or AAV-DIO-NgR (AAV-NgR), with or without Nogo-p4, and subsequently received fibrillar Aβ42 (fAβ) injections into the hippocampus at the age of 2 months. To investigate the efficiency of tamoxifen, we first analyzed the pattern of hippocampal green fluorescent protein (GFP) fluorescence in mice 30 days after tamoxifen treatment. In Cx3cr1CreER mice administered tamoxifen, GFP+staining expressed only of microglial cells (Iba-1+) in brain sections, but not astrocytes (GFAP+) or neurons (NeuN+) (Figure 5a). Subsequently, to test whether it is microglia-specific expression of NgR, NgR was stained in the brain sections after AAV-NgR administration. As shown in Figure S2a, almost all of the GFP+cells colocalized with NgR+cells, which revealed that microglia cells were the primary cell type transfected by the AAV-NgR vectors.
We intraperitoneally administered methoxy-X04 at 3 h prior to microglial isolation and analyzed the proportion of (Figure 5g-h). Therefore, these results demonstrate that NgR acutely decreased microglial Aβ uptake in the AD model after intra-hippocampal fAβ injection by regulating ROCK-Smad2/3-CD36 signaling.
| DISCUSS ION
AD is a neurodegenerative brain disorder characterized by the formation of Aβ plaques, predominantly in hippocampal and cortical regions (Mattson, 2004). Impaired cerebral Aβ clearance has been proposed as the cause of sporadic AD in vast majority of cases (Mawuenyega & Sigurdson, 2010). Microglial activities are critical in the defense against the development of neurodegenerative disease. In AD, activated microglia are in close proximity to Aβ deposits, and produce a wide range of cytokines and chemokines (Rogers & Lue, 2001). The first responders to Aβ accumulation are microglia, which are recruited to amyloid plaques. Furthermore, clearance of Aβ is controlled by complex mechanisms, and dysregulation thereof is suggested to be a major reason for disease onset (Wildsmith & Basak, 2012). Aging is one of the major risk factors for the development of various diseases, including AD, and recent studies have noted a steady decline in normal microglial functions in aging and AD (Johansson & Woodling, 2015). Interestingly, the Aβ-clearing capacity of microglia in APP/PS1 mice is impaired with aging, as indicated by the reduced expression of Aβ-binding receptors and Aβ-degrading enzymes (Hickman & Allison, 2008), but the exact mechanism is unclear. In this study, we found that the effects of microglial Aβ phagocytosis were reduced, in contrast to the upregulation of NgR seen in aging microglia from APP/PS1 mice. This interesting age-dependent effect suggests that microglial NgR maybe exerted harmful effects in response to Aβ phagocytosis and accumulation in APP/PS1 mice.
We all know that NgR plays an important role not only in inhibition of neurite/axon growth (Fournier & GrandPre, 2001), but also in AD pathogenesis (Masliah et al., 2010;Park & Gimbel, 2006). It has been demonstrated that Nogo-A expression is increased in the hippocampus of patients with AD and is also localized in senile plaques around amyloid deposits (Gil & Nicolas, 2006). In addition, deleting Nogo improves the learning and memory deficits of APP transgenic mice (Masliah et al., 2010). Furthermore, the expression of NgR and downstream signaling proteins are elevated in patients with AD and aged rats with deficits of spatial cognition (VanGuilder F I G U R E 4 NgR inhibits CD36 expression by activating ROCK-Smad2/3 signaling in microglia. (a) The primary microglia were treated with Rtn-p4 (100 μg/ml) or Nogo-P4 (100 μg/ml) for 12 h after transfection with control-siRNA or NgR-siRNA for 24 h, and then, the Chip-qPCR analysis was used to measure Smad2/3 occupancy at the Smad2/3 binding site on CD36 promoter. (b-e) The primary microglia were treated with Rtn-p4 (100 μg/ml) or Nogo-P4 (100 μg/ml) for 12 h after treatment with PBS or SIS3 (10 μM) for 12 h. (b-c) The relative CD36 protein and mRNA levels were quantitatively analyzed. (d) Representative images of phagocytosed by primary microglia phagocytosis (Iba-1, red) after incubated with 0.5μM FAM-Aβ (Anaspec, green) for 4 h. Scale bar: 20 μm. (e) The quantification of the phagocytosis assay. n = 3wells/ group, and N = 3 independent experiments. (f-h) The primary microglia were treated with Rtn-p4 (100 μg/ml) or Nogo-P4 (100 μg/ml) for 12h after treatment with PBS or Y27632 (50 μM) for 30 min. (f) The relative p-Smad2/3 protein expression levels were quantitatively analyzed.
(g) Co-IP assay confirms the protein binding between ROCK and Smad2/3. Total protein was extracted and subjected to INPUT and IP study with indicated antibodies. (h) Representative images of primary microglia phagocytosis (Iba-1, red) after incubated with 0.5μM FAM-Aβ (Anaspec, green) for 4 h and the quantification of the phagocytosis assay. Scale bar: 20 μm. n = 3wells/group, and N = 3 independent experiments. (i) The primary microglia were treated with Rtn-p4 (100 μg/ml) or Nogo-P4 (100 μg/ml) for 12 h after treatment with PBS, JSH-23 (10 μM) or Pioglitazone (1 μM) for 6 h, and the relative p-Smad2/3 protein expression levels were quantitatively analyzed. Values were reported as mean±SEM. n = 3-5 samples/group in every experiment, and N = 3 independent experiments. Statistical analyses were performed using a two-way ANOVA with Bonferroni's multiple-comparison post hoc test, *p < 0.05, **p < 0.01 for vehicle+Nogo-p4 vs. vehicle+Rtn-p4; and #p < 0.05, ##p < 0.01 for NgR-siRNA/SIS3/Y27632+Nogo-p4 vs. control-siRNA/vehicle+Nogo-p4; ns, not significant & Sonntag, 2013; Zhu & Guo, 2007), and neuronal overexpression of NgR impairs cognitive function in APP/PS1 mice (Karlsson et al., 2013). However, Park and Gimbel found a protective role for NgR in AD, in which NgR, presumably on the neuronal cell surface, interacts with APP on the cell membrane to block its processing by secretases, thereby reducing Aβ, amyloid plaque deposition, and dystrophic neurites (Park & Gimbel, 2006). Thus, above studies imply that the role of NgR in AD pathology is very complicated. In our previous and other researcher's studies, NgR is not only expressed on the neuron (Fournier & GrandPre, 2001), but also expressed on the microglia F I G U R E 5 AAV-NgR treatment causes a decrease in microglial Aβ phagocytosis after intra-hippocampal fAβ injection (a) Confocal microscope images from brain sections of mice treatment with tamoxifen-induced GFP (green) stained with astrocytes markers GFAP, neuronal markers NeuN, and microglia markers Iba-1(red) 30 days after injection. Scale bar, 50 μm. (b) Representative FACS dot plots showing the microglial phagocytosis of Aβ in intra-hippocampal fAβ injection mice. (c-h) ROCK (c), P-Smad2/3 (e), and CD36 (g) proteins from the hippocampus were analyzed by Western blotting 2 days after Rtn-p4 or Nogo-p4 injection to the Cx3cr1CreER mice after treatment with AAV-control or AAV-NgR for another 26 days. The quantitation of Western blots in ROCK (d), P-Smad2/3 (f), and CD36 (h). Data were shown as mean ±SEM. n = 6-9 mice per group. Statistical analyses were performed using a two-way ANOVA with Bonferroni's multiplecomparison post hoc test, **p < 0.01 for AAV-NgR+Nogo-p4 vs. all three other groups and astrocyte (Wang & Sun, 2018;Yan & Zhou, 2012). In view of the distribution of the NgR+cells around the periphery of the plaques (VanGuilder & Sonntag, 2013;Zhu & Guo, 2007) and the colocalization of NgR and microglia was detected in hippocampus and cerebral cortex in the 8-month-old AD transgenic mice (Fang & Yao, 2016), we dissected the role of NgR expressed on the microglia in AD transgenic model mice. We found that the adhesion and migration of microglia to Aβ were inhibited by Nogo/NgR pathway in vitro and in vivo (Fang & Wang, 2018) and blocking Nogo/NgR signal pathway in microglia improved the neuroinflammatory environment in APP/PS1 mice and alleviated the formation of Aβ plaques and tau phosphorylation in APP/PS1 transgenic mice (Fang & Yao, 2016). The work by Jiang et al (Jiang et al., 2020) showed that NgR plays a critical role in APP processing through Rho/ROCK signaling pathway, and NgR knockdown in the perforant path reduces the APP level and Aβ production, which might account for the improved synaptic and cognitive function in the APP/PS1 mice. Differing from the research on Aβ production by Jiang (Jiang et al., 2020) in perforant path, the functions of NgR in microglial Aβ phagocytosis were explored in this paper. Our results showed that NgR participated in Aβ pathology by modulating the microglial phagocytosis of Aβ in APP/ PS1 mice. Conditional knockdown of NgR in microglia reduced the amyloid burden and improved learning and memory by enhancing Aβ phagocytosis in the brain. In addition, we elucidated the molecular mechanism by which NgR deficiency promotes the phagocytosis of Aβ by upregulating ROCK-Smad2/3-CD36 signaling in microglia, both in vitro and in vivo. In conclusion, NgR expressed in microglia impaired the clearance of Aβ and accelerated Alzheimer's-like disease progression. Therefore, these results suggest that NgR has different roles mainly based on its expression pattern in microglia and neuronal cells and imply the function of NgR in the pathogenesis of AD is relatively complicated and remains to be elucidated furthermore.
To better understand the role of microglial NgR in response to Aβ phagocytosis in vivo, we used the LV-shNgR technique under control of the CD11b promoter to conditionally delete the protein of interest in APP/PS1 transgenic mice (Gowing & Vallieres, 2006), in which the NgR gene was decreased only in endogenous microglial cells, thereby excluding any confounding effects of neuronal NgR (which reportedly regulate Aβ production) (Zhou & Hu, 2011). We observed strong attenuation of Aβ plaque formation and strikingly diminished Aβ peptide concentrations in brain tissue from NgR-deficient APP/ PS1 mice, which correlated with less severe behavioral impairment, illustrating the critical role of NgR in the improvement of AD-like pathology in this model. Furthermore, more CD68+ phagocytic microglial cells in APP/PS1 mice contain amyloid plaques after LV-shNgR treatment, suggesting that a deficiency in NgR restores the lysosomal activity of microglia and promotes Aβ clearance.
We were interested in determining the mechanisms underlying the ability of NgR to attenuate Aβ phagocytosis. CD36 is an important receptor for microglial Aβ phagocytosis (Coraci & Husemann, 2002). Therefore, we tested whether knockdown of NgR using siRNA would result in a functional improvement in CD36 expression and, subsequently, Aβ phagocytosis. The results showed that NgR knockdown induced the upregulation of CD36, and subsequently enhanced phagocytosis of Aβ. The NF-кB pathway plays a role in regulating CD36 expression and influences Aβ phagocytosis (Liu and Liu et al., 2014), while PPARг pathway activation increases Aβ phagocytosis (an effect mediated by upregulated CD36 expression) (Yamanaka & Ishikawa, 2012). However, in our study, neither the NF-кB nor PPARг pathway played a role in NgR knockdown-induced upregulation of CD36. Moreover, we used the ChIP assay to determine the mechanism by which NgR regulates the expression of CD36, and found that NgR siRNA treatment increased the binding of Smad2/3 to the CD36 promoter; in other words, NgR inhibited the transcription of CD36 in microglia regulated by Smad2/3. Similarly, it has been reported that Smad2/3 signaling downregulates the scavenger receptor CD36 and reduces Aβ phagocytosis (Town and Laouar et al., 2008;Rustenhoven and Aalderink et al., 2016). Our previous study showed that Nogo-66 binding to NgR can inhibit the adhesion and migration of microglia through the ROCK pathway in AD (Fang and Yao et al., 2016). We hypothesized that ROCK might be a key factor in the intracellular signal transduction of NgR, so we tested the interaction between ROCK and Smad2/3 using Co-IP assays.
Nogo-p4 significantly disrupted the interaction between ROCK and Smad2/3, which was reversed by Y27632 treatment. Ultimately, we demonstrated that NgR deficiency enhanced microglial Aβ phagocytosis through ROCK-Smad2/3-CD36 signaling. To better understand the role of NgR in vivo microglial Aβ uptake, we used intra-hippocampal fAβ injection mouse models that recapitulate the acute aspects of microglial responses to Aβ peptides (Johansson & Woodling, 2015;Krauthausen & Kummer, 2015). We found that NgR decreased microglial Aβ uptake in the AD model after intrahippocampal fAβ injection via regulating ROCK-Smad2/3-CD36 signaling. Taken together, these data suggest that NgR decreases Aβ phagocytosis through ROCK-Smad2/3-CD36 signaling.
In summary, we showed that NgR participates in Aβ pathology by modulating the microglial phagocytosis of Aβ in APP/PS1 mice.
Conditional knockdown of NgR in microglia reduces the amyloid burden and improves learning and memory, by enhancing Aβ phagocytosis in the brain. In addition, we elucidated the molecular mech-
| Western blot analysis
Equal amounts of protein (40 µg) were separated electrophoretically using denaturing gels and transferred to nitrocellulose membranes.
Subsequently, the membranes were blocked for 1 h at room temperature with 5% BSA in TBST, and incubated with following primary antibody overnight at 4°C: mouse antiβ-actin monoclonal antibody Cell Signaling Technology Inc., #8828). After washed with TBST thrice, the membranes were incubated with horseradish peroxidaseconjugated secondary antibodies anti-mouse IgG (1:10000; Sigma) or anti-rabbit IgG (1:5000; Cell Signaling Technology Inc.). The immunoreactive bands were visualized using chemiluminescence reagents (ECL; Millipore) and captured with Bio-Rad Gel Doc XR documentation system. Pixel density of bands was performed using Quantity One software.
| Quantitative RT-PCR
Total RNAs were extracted from primary microglia using TRIzol reagent (Life Technologies, USA) according to the manufacturer's instructions. RNA concentrations were equalized and converted to cDNA using Hiscript Ⅱ reverse transcriptase kit (Vazyme, Nanjing, China). Gene expression was measured by quantitative PCR system (Roche, Basel, Switzerland) using SYBR-green (Roche, Basel, Switzerland). The amount of double-stranded PCR product synthesized in each cycle was measured by detecting the SYBR green dye, which binds to double-stranded DNA. Threshold cycle (Ct) values for each test gene from the replicate PCRs were normalized to the Ct values for GAPDH RNA control from the same cDNA preparations. Transcription ratios were calculated as 2 (ΔCt) , where ΔCt is the difference between Ct (GAPDH) and Ct (test gene). In addition, we calculated the fold change expression of target genes to the indicated control group. Triplicate PCRs were performed for each of three independently purified RNA samples.
The following PCR primer sequences were used for detecting
| Intracerebral Administration of lentiviral Vectors
At the age of 12 months, when the deposits are readily detected in the brain, APP/PS1 mice and their WT littermates were anesthetized with isoflurane and placed in a stereotactic frame. APP/ PS1 mice were administered a lentivirus vector encoding shNgR under control of the CD11b promoter (LV-shNgR), bilaterally in the hippocampus and cortex, and subsequently sacrificed at 14 months of age. Before injections, lentiviral vectors were diluted with sterile PBS (pH 7.4) to achieve a titer of 1.71 × 10 9 .
Viral preparations in 2-μL volume were injected bilaterally into the hippocampus (±3.2 mm medial/lateral, −2.7 mm anterior/ posterior, and −2.7 mm dorsal/ventral from the bregma) and the cerebral cortex (±2.5 mm medial/lateral, −2.0 mm anterior/ posterior, and −1.0 mm dorsal/ventral from the bregma). The preparation was injected with a speed of 0.5 μl/min over a period of 4 min by using a Hamilton 5-μL syringe and a 27 G needle. Before waking, mice were allowed to recover in a heated chamber.
In the brain of 14-month-old APP/PS1+LV-shNgR mice, Western blotting showed that the amount of NgR in CD11b+ cells isolated from APP/PS1+LV-shNgR mice was significantly lower than that in cells isolated from APP/PS1+LV-control mice in the hippocampus and cortex (Figure S3a-b). Similarly, qPCR analysis showed that the level of NgR transcripts in CD11b+ brain cells from APP/PS1+LV-shNgR mice was 39.30 ± 5.59% of the level of NgR transcripts from APP/PS1+LV-control mice ( Figure S3c). By contrast, in CD11b-brain cells, including neurons, astrocytes, and oligodendrocytes, the levels of general NgR transcripts were not affected by LV-shNgR ( Figure S3c).
| Aβ ELISA
Levels of Aβ peptides were quantified using human Aβ1-40 and Aβ1-42 ELISA kits (Millipore) according to the manufacturer's protocol. Snapfrozen forebrain hemispheres were homogenized in PBS with protease inhibitor mixture (Sigma). Protein was extracted in RIPA (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% sodium desoxycholate, 1% NP-40, and 0.1% SDS) for 30 min on ice. After centrifugation at 100,000 g for 30 min at 4°C, the resulting supernatant (RIPA-soluble fraction) was saved, and the pellet was sacrificed in 25 mM Tris-HCl, pH 7.5, and 2% SDS (RIPA-insoluble fraction). RIPA and SDS fractions were analyzed for Aβ1-40 and Aβ1-42 peptide concentrations. Samples were analyzed in duplicate. Results were normalized on the basis of the sample's protein concentration.
| Morris water-maze test (MWM)
MWM test was performed to detect spatial memory as previously described (Krauthausen & Kummer, 2015). Spatial memory testing was conducted in a pool consisting of a circular tank (1 m diameter) filed with opacified water at 24°C. The water basin was dimly lit (20-30 lux) and surrounded by a white curtain. The maze was virtually divided into 4 quadrants, 1 containing a hidden platform (15 × 15 cm) present 1.5 cm below the water surface. Mice were trained to find the platform using 3 extra maze cues placed asymmetrically as spatial references. Mice were placed into the water in a quasi-random fashion to prevent strategy learning. Mice were allowed to search for the platform for 60 s and were placed onto it manually if they did not reach the platform in the allotted time. Mice were allowed to stay on the platform for 15 s before the initiation of the next trial.
After 4 trials, mice were dried and placed back into their home cages.
Mice were trained for 5 consecutive days with 4 trials per day. In spatial probe trials (Day 6), which were conducted 24 h after the last training session, the platform was removed and mice were allowed to swim for 60 s. Data are given as percentage of time spent in quadrant Q1, where the platform was located previously, which was compared with the average time the animals spent in the remaining quadrants. All movements were recorded by a computerized tracking system that calculated distances moved and latencies required for reaching the platform.
| Novel object recognition test (NOR)
The NOR task, based on the ability of mice to show preference for novel versus familiar objects when allowed to explore freely. NOR was performed during the light cycle. Mice were individually habituated to an open arena (50 cm ×50 cm, dim light, 24°C) on Day 1. During the subsequent training session, 2 identical objects were placed into the arena, and exploratory behavior was monitored for 5 min. On Day 2, mice were placed back into the same arena, in which one of the objects used during training was replaced by a novel object of similar dimensions but a different shape/color, and exploratory behavior was monitored for 5 min. Exploration behavior was assessed by calculating DI, the ratio of time spent exploring the old object to time spent exploring both objects (expressed as a percentage). A DI of ~50% is associated with correct training and no object preference; a significant decrease in DI is characteristic of recognition of the novel object. To evaluate memory, comparisons were made for each group between the recognition (24 h) and training (0 h) sessions.
| Immunohistochemistry
Coronal cryosections (20 μm) of perfused mouse brains were used for immunohistochemistry. Sections obtained were stored in 0.1% NaN 3 and PBS in a cold room. For immunohistochemistry, sections were treated with 50% methanol for 15 min. Then, sections were washed three times for 5 min in PBS and blocked in 3% BSA, 0.1% Triton X-100, and PBS (blocking buffer) for 30 min, followed by overnight incubation with the primary antibody in blocking buffer. Next, sections were washed three times in 0.1% Triton X-100 and PBS and incubated with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (1:500; Invitrogen) for 90 min, washed three times with 0.1% Triton X-100 and PBS for 5 min. Finally, the sections were mounted on glasses in tap water and embedded. The following primary antibodies were used with respective concentrations: rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1:500; 019-19741, Wako), and rat anti-CD68 (1: 400; MCA1957, Bio-Rad), mouse anti 6E10 (1:500; SIG-39300, Covance). Fluorescence microscopy was done on an BX61 equipped with a disk-spinning unit (Olympus) or an A1-MP (Nikon) laser-scanning microscope, and images were processed in Cell-P (Olympus) or NIS elements (Nikon).
| Primary mouse neuronal culture
Primary microglia were prepared from cerebral cortex of neonatal C57BL/6J mice as previously described. Briefly, neonatal mice were ice-anaesthetized and decapitated. After removing the meninges from the brains, the cortex was enzymatically dissociated (0.25% trypsin-EDTA, Sigma). Then, cells were suspended in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12, Gibco) supplement with 10% FBS (Gibco) and seeded into poly-L-lysine (PLL, 0.01 mg/ml, Sigma) pre-coated T75 tissue culture flasks. Flasks were incubated at 37 °C and 5% CO 2 until mixed glial cultures were completely confluent. After 2 weeks in culture, cells were isolated by gently shaking of the flask. Microglia in the medium were collected and seeded into new dishes or plates with culture media.
4.9.6 | AAV administration and establishment of the AD model of intra-hippocampal fAβ As showed in Figure S4, tamoxifen (Sigma) was dissolved in corn oil (Sigma) at a concentration of 20 mg/ mL, and Cx3cr1CreER mice were administered the mixture (9 mg/40 g body weight; i.p.) every other day 5 times starting at 2-3 months old. After 2 days, the mice received unilateral injections to allow comparisons against the contralateral side, with AAV-control or AAV-NgR.
The pAAV-CMV-DIO-NgR-GFP (AAV-NgR) and a AAV vector that expressed GFP alone (AAV-Control) were purchased from BrainVTA (Wuhan). Mice were anesthetized with ketamine/xylazine (30 mg/kg, 4 mg/kg) and immobilized using a stereotactic device. Before injections, AAV vectors were diluted with sterile PBS (pH 7.4) to achieve a titer of 2 × 10 11 . Viral preparations in 2-μL volume were injected unilaterally into the hippocampus (anteroposterior -3.2, lateral 2.7 at 2.5 mm relative to the bregma). The preparation was injected with a speed of 0.5 μl/ min over a period of 4 min by using a Hamilton 5-μL syringe and a 27 G needle. 21 days after AAV injection, mice were received intra-hippocampal injection of fibrillar Aβ. A 0.5-mm burr hole was drilled in the skull, and 1 μl of fibrillar Aβ1-42 (2 μg/μl) with Nogo-p4/Rtn-p4 (5 μmol/μl) solution injected intra-hippocampal into the right hemisphere at a rate of 1 μl/min using a 5μl Hamilton syringe. Mice were sacrificed 2 days after injection and prepared for vivo Aβ phagocytosis.
| Microglial in vivo phagocytosis of methoxy-X04-labeled Aβ
In vivo Aβ phagocytosis was determined using mice intraperitoneally injected with 10 mg/kg methoxy-X04 (50% DMSO/50% NaCl [0.9%], pH 12) 3 h before sacrifice. Mice were perfused with ice-cold PBS, and the brains were removed, minced using scalpels, and incubated in HBSS and 10% FCS containing 0.144 mg/ml collagenase type IV (Sigma, Aldrich) for 1 h at 37°C. Homogenization was achieved by pipetting through a 19-gauge needle. The homogenate was filtered through a cell strainer (70 μm) and centrifuged at 155 g at RT for 10 min without using the brake. The pellet was resuspended in 37% percoll in PBS, underlayered with 70% percoll in PBS, and overlayered with ice-cold PBS. The gradient was centrifuged at 800 g at 4°C for 25 min without using the brake. Microglial cells were recovered from the 37%/70% percoll interphase, diluted with 3 vol PBS, and centrifuged at 880 g at 4°C for 25 min without a break. The pellet containing the microglial cells was resuspended in 200 μl PBS. For flow cytometry analysis, 50μl of cells was diluted with 0.5 ml HBSS and centrifuged at 250 g for 5 minutes at 4°C. Binding of antibodies to Fc receptors was prevented by adding 1 μg of Fc block (BD Bioscience).
Cells were taken up in 50 μl of primary antibody mix (CD11b-APC, 1:100, BioLegend and CD45-FITC, 1:100, eBioscience) and incubated for 30 min on ice. Cells were centrifuged at 250 g for 5 min at 4°C and resuspended in 200 μl HBSS. Corresponding isotype control antibodies were used for control and compensation. Cells were measured on a FACSCanto II Flow Cytometer (BD Bioscience), and data were analyzed using the flow cytometry software FlowJo (TreeStar). For analysis, the CD11b+CD45-population was gated. WT mice injected with methoxy-X04 were used to determine the methoxy-X04 threshold for non-phagocytosing cells, and unstained WT cells were used to determine background fluorescence.
| Statistical analysis
Data shown are mean ±standard error of the mean (SEM) of at least three independent experiments. The differences were considered to be significant at p < 0.05. Statistical comparisons were made with GraphPad Prism 7.0 software using 2-tailed Student's t-test (for 2 groups meeting normal distribution criteria) or 2-way ANOVA with Bonferroni's multiple-comparison post hoc test (for groups across 2 variables, with multiple comparisons between groups). The behavioral experiments as summarized in Figure 2 were statistically analyzed using 2-tailed Student's t-test (NOR) and 2-way ANOVA with Bonferroni's multiple-comparison post hoc test (MWM). Where statistical significance is evaluated, variance between groups is confirmed to be similar between comparison groups and the statistical analysis is deemed appropriate. For data sets with sufficient n to analyze population distribution, tests for normality were adminis-
ACK N OWLED G EM ENTS
This work was supported by grants of National Natural Science We thank Dr. Qianming Du from Nanjing First Hospital, Nanjing Medical University for English proof reading.
CO N FLI C T O F I NTE R E S T
The authors declare no financial or commercial conflict of interest.
AUTH O R CO NTR I B UTI O N S
HL, JW, and XQ conceived and participated in the design of the study. JW, XQ, and MH mainly wrote the manuscript, performed experiments, and confirmed all data analyses in all figures and supporting information. HS and QL participated in preliminary manuscript preparation and experiments. JW, QL, CG, and XH performed molecular/cellular and in vivo experiments. All authors read and approved the final manuscript.
DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated and/or analyzed during this study are included in this article, and the data that support the results of this study are available from the corresponding author upon reasonable request.
The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textc heck.com/certi ficat e/TIG3Ou | 2021-11-26T06:17:34.785Z | 2021-11-24T00:00:00.000 | {
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260920336 | pes2o/s2orc | v3-fos-license | Statistical Study of Bisection Method for Cubic Equations with Random Coefficients
: The bisection method is an iterative approach used in numerical analysis to find nonlinear equations' solutions. It is an easily understandable and simple method that assured convergence. The main purpose of this paper is to study how the parameters of a probability distribution which covers both discrete and continuous distribution that characterizes the coefficient of a cubic polynomial influence the convergence of the bisection method. Distributions covered among discrete and continuous are discrete uniform distribution, continuous uniform distribution and normal distribution. In case of both discrete and continuous uniform distribution inputs, the parameter r indicates the distribution interval [-r, r]. In case of normal distribution input, the parameters m and sigma implies mean and standard deviation respectively. The parameter values are gradually changed. In discrete as well as continuous uniform distribution inputs, and it is found that a second degree polynomial equation can be used to predict the average iteration for a given parameter value, i.e., second-degree polynomial is the best fit. On comparison between discrete and continuous uniform distributions, even the coefficients of both the second-degree polynomial are almost same. For normal distribution input, the average iteration does not depend upon the standard deviation when the mean is fixed and the standard deviation is varying whereas second degree polynomial is again the best fit when the standard deviation is fixed and the mean is varying. This means the average iteration depends upon mean of the normal distribution. Overall, our paper concludes that: I
Introduction 1.1 Introduction to bisection method
It is sometimes difficult to locate the solution of equations f(x)=0 in scientific and technical inquiries. Either f(x) is a quadratic or cubic or biquadratic equation, there are algebraic approach for locating the roots in terms of the coefficients. However, if f(x) is a higher-degree polynomial or an equation with transcendental functions, algebraic approaches are not available. For instance, when 0 , g, u, 0 , and are supplied, the equation is a nonlinear equation for t. This kind of equation is used in rocket research. It is difficult to locate the roots of such nonlinear equations which is frequently encountered in engineering. As a result, several numerical methods have been developed with the goal of offering effective ways to discover numerical solutions to such issues, among which the bisection method was one of the first numerical methods to be developed. The next section will include the explanation of the bisection method.
Bisection method
To "bisect" something implies to slice it down the middle. The bisection method reduces the search area by half at each stage while looking for a solution. The bisection method is thought to possess a linear rate of convergence and provides acceptable accuracy.
Literature review
The fundamental approach to finding a root is the bisection method. Every loop implies a halving of the interval. Since f(a) and f(b) should have different signs and "f" is a continuous function in the interval [a, b], then method will surely converge to a root of "f". The role of bisection method by C Solanki, P Thapliyal, and K Tomar [1] helped us to learn more about the area of the bisection method. In addition to focusing on the bisection method's importance in computer science research, this work also offered a novel method that combines bisection with other methods, such as the Newton-Raphson method which keeps the root bracketed while enabling us to take advantage of the Newton-Raphson method's speed. To further address the issue of nonlinear unconstrained minimization, E.Y. Morozova [2] suggested an adaptation of a new multidimensional bisection method for minimizing function over the simplex. This method does not need the function to be differentiable and is guaranteed to converge to the minimizer for the class of strictly unimodal functions.
Another interesting research study on the bisection method for triangles was conducted by Andrew Adler [3]. According to this study, the longest edge of a triangle was picked and bisected to give birth to two daughter triangles, and continued the bisection procedure indefinitely. He established precise estimates for the longest j th generation edge and demonstrated that the infinite family of triangles so formed falls into a finite number of similarity classes. A geometrical approach to the bisection method by Cl audio Gutierrez, Flavio Gutierrez and Maria-Cecilia Rivara [4] helped us to know that how the behavior of the bisection method depends on the classification of triangles which is to be bisected. Since, the bisection method is the consecutive bisection of a tri angle by the median of the longest side. Therefore, partition of the triangles into classes reflects this behavior by taking into account some fundamental geometrical properties. Its main finding is an asymptotic upper constraint on the total number of triangle similarity classes that may be established on an iterative bisection-created mesh, that was previously unknown. It shows that there are finite number of directions on the plane that may be given by the sides of the resultant triangles. In addition to these research articles, we made use of other books and works that are cited in the reference section ([5]-[10]).
For further literature on bisection method, the reader is referred to [11]- [21].
Motivation and problem statement
Sometimes we can solve problems in a good, easy, or accurate way. For instance, equations like quadratic and linear equations may be solved precisely. However, certain equations might be considerably trickier to solve than others precisely. In rare cases, it may even be difficult to pen down an accurate expression for a solution.
Exercises in mathematics textbooks for schools are frequently purposefully made to provide exact solutions. However, there is no reason to anticipate a particularly good outcome when solving many mathematical equations derived from real-life situations. Most of the time, what we can expect is an approximate solution with the required level of precision. We can use approximate numerical methods to obtain a solution when equations are challenging to solve. Sometimes getting an approximate solution is more effective.
This paper primarily focuses on the bisection method, one of the widely used numerical methods for locating roots. It determines how the parameter of a probability distribution which characterizes the coefficient of a cubic polynomial influence the convergence of the bisection method. Therefore, this study would help us in taking a decision whether to recommend or not to recommend bisection method for a solution if we have prior knowledge that the coefficient of the equations to be solved coming from a particular probability distribution. Further if we have knowledge of parameters of the underlined distributions we may be able to predict the average iterations as the function of the distribution parameter. Research in this direction might be important in tackling many issues emerging in diverse fields of higher mathematics. Such a statistical analysis of the bisection method speeds up the process of solving a problem.
Paper alignment
The alignment of this paper is as follows: Section 1: This section presents the background and basis for the paper. It will provide an overview of bisection method, it's methodology and research problem. Section 2: This section examines the performance of bisection method for a cubic equation with Discrete Uniform coefficients through a statistical approach.
Section 3: This section examines the performance of bisection method for a cubic equation with Continuous Uniform coefficients through a statistical approach.
Section 4: This section examines the performance of bisection method for a cubic equation, with coefficients normally distributed, through a statistical approach.
Section 5: This section provides a summary of the results and makes recommendations for further study.
Study on Cubic Equation for Discrete Uniform Coefficients 2.1 Overview
The term "uniform distribution" in statistics refers to a kind of probability distribution wherein each potential outcome has an equal chance of occurring, i.e., the probability is constant while every variable has an equal number of chances of being the outcome.
For illustration, each person who passes by has an equal chance of receiving the 100-rupee note, if you were to start randomly handing out the note while standing on a street corner. The probability as a percentage is equals to 1/ entire number of possibilities (the number of onlookers). On the other hand, the chances of short persons or women receiving the 100-rupee note are higher than those of other onlookers if you favour them. But this is not what is meant by uniform probability.
Based on the types of probable outcomes, uniform distribution can be divided into two categories: Discrete and Continuous. This section will cover the discrete uniform distribution in detail.
Discrete uniform distribution
The discrete uniform distribution is a statistical distribution in probability theory and statistics where the probability of outcomes is equal and has finite values. For instance, the possible results of throwing a 6-sided die. 1, 2, 3, 4, 5 and 6 are the possible values. Each of the six numbers has a similar chance of appearing in this scenario. Consequently, each time the 6-sided die is thrown, each side gets a chance of 1/6.
There is a finite number of values. When rolling a fair die, it is impossible to obtain a value of 1.3, 4.2, or 5.7. The distribution, however, is no longer uniform if a second die is added, and they are both thrown, as the likelihood of the sums is not the same. The likelihood of tossing a coin is another straightforward illustration. There can only be two outcomes in such a situation. Consequently, 2 is the finite value.
To be more specific, take x to be a discrete random variable with ɱ values in interval [a,b]. Let X has a discrete uniform distribution if its probability mass function (pmf) can be expressed as follows:
Expected value and variance
Two statistics which are often obtained are the expected value and the variance. The discrete uniform random variable's expected value is given by: which, for discrete uniform variate X is, The expression for variance is given by: Where, ℰ( 2 ) is given by: which, in our case, gives Where, is standard deviation.
Methodology
This section will provide the method that has been used to fulfil the objective of this section. Here, coefficients of cubic equation have been generated using the function dis_unirand() which includes inbuilt function rand and rng.
dis_unirand()
u=dis_unirand(r) generates random numbers from the discrete uniform distribution specified by r which implies the range upto which random number is generated i.e.
[-r, r]. The function dis_unirand() includes following functions: rand: X=rand() generates a random scalar in the range (0,1) that is chosen at random from the uniform distribution.
Rng:
In random number generator rng is used to change the seed in MATLAB to prevent using the same random numbers repeatedly. By producing a seed based on the current time, rng provides a simple method for doing that. 'Shuffle' reseeds the generator with a different seed each time you use it. rng can be called without any inputs to reveal the exact seed that was used. Example:
INPUT ARGUMENTS
In MATLAB, the `rand()` function is used to obtain random numbers from a uniform distribution. The function can take one or two input arguments. When called with a single argument, ` ŋ`, `rand(ŋ)` obtain a ` ŋ`-by-` ŋ` matrix of random numbers uniformly distributed between 0 and 1.
Alternatively, when called with two arguments, ` ŋ1` and ` ŋ2`, `rand(ŋ1, ŋ2)` generates a ` ŋ1`-by-` ŋ2` matrix of random numbers with same distribution. In both cases, the resulting matrix of random numbers is returned as the output of the `rand()` function.
MATLAB CODE -REFER APPENDIX A
Steps to follow in matlab code
1.Enter the range of the random number as r and the value of tolerance is assumed to be 0.001. 2.Using the function dis_unirand(), the cubic equation's coefficients are generated at random from a discrete uniform distribution.
Results
Following the above instructions in the MATLAB code stated in APPENDIX A, the mean and standard deviation of iteration (k) for a certain range [-r, r] of parameters over 100 trials were obtained, as shown in Table 1. From fig. 1, it is clear that resultant second degree polynomial equation can be used to predict the average iteration for a given parameter.
Study on Cubic Equation for Continuous Uniform Coefficients 3.1. Overview
Instead of being discrete, some uniform distributions are continuous. The most commonly used distribution among the two is a continuous uniform distribution. Every outcome in this distribution has an equal chance of appearing though the number of outcomes is infinite. In the previous section, discrete uniform distribution has been discussed broadly. Now, this section contains a detailed analysis of continuous uniform distribution.
Continuous uniform distribution
The random variable in a continuous uniform distribution, X, can have values between γ and δ (lower and upper bounds). Both γ and δ are referred to as the continuous uniform distribution parameters in the discipline of statistics. We cannot have a result that is either bigger than δ or smaller than γ. Every variable has an identical chance of appearing in a continuous uniform distribution, also known as a rectangle distribution, where the density function is constant or flat. It has infinite number of outcomes in a given range.
For example: The time it takes a student to finish a mathematics test ranges evenly between 30 and 60 minutes, despite the fact that there are an unlimited number of points between 30 and 60. To be more specific, take x as a continuous random variable within the interval [γ, δ], X ~ U(γ, δ), if its probability distribution function (pdf) can be expressed as follows:
Expected value and variance
The continuous uniform random variable's expected value is given by: Hence, the expected value is The expression for continuous uniform distribution's variance is given by: Where, ℰ( 2 ) is given by: Hence, the variance is Where, is standard deviation.
Methodology
This section will provide the method that has been used to fulfil the objective of this section. Here, coefficients of the cubic equation have been generated using the function con_unirand() which includes inbuilt function rand and rng.
con_unirand()
u=con_unirand(r) generates random numbers from the continuous uniform distribution specified by r which implies the range upto which random number is generated i.e.
Rng:
In random number generator rng is used to change the seed in MATLAB to prevent using the same random numbers repeatedly. By producing a seed based on the current time, rng provides a simple method for doing that. 'Shuffle' reseeds the generator with a different seed each time you use it. rng can be called without any inputs to reveal the exact seed that was used.
INPUT ARGUMENTS
In MATLAB, the `rand()` function is used to obtain random numbers from a uniform distribution. The function can take one or two input arguments. When called with a single argument, ` ŋ`, `rand(ŋ)` obtain a ` ŋ`-by-` ŋ` matrix of random numbers uniformly distributed between 0 and 1.
Alternatively, when called with two arguments, ` ŋ1` and ` ŋ2`, `rand(ŋ1, ŋ2)` generates a ` ŋ1`-by-` ŋ2` matrix of random numbers with same distribution. In both cases, the resulting matrix of random numbers is returned as the output of the `rand()` function.
MATLAB CODE -REFER APPENDIX B
Steps to follow in matlab code
1.Enter the range of the random number as r and the value of tolerance is assumed to be 0.001. 2.Using the function con_unirand(), the cubic equation's coefficients are generated at random from a continuous uniform distribution.
Results
Following the above instructions in the MATLAB code stated in APPENDIX B, the mean and standard deviation of iteration (k) for a certain range [-r, r] of parameters over 100 trials were obtained, as shown in Table 2. From fig. 2, it is clear that resultant second degree polynomial equation can be used to predict the average iteration for a given parameter.
Comparison between discrete and continuous uniform distribution results
In discrete as well as continuous uniform distribution, second degree polynomial equation can be used to predict the average iterations for a given parameter .i.e., second degree polynomial is the best fit. On comparison, even the coefficients of both the y = -5E-08x 2 Mean iterations Parameter r of continuous uniform distribution second-degree polynomial are almost same which implies that the average iteration does not depend on whether the distribution is discrete or continuous but rather depend on the range of the distribution which is a parameter. Hence, in uniform distribution second degree polynomial is the best fit.
Study on Cubic Equation for Coefficients Normally Distributed 4.1. Overview
The normal distribution, sometimes known as the Gaussian distribution, is the most important probability distribution in statistics for independent, random variables. Its well-known bell-shaped curve is readily noticed in statistics reports.
Most of the observations tend to cluster around the central peak of a normal distribution, which is a type of continuous probability distribution that is symmetrically distributed around its mean. The probability of obtaining values that are further away from the mean decreases at an equal rate in both directions, and extreme values in the tails of the distribution are also infrequent. It's worth noting that while the normal distribution is symmetrical, not all symmetrical distributions are normal.
The normal distribution is a probability distribution that describes how a variable's values are distributed, similar to other probability distributions. Due to its ability to accurately represent the distribution of values for many natural phenomena, it is considered the most important probability distribution in statistics. Normal distributions are commonly used to describe characteristics that are the result of multiple independent processes. For example, the normal distribution is commonly observed in traits such as height, blood pressure, measurement error, and IQ scores.
Parameters of normal distribution
The normal distribution's parameters ultimately determine its structure and probabilities, just like with any other probabili ty distribution. The mean and standard deviation are the two variables that jointly form the normal distribution. There is not just one variant of the Gaussian distribution. Instead, the form alters according to the values of the parameter.
Mean:
The mean of the normal distribution represents its center of tendency and identifies the location of the peak of the bell curve. The majority of the data is clustered around the mean. When the mean is changed on a graph, the entire curve shifts to the left or right on the X-axis, as illustrated in
Standard deviation:
The standard deviation is a measure of variability that determines the spread of the normal distribution. It quantifies how much the data typically deviates from the mean and represents the typical distance between observations and the mean. It is used to display the normal separation between the average and the observations, with a larger standard deviation indicating that the data is more spread out, while a smaller standard deviation indicates that the data is more tightly clustered around the mean.
As shown in fig 4, altering the standard deviation causes the width of the distribution along the X-axis to either tighten or spread out. Wider distributions are produced by higher standard deviations. When distributions are narrow, there is a higher probability that values will not deviate significantly from the mean. Conversely, as the dispersion of the bell curve widens, there is a greater likelihood that observations will deviate further away from the mean. In other words, the risk of significant deviations from the mean increases as the normal distribution becomes more spread out.
Normal distribution probability density function
Normal distribution is obtained as a limiting case of Binomial distribution. Let X be a Binomial variate with parameters n and p. Let where, np=θ and √ =σ, which means Naturally Z is a standardized form of Binomial variate. When the two limiting conditions: i. n→∞ ii. p is neither small nor large are applied on Z, then it can be shown that Z becomes a standard normal variate with probability density function (pdf) given by: is probability density function of Z; φ(z)dz is called probability differential of Z. X becomes a normal variate with probability density function given by: f(x)is probability density function of X; ( ) is called probability differential of X. X is a normal variate with mean θ and variance σ 2 . In symbols, X~N(θ, σ 2 ). Z is a standard normal variate. In symbols, Z~N(0,1). Normal distribution is so called because there was an attempt to project this distribution as the benchmark for all continuous probability distribution. The attempt failed as many continuous distributions turned out to be very different from normal. However, the nomenclature "normal" stayed.
Methodology
This section will provide the method that has been used to fulfil the objective of this section. Here, coefficients of the cubic equation have been generated using the function normal() which includes inbuilt function rand and rng.
normal()
u=normal(m, sigma) generates random numbers from the normal distribution specified by m and sigma which implies the mean and standard deviation respectively. The function normal() includes following functions: rand: X=rand() generates a random scalar in the range (0,1) that is chosen at random from the uniform distribution. (ŋ1, ŋ2..., ŋn) generates an array of random numbers of size ŋ1- by-ŋ2-by-....-by-ŋn, where ŋ1, ŋ2..., ŋn indicate the size of each dimension.
Rng:
In random number generator rng is used to change the seed in MATLAB to prevent using the same random numbers repeatedly. By producing a seed based on the current time, rng provides a simple method for doing that. 'Shuffle' reseeds the generator with a different seed each time you use it. rng can be called without any inputs to reveal the exact seed that was used.
Example: rng shuffle r=rand() r=0.1929 INPUT ARGUMENTS In MATLAB, the `rand()` function is used to obtain random numbers from a uniform distribution. The function can take one or two input arguments. When called with a single argument, ` ŋ`, `rand(ŋ)` obtain a ` ŋ`-by-` ŋ` matrix of random numbers uniformly distributed between 0 and 1.
Alternatively, when called with two arguments, ` ŋ1` and ` ŋ2`, `rand(ŋ1, ŋ2)` generates a ` ŋ1`-by-` ŋ2` matrix of random numbers with same distribution. In both cases, the resulting matrix of random numbers is returned as the output of the `rand()` function.
MATLAB CODE -REFER APPENDIX C
Results
Following the above instructions in the MATLAB code stated in APPENDIX C, the mean and standard deviation of iteration (k), once for a fixed parameter-mean(m) and varied parameter-standard deviation(sigma) of normal distribution, as shown in Table 3(a) and Table 3(b) and another for a fixed parameter-standard deviation(sigma) and varied parameter-mean(m) of normal distribution, as shown in Table 4(a) and Table 4(b) over 100 trials were obtained.
Conclusion and Future Scope 5.1 Conclusion
Having knowledge of statistics allow us to make informed decisions about the most appropriate data collection techniques, apply suitable statistical analyses, and effectively communicate the resulting findings. Making decisions based on data, predicting future outcomes, and making scientific discoveries all rely on statistical knowledge and skills. In other words, statistics is a crucial tool that allows us to draw meaningful conclusions from data and make evidence-based decisions, therefore applying statistical analysis on bisection method to analyse its performance in a cubic equation when coefficients are coming from particular distribution.
The convergence of the bisection method for solving a cubic equation will depend on the coefficients of the equation. Now, if the coefficients are coming from some probability distribution, then it is logical that the parameters of that probability distribution will influence the convergence. Hence, our study is to investigate in what way the parameter influences the convergence and considered both uniform and non-uniform distribution which includes discrete uniform distribution, continuous uniform distribution and normal distribution. In order to generate random number from these distribution various functions have been created like dis_unirand(), con_unirand(), normal() with the help of inbuilt functions rand and rng in MATLAB.
After analysis, in all the three distributions second degree polynomial equation can be used to predict the average iteration for a given parameter. On the other hand, coefficients of the polynomial are almost same for discrete and continuous uniform distribution whereas in the normal distribution the coefficients are different, which conclude the following: i. In case of uniform distribution input, the average iteration does not depend on whether the distribution is discrete or continuous but rather depend on the range of the distribution which is a parameter.
ii. In case of non-uniform distribution input, the average iteration depends on the mean of the distribution but not on the standard deviation. Thus, it depends on the location parameter but not on the scale parameter.
Our statistical study of bisection method for cubic equations is intended to inspire other scholars to conduct related research.
Future scope of this work
Other distributions can also be used to study bisection method like Bernoulli distribution, exponential distribution, etc. Since, bisection method is guaranteed to converge though it is slow therefore, a new improvised method can be generated to improve the rate of convergence of bisection method by combining it with regula falsi method or Newton-Raphson method. Even the statistical study of new improvised method can also be done along with comparisons to the traditional method but new methods add new limitations to the data set, for instance, in case of Newton-Raphson method 1 st derivative of polynomial should not be equal to 0, therefore demands new data set for resultant output and comparison. Hence, it provides new scope to this work in future with more improvised algorithm and appropriate data set and make it more reliable to find the root of any equation in real life for wider applications. | 2023-08-16T15:16:32.110Z | 2023-08-14T00:00:00.000 | {
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7616175 | pes2o/s2orc | v3-fos-license | Poincar\'e-Verdier duality in o-minimal structures
Here we prove a Poincar\'e-Verdier duality theorem for the o-minimal sheaf cohomology with definably compact supports of definably normal, definably locally compact spaces in an arbitrary o-minimal structure.
Introduction
We fix an arbitrary o-minimal structure M = (M, <, . . .) and work in the category of definable spaces, X, in M with the o-minimal site on X, with morphisms being definable continuous maps. The o-minimal site on X is the site whose underlying category is the set of all relatively open definable subsets of X (open in the strong, o-minimal topology) with morphisms the inclusions and admissible coverings being covers by open definable sets with finite subcoverings.
The o-minimal setting generalizes the semi-algebraic and globally sub-analytic contexts ( [11]), and so our first main theorem (on Subsection 2.2) generalizes the existence of sheaf cohomology with supports in semi-algebraic geometry, as described in the book [8]. This o-minimal sheaf cohomology with supports satisfies the Eilenberg-Steenrod axioms adapted to the o-minimal site -for the homotopy axiom we need to assume that M has definable Skolem functions and the definable space X involved is definably normal such that for every closed interval [a, b] ⊆ M the projection X × [a, b] −→ X maps closed definable subsets into closed definable subsets. Other cohomology theories have been constructed for o-minimal structures of special types in the past. Simplicial and singular cohomologies were constructed in o-minimal expansions of fields by A.Woerheide in his doctoral thesis, a report of which can be found in [15]. A sheaf cohomology without supports has been constructed in [13] for o-minimal structures (with the extra technical assumptions for the homotopy axiom given above), which generalised the sheaf cohomology without supports for real algebraic geometry of Delfs, for which he proved the homotopy axiom in [7]. The theory presented here generalises all of these and is an extension of the corresponding theory in topological spaces ( [4], [17], [18] and [19]).
Following the classical proof of the Poincaré -Verdier duality for topological spaces we prove here a version Verdier duality theorem for the o-minimal sheaf cohomology with definably compact supports of definably normal, definably locally compact spaces in an arbitrary o-minimal structure (Theorem 4.5). This result is new even in the semi-algebraic context. We do not develop yet the full theory of proper direct image and its dual in the o-minimal context but nevertheless we prove our version of Verdier duality by considering inclusions of definably locally closed definable subsets. The theory of proper direct image is partially developed in the semi-algebraic case in the book by Delf's ( [8]). In the sub-analytic context there are several approaches to this theory by Kashiwara and Schapira ([20]) and also L. Prelli ([23]).
From Verdier duality we derive the Poincaré and Alexander duality theorems (Theorems 4.11 and 4.14). The later results are based on a general and new orientation theory for definable manifolds which we show to be the same as the orientation theory in o-minimal expansions of fields defined in [1] and [2] using o-minimal singular homology. (See subsection 4.4).
Our Poincaré -Verdier duality theory relays heavily on the theory of normal and constructible supports and o-minimal cohomological Φ-dimension. This rather technical theory in presented in Section 3 and is the o-minimal version of the topological theory of paracompactifying families of supports and cohomological Φ-dimension and generalizes the corresponding theory in the semi-algebraic context ( [8]).
The motivation for developing this general o-minimal Poincaré -Verdier duality is to be able to apply it to compute the o-minimal cohomology of definably compact definable groups defined in arbitrary o-minimal structures generalizing in this way the computation of the o-minimal singular cohomology of definable groups in ominimal expansions of fields already presented in [14]. We hope do this in a different paper.
Acknowledgement: The contents of Subsection 2.2 and most of Subsection 3.1 were previously worked out jointly with Gareth O. Jones and Nicholas J. Peatfield. The first author would like to thank them for allowing him to include this material here.
Notations and review
In this section we recall some preliminaries notions about sheaves on topological spaces and the previous results about sheaves on the o-minimal spectrum of a definable space. For further details about classical sheaf theory, see for example [4], [17], [18], [19] and [21]. Good references on o-minimality are, for example, the book [11] by van den Dries and the notes [5] by Michel Coste. For semi-algebraic geometry relevant to this paper the reader should consult the work by Delfs ([7] and [8]), Delfs and Knebusch ( [9]) and the book [3] by Bochnak, Coste and M-F. Roy.
2.1. Sheaves on topological spaces. Let X be a topological space and let k be a field. As usual, we will set Mod(k X ) the category of sheaves of k-modules on X. This is a Grothendieck category, hence it admits enough injectives and a family of generators (the sheaves k U defined below). Moreover filtrant inductive limits are exact.
Let f : X → Y be a morphism of topological spaces. As usual we denote by f * and f −1 the functors of direct and inverse image. In particular, when Y is a subset of X we will denote by i Y : Y ֒→ X the inclusion.
When S is closed and F ∈ Mod(k X ) one sets F S = i S * i −1 S F , and when U is open F U = ker(F → F X\U ) (or equivalently F U is the sheaf associated to the presheaf V → Γ(V ; F U ) which is Γ(V ; F ) if V ⊆ U and 0 otherwise). When Z = U ∩ S set F Z = (F U ) S . The functor (•) Z is exact and F Z is characterized by F Z|Z = F |Z and F Z|X\Z = 0. If Z ′ is another locally closed subset of X, then (F Z ) Z ′ = F Z∩Z ′ . When F = k X is the constant sheaf on X we just set k Z instead of (k X ) Z . If Z 1 , Z 2 are locally closed and Z 1 is closed in Z 2 we have an exact sequence Let Z be a locally closed subset of X. We are going to define the functor i Z! such that for F ∈ Mod(k Z ), i Z! F is the unique k-sheaf in Mod(k X ) inducing F on Z and zero on X \ Z. First let U be an open subset of X and let F ∈ Mod(k U ). Then i U! F is the sheaf associated to the presheaf V → Γ(V ; i U! F ) which is Γ(V ; F ) if V ⊆ U and 0 otherwise. If S is a closed subset of X and F ∈ Mod(k S ), then i S! F = i S * F. Now let Z = U ∩ S be a locally closed subset of X, then one defines The functor i Z! is exact and has a right adjoint, denoted . Let X be a topological space and Φ a family of supports on X (i.e. a collection of closed subsets of X such that: (i) Φ is closed under finite unions and (ii) every closed subset of a member of Φ is in Φ). Recall that for G ∈ Mod(k X ), an element s ∈ Γ(X; G) is in Γ Φ (X; G) if and only if its support, The following fact (see [4], Chaper I, Proposition 6.6) will also be useful later: Proposition 2.1. Let X be a topological spaces, Φ a family of supports on X, Z a locally closed subset of X and let i Z : Z → X be the inclusion. Let F be a sheaf in Mod(k Z ). Then A definable space has a topology such that each X i is open and the φ i 's are homeomorphisms: a subset U of X is an open in the basis for this topology if and only if for each i, φ i (U ∩ X i ) is an open definable subset of φ i (X i ). We also say that a subset A of X is definable if and only if for each i, φ i (A∩X i ) is a definable subset of φ i (X i ). A map between definable spaces is definable if when it is read through the charts it is definable. Thus we have the category of definable spaces with definable continuous maps.
We say that a definable space X is: • definably connected if it is not the disjoint union of two open and closed definable subsets; • definably compact if for every continuous definable map σ : (a, b) ⊆ M ∪ {−∞, +∞} −→ X, the limits lim t→a + σ(t) and lim t→b − σ(t) exist and belong to X. • definably locally compact if for every definably compact subset Z with open definable neighborhood U in X, there is a definably compact neighborhood of Z in U . • definably normal if for every disjoint closed definable subsets Z 1 and Z 2 of X there are disjoint open definable subsets U 1 and U 2 of X such that is an open definable subset of M n for every i = 1, . . . , k. The o-minimal site on a definable space X is the category whose objects are open definable subsets of X, the morphisms are the inclusions and the admissible covers are covers by open definable subsets with finite subcoverings.
The following results are an easy adaptation of Propositions 6.4.1 and 6.3.3 of [20], replacing T c with open definable (indeed we just need the site generated by a family of open subsets closed under finite intersections and whose coverings admit a finite subcover). The first result gives an easy way to construct o-minimal k-sheaves: Suppose that X be a definable space. Let F be a k-presheaf on X relative to the o-minimal site on X and assume that: (1) F (∅) = 0; (2) for any U and V open definable subsets of X the canonical sequence is exact. Then F is a k-sheaf on X relative to the o-minimal site on X.
The second result shows that in this setting the global sections functor commutes with filtrant inductive limits: Proposition 2.3. Let U be a open definable subset of X and let (F i ) i∈I be a filtrant inductive family of sheaves on the o-minimal site associated to X. Then We define the o-minimal spectrum X of a definable space X as in [5], [6] and [22]: it is the set of ultrafilters of definable subsets of X. The o-minimal spectrum X of a definable space X is T 0 , quasi-compact and a spectral topological space when equipped with the topology generated by the open subsets of the form U , where U is an open definable subset of X. That is: (i) it has a basis of quasi-compact open subsets, closed under taking finite intersections; and (ii) each irreducible closed subset is the closure of a unique point.
The dimension of the o-minimal spectrum X of a definable space X is defined as dim X = dim X. By a constructible subset of X we mean a subset of the form A where A is a definable subset of X.
We also have the o-minimal spectrum f : X −→ Y of a continuous definable map f : X −→ Y between definable spaces: given an ultrafilter α ∈ X, f (α) is the ultrafilter in Y determined by the collection {A : We now recall some results from [13] about this tilde functor. Note that these results were stated in [13] in the category of definable sets but are true in the category of definable spaces with exactly the same proofs.
As we saw in [13] we have: The tilde functor is an isomorphism between the boolean algebra of definable subsets of a definable space X and the boolean algebra of constructible subsets of its o-minimal spectrum X and it commutes with image and inverse image under definable maps.
Another useful property is the following result: . Let X be a definable space. Then the following hold: (i) X is definably connected if and only if its o-minimal spectrum X is connected. (ii) X is definably normal if and only if its o-minimal spectrum X is normal.
Also we have the following shrinking lemma: Proposition 2.6 ([13], The shrinking lemma). Suppose that X is a definably normal definable space (resp. a normal o-minimal spectrum of a definable space). If Since the o-minimal spectrum of a definable space is quasi-compact, as in the proof of Propositions 2.2 and 2.3, we have: Suppose that X is an object in the category of o-minimal spectra of definable spaces. Let F be a k-presheaf on X and assume that: (2) for any U and V open constructible subsets of X the canonical sequence Moreover sections on open constructible subsets commute with filtrant lim − → .
We have a morphism of sites naturally induced by the above tilde functor from the category of definable spaces with continuous definable maps into the category of o-minimal spectral spaces with the o-minimal spectra of continuous definable maps. This morphism of sites induces the following isomorphism: . Let X be a definable space. Then there is an isomorphism between the category of k-sheaves on X relative to o-minimal site on X and the category of k-sheaves on the o-minimal spectrum X of X relative to the spectral topology on X.
The isomorphism of Theorem 2.8 allowed the development of o-minimal sheaf cohomology without supports in [13] by defining concepts and also sometimes proving results via this tilde isomorphism. In this paper we will continue to use this technique but allowing now the presence of supports.
We will now define the notion of family of supports on a definable set. Our treatment of this will follow the corresponding theory in semi-algebraic geometry in [8] (Chapter II, Sections 1 -5) and in topological spaces in [4] (Chapter I, Section 6 and Chapter II, Sections 9 -13). Note also that since, as we saw in [13], the role of paracompactness in sheaf theory on topological spaces has to be replaced by normality in sheaf theory on o-minimal spectral spaces, we will continue to do this here.
Definition 2.9. Let X be a definable space. A family of definable supports is a family of closed definable subsets of X such that: (1) every closed definable subset of a member of Φ is in Φ; (2) Φ is closed under finite unions.
Φ is said to be a family of definably normal supports if in addition: Example 2.10. Let X be a definable space and let c be the collection of all definably compact definable subsets of X. Then c is a family of definable supports on X. Moreover, if X is definably normal and definably locally compact, then c will be a family of definably normal supports on X.
If Y is a definable subset of the definable space X and Φ a family of definable supports on X, then we have families of definable supports If f : X −→ Z is a continuous definable map between definable spaces and Φ is a family of definable supports on Z, then we have a family of definable supports Remark 2.11. Note that a family of definable supports Φ on a definable space X determines a family of supports We will say that the family of supports on X is constructible if it is obtained by applying tilde to some family of definable supports on X.
By theorem 2.5 it follows that Φ is definably normal if and only if Φ is normal.
Here, we say that Ψ is a family of normal supports on the spectral topological space X if is a family of supports and: (1) each element of Ψ is normal; Definition 2.12. Let X be a definable space, Φ a family of definable supports in X and F a k-sheaf on X relative to the o-minimal site on X. We define the o-minimal sheaf cohomology groups with definable supports in Φ via the tilde isomorphism of Theorem 2.8 by where F is the image of F via the isomorphism between the category of k-sheaves on X relative to o-minimal site on X and the category of k-sheaves on the o-minimal spectrum X of X.
The proof of the o-minimal Vietoris-Begle theorem with supports below is similar to its analogue without supports ([13] Theorem 4.3) using classical arguments: Theorem 2.13 (Vietoris-Begle theorem). Let f : X −→ Y be a surjective morphism in the category of o-minimal spectra of definable spaces that maps constructible closed subsets of X onto closed subsets of Y . Let F ∈ Mod(k Y ), Φ a constructible family of supports on Y and suppose that Y is a subspace of a normal space in the category of o-minimal spectra of definable spaces. Assume that We have in this context the Eilenberg-Steenrod axioms with definable supports adapted to the o-minimal site. Indeed, once we pass to the category of o-minimal spectra of definable spaces the proofs of the exactness and excision axioms are purely algebraic. See [4]. The dimension axiom is also immeadiate. On the other hand, from the Vietoris-Begle theorem (Theorem 2.13) we obtain: Theorem 2.14 (Homotopy axiom). Suppose that X is a definable space and F is a k-sheaf on X relative to the o-minimal site on X. Let [a, b] ⊆ M be a closed interval. Assume that M has definable Skolem functions, X is definably normal and the projection π : Proof. The homotopy axiom will follow once we show that the projection map for all n ∈ N. Equivalently we need to show that
Φ-soft sheaves
The results we present below are in the category of o-minimal spectra of definable spaces but by the isomorphism of Theorem 2.8 they have a suitable, but more restrictive, analogue in the category of definable spaces. In fact these results are the analogue of classical results on paracompactifying families of supports on topological spaces ( [4]) adapted to normal and constructible families of supports on spectral spaces.
3.1. Normal and constructible supports. We start the subsection with the following useful result: Assume that X is an object in the category of o-minimal spectra of definable spaces and let (F i ) i∈I be a filtrant inductive family of sheaves in Mod(k X ) and Φ a constructible family of supports on X. Then Proof. First observe that by definition for any G ∈ Mod(k X ) we have Thus it is enough to show that for each S ∈ Φ constructible we have For this consider the following commutative diagram where the vertical arrows are the canonical maps , it follows that the two vertical arrows on the right are isomorphisms. Hence the first vertical arrow is also an isomorphism as required.
The following lemma is fundamental in this Subsection: Assume that Z is a subspace of a normal space X in the category of o-minimal spectra of definable spaces, G is a sheaf in Mod(k Z ) and Y is a quasicompact subset of Z. Then the canonical morphism where U ranges through the family of open constructible subsets of X, is an isomorphism.
Proof. Since Y is quasi-compact, the family of open neighborhoods of Y in Z of the form V ∩ Z where V is an open constructible subset of X is a fundamental system of neighborhoods of Y in Z. Hence, the morphism of the lemma is certainly injective.
To prove that it is surjective, consider a section s ∈ Γ(Y ; G |Y ). There is a covering {U j : j ∈ J} of Y by open constructible subsets of X and sections s j ∈ Γ(U j ∩ Z; G |Uj ∩Y ), j ∈ J, such that s j|Uj ∩Y = s |Uj ∩Y . Since Y is quasi-compact, we can assume that J is finite, and so ∪{U j : j ∈ J} is an open constructible subset of X. Since X is normal, by the shrinking lemma (Proposition 2.6), there are open constructible subsets {V j : j ∈ J} of this union such that V j ⊆ U j for every j ∈ J and Y ⊆ ∪{V j : j ∈ J}. For x ∈ Z set J(x) = {j ∈ J : x ∈ V j }. Each x has a constructible neighborhood W x with J(y) ⊆ J(x) for each y ∈ W x . This is defined by Observe that for all i, j ∈ J(x) we have that W x is an open subset of both U i and U j . Hence, for every i, j ∈ J(x) we have s i|Wx∩Y = s |Wx∩Y = s j|Wx∩Y . So, for y ∈ W x ∩ Y , we have (s i ) y = (s j ) y for any i, j ∈ J(x). This implies that the set On the other hand, the condition (s i ) z = (s j ) z for any i, j ∈ J(x) and the fact that J(x) is finite implies that z has an open neighborhood in Z on which s i = s j for any i, j ∈ J(x). Thus W is an open neighborhood of Y in Z. Since Y is quasi-compact we may assume that W is of the form U ∩ Z for some open constructible subset U of X. Since s i |W ∩V i ∩V j = s j |W ∩V i ∩V j there exists t ∈ Γ(W ; G) such that t |W ∩Vj = s j|W ∩Vj . This proves that the morphism is surjective. A general form of Lemma 3.2 is: Assume that X is an object in the category of o-minimal spectra of definable spaces, Z is a subspace of X, G is a sheaf in Mod(k Z ), Φ is a normal and constructible family of supports on X and Y is a subset of Z such that D ∩ Y is a quasi-compact subset for every D ∈ Φ. Then the canonical morphism where U ranges through the family of open constructible subsets of X, is an isomorphism.
Proof. Let us prove injectivity. Let s ∈ Γ D∩U∩Z (U ∩ Z; G), with D ∈ Φ and U ⊃ Y open constructible subset of X and such that s |D∩Y = 0. Since Φ is a normal and constructible family of supports on X, there is a constructible and normal E ∈ Φ which is a closed neighborhood of D in X. Thus D ∩ Z is a subspace of a normal space E in the category of o-minimal spectra of definable spaces and D ∩ Y is a quasi-compact subset of D ∩ Z. By Lemma 3. Let us prove that the morphism is surjective. Let s ∈ Γ Φ∩Y (Y ; G |Y ) and consider normal constructible sets C, D and E in Φ such that D is a closed neighborhood of C in X, E is a closed neighborhood of D in X and the support of s is contained in C ∩Y . We shall find t ∈ Γ D (U ∩Z; G) such that t |Y = s. After applying Lemma 3.2 above to E, D ∩ Z and D ∩ Y we see that there exists an open in E \ ∂E (and hence in X) constructible neighborhood V of D ∩ Y and a section t ∈ Γ(V ∩ D ∩ Z; G) such that t |D∩Y = s |D∩Y . Since t |∂D∩Y = 0, then each point x of ∂D ∩ Y has an open constructible neighborhood W x ⊂ V such that t |Wx∩D∩Z = 0. Using quasicompactness of ∂D ∩ Y (it is closed on the quasi-compact set D ∩ Y ), there exists a finite number of points and we can define t ∈ Γ(U ∩ Z; G) in the following way: t |U1∩Z = t ′ |U1∩Z , t |U2∩Z = 0. It is well defined since t ′ |W ∩Z = 0 and U 1 ∩ U 2 ⊂ W . Moreover supp t ⊆ D and t |Y = s as required.
Recall that a sheaf F on a topological space X with a family of supports Φ is Φ-soft if and only if the restriction Γ(X; F ) −→ Γ(S; F |S ) is surjective for every S ∈ Φ. If Φ consists of all closed subsets of X, then F is simply called soft. Proposition 3.4. Let X be a topological space and F is a sheaf in Mod(k X ). If Φ is a family of supports on X such that every C ∈ Φ has a neighborhood D in X with D ∈ Φ. Then the following are equivalent: If in addition X is an object in the category of o-minimal spectra of definable spaces and Φ is a constructible family of supports on X, then the above are also equivalent to: If moreover Φ is a normal and constructible family of supports on X, then the above are also equivalent to: Proof. The equivalence of (1), (2) and (3) is shown in [4] Chapter II, 9.3. (Our hypothesis is sufficient in the proof given there). The equivalence of (2) and (4) is obvious since every S ∈ Φ is contained in some constructible subset of X which is in Φ.
Clearly (1) implies (5). Assume (5) and let S ∈ Φ and s ∈ Γ(S; F |S ). Since Φ is normal and constructible, there is a normal closed and constructible neighborhood D of S which is in Φ. By Lemma 3.2, s can be extended to a section t ∈ Γ(W ; F ) of F over a neighborhood W of S in D. Applying the shrinking lemma we find a closed constructible neighborhood Z of S in W . Since D ∈ Φ we have Z ∈ Φ. So t |Z ∈ Γ(Z; F |Z ), (t |Z ) |S = s and t |Z can be extended to X by (5). Hence, (5) implies (1).
Corollary 3.5.
Assume that X is an object in the category of o-minimal spectra of definable spaces and Φ is a normal and constructible family of supports on X. Then filtrant inductive limits of Φ-soft sheaves in Mod(k X ) are Φ-soft.
Proof. It follows combining Propositions 3.1 and 3.4 (5) and the exactness of filtrant inductive limits.
The following topological result will also be useful below: Proposition 3.6. Let X be a topological space and Φ is a family of supports on X such that every C ∈ Φ has a neighborhood D in X with D ∈ Φ. Let W be a locally closed subset of X. The following hold: Proof. (i) If W is open it is obvious. If W is closed it follows from Proposition 3.4 (3). Combining these two cases (i) follows.
(ii) The "if " part follows from Proposition 3.4 (2). For the "only if" part note that by Proposition 2.1 (applied to X and S respectively and using the fact that (Φ |S ) |W = Φ |W ∩S ) we have Γ Φ (X; i W ! G) ≃ Γ Φ|W (W ; G) and Γ Φ|S (S; i W ! G) = Γ Φ|W ∩S (S ∩ W ; G) for any closed subset S of W . Then apply Proposition 3.4 (3).
(iii) The result follows from (i) and (ii), since F W = i W ! F |W . A special and useful case of Proposition 3.6 is when X is an object in the category of o-minimal spectra of definable spaces and Φ is a normal and constructible family of supports on X.
Proposition 3.7. Assume that X is an object in the category of o-minimal spectra of definable spaces, Φ is a normal and constructible family of supports on X and Y is a subspace of X such that D ∩ Y is a quasi-compact subset for every D ∈ Φ. Then the full additive subcategory of Proof. The result for the full additive subcategory of Mod(k Y ) of injective (and flabby) k-sheaves is classical for topological spaces (see for example [19], Proposition 2.4.3). Thus (1) holds for the Φ∩Y -soft case since injective k-sheaves are Φ∩Y -soft.
We now prove (2). Let s ′′ ∈ Γ Φ∩Y (Y, F ′′ ). Then since Φ is normal and constructible, supp s ′′ ⊂ V , with V open constructible in X and V ∈ Φ. Now, let us consider the exact sequence be a finite covering of Y such that there exists s i ∈ Γ(D i ; F ) whose image is s ′′ | Di . There exists such a covering since Φ is normal and Y ∩ V is quasi-compact. For n ≥ 2 on D 1 ∩ D 2 s 1 − s 2 defines a section of Γ(D 1 ∩ D 2 ; F ′ ) which extends to s ′ ∈ Γ(Y ; F ′ ) since F ′ is Φ ∩ Y -soft. Replace s 1 with s 1 − s ′ . We may suppose that s 1 = s 2 on D 1 ∩ D 2 . Then there exists s ∈ Γ(D 1 ∪ D 2 ; F ) such that s| Di = s i , i = 1, 2. Thus the induction proceeds.
Finally, (3) follows at once from (2) by a simple diagram chase using Proposition 3.4 (3): let Z be a set in Φ ∩ Y and consider the following commutative diagram . By hypothesis on F , α is surjective. By (2) β is surjective. Therefore, γ is surjective as required.
Hence, if X is an object in the category of o-minimal spectra of definable spaces, Φ is a normal and constructible family of supports on X and Y is a subspace of X such that D ∩ Y is a quasi-compact subset for every D ∈ Φ. Then one can take a Φ ∩ Y -soft resolution of F to compute H * Φ∩Y (Y ; F ).
Example 3.8. Some particular cases of Proposition 3.7 are: Corollary 3.9. Assume that X is an object in the category of o-minimal spectra of definable spaces. Suppose either that Φ is a normal and constructible family of supports on X and W is a (constructible) locally closed subset of X or that Φ is any family of supports on X and W is a closed subset of X. If F ∈ Mod(k W ), then The second case is covered by [4] Chapter II, 10.1. If W is closed in an open subset U of X, then Φ |U is a normal and constructible family of supports on U and Φ |W = Φ |U ∩ W. And the result follows from Propositions 3.7, 3.6 (ii) and 2.1.
The following will be useful in the next subsection: Assume that X is an object in the category of o-minimal spectra of definable spaces, F is a sheaf in Mod(k X ) and Φ is a normal and constructible family of supports on X. The following are equivalent:
3.2.
Cohomological Φ-dimension. Recall that for a topological space X and Φ a family of supports on X, the cohomological Φ-dimension of X is the smallest n such that H q Φ (X; F ) = 0 for all q > n and all sheaves F in Mod(k X ). The following holds: Proposition 3.11. Assume that X is an object in the category of o-minimal spectra of definable spaces and Φ is a normal and constructible family of supports on X. Let F be a sheaf in Mod(k X ). Then the following are equivalent: (1) If 0 −→ F −→ I 0 −→ I 1 −→ · · · −→ I n −→ 0 is an exact sequence of sheaves in Mod(k X ) such that I k is Φ-soft for 0 ≤ k ≤ n − 1. Then I n is Φ-soft. (2) F has a Φ-soft resolution of length n; Proof. The result follows from Proposition 3.10 (2) and is a particular case of a general result of homological algebra ( [19], Exercise I.19): let F be a left exact functor and let J be the family of F -acyclic objects. Suppose that J is cogenerating. Then (1) ⇔ (2) ⇔ (3) with J instead of Φ-soft and F instead of Γ Φ (X; (•) U ).
Theorem 3.12. Let X be an object in the category of o-minimal spectra of definable spaces and let Φ be a normal and constructible family of supports on X. Then the cohomological Φ-dimension of X is bounded by dim X.
Proof. To prove our theorem we will use (1) of Proposition 3.11. Let n = dim X. Then, in this situation it suffices to prove that I n |Z is soft for every constructible subset Z of X which is in Φ (Proposition 3.4 (4)). Since Φ is normal, there is a constructible neighborhood Y of Z in X which is in Φ. If we show that I n |Y is soft, then it will follow that I n |Z is soft (Proposition 3.4 (2)). Let U be an open and constructible subset of Y . By hypothesis and Proposition 3.10 each ( . Then the long exact cohomology sequences of the short exact sequences 0 Since Y is normal, constructible and dim Y = n we have H q (Y ; G) = 0 for q > n and every sheaf G on Y ([13] Proposition 4.2). Thus H 1 (Y ; (I n |Y ) U ) = 0. Since U was an arbitrary open and constructible subset of Y , it follows from Proposition 3.10 that I n |Y is soft as required. Proposition 3.13. Assume that X is an object in the category of o-minimal spectra of definable spaces and Φ is a normal and constructible family of supports on X. If G ∈ Mod(k X ) is Φ-soft, then for every F ∈ Mod(k X ) we have that G⊗F ∈ Mod(k X ) is Φ-soft.
Proof. By Theorem 3.12, X has finite cohomological Φ-dimension. Suppose that the cohomological Φ-dimension of X is n. Since the family of the constant sheaves {k U }, U constructible open subset of X is generating, there is a resolution of F P n−1 ∂n−1 → P n−2 · · · P 1 ∂1 → P 0 → F → 0 where the P i 's are direct sums of sheaves of the form k U , U constructible (see [19], Proposition 2.4.12). From Proposition 3.6 (iii) it follows that From the resolution above we obtain an exact sequence of sheaves By Proposition 3.11, since G ⊗ P i is Φ-soft for i = 0, . . . , n − 2, we conclude that G ⊗ F is Φ-soft.
Duality with coefficient in a field
In this section we will work in the category of definable spaces with continuous definable maps and k-sheaves on such spaces will be considered always relative to the o-minimal site. In our results we will have a definably normal, definably locally compact definable space X and the family of definable supports c on X of definably compact definable subsets of X. By Example 2.10 and Remark 2.11 the corresponding constructible family of supports on the o-minimal spectra of X will be a normal and constructible family of supports. Hence, by the tilde isomorphism in the category of k-sheaves given by Theorem 2.8 and our Definition 2.12, in our proofs we will apply the results of Section 3 since they transfer to this definable setting.
Remark 4.1. We observe that since all the results of this section depend only on Section 3, they hold on an arbitrary definable space X replacing c by a definably normal family of definable supports Φ on X. In particular, these results hold on any definable space X on which c is a definably normal family of definable supports.
Sheaves of linear forms.
Here we shall work with a fixed field k. For a kvector space N we let N ∨ denote the dual k-vector space, i.e. N ∨ = Hom k (N, k).
Let X be a definably normal, definably locally compact definable space and F a k-sheaf on X. From now on, given a locally closed subset Z of X, we will write Γ c (Z; F ) instead of Γ c|Z (Z; F ) for short. The inclusion V −→ U of two open definable subsets of X will induce a map Proposition 4.2. Let X be a definably normal, definably locally compact definable space. For every c-soft k-sheaf F on X, the presheaf F ∨ is a sheaf.
Proof. By Proposition 2.2, it is enough to show that for any two open definable subsets W and V of X the sequence formed by the sum and difference between two restriction maps is exact.
Consider the Mayer-Vietoris sequence Proof. Let U be an open definable subset. Consider the natural maps The dual of the composite can be written By variation of U this defines a map which we must show that it is an isomorphism.
(i) First we consider the case where F = k U where U is an open definable subset. We have These identifications transform the map (1) into the identity.
(ii) For the general case, consider a presentation of F of the form where P and Q are direct sums of sheaves of the form k U as above (see [19], Proposition 2.4.12). Let us consider the following diagram with exact rows The two functors of (1) transform direct sums into direct products. It follows that the two vertical maps to the right are isomorphisms. Then it follows from the five lemma that the first vertical arrow is an isomorphism.
Corollary 4.4. Let X be a definably normal, definably locally compact definable space. Let G be a c-soft k-sheaf on X. Then the sheaf G ∨ is injective in the category of k-sheaves on X.
Proof. By Proposition 4.3, we must show that is an exact functor. But this follows from Propositions 3.13 and 3.7 and the exactness of ∨ in Mod(k).
4.2.
Verdier duality. If X is a definably normal, definably locally compact definable space we will let D + (k X ) denote the derived category of bounded below complexes of k-sheaves on X. We are now ready to prove our main result: Theorem 4.5 (Verdier duality). Let X denote a definably normal, definably locally compact definable space. Then there exists an object D * in D + (k X ) and a natural isomorphism as F * varies through D + (k X ). and the result follows since H −p D * is the k-sheaf associated to the k-presheaf U → H −p (U ; D * ).
Corollary 4.8. On a definably normal, definably locally compact definable space X of cohomological c-dimension n, the k-presheaf definable sub-balls, it follows that X has an orientation k-sheaf. Observe that the result on coverings by definable sub-balls is related to [1] Let X be a definably normal, definably locally compact definable manifold of dimension n with an orientation k-sheaf Or X . Then the k-sheaf Or X is locally isomorphic to k X . Theorem 4.11 (Poincaré duality). Let X be a definably normal, definably locally compact definable manifold of dimension n with an orientation k-sheaf Or X . There exists an isomorphism Proof. Proposition 4.7 and the fact that X has an orientation k-sheaf, imply that H −p D * = 0 ; p = n. On the other hand, by Corollary 4.8 we have H −n D * = Or X . Thus we have a quasi-isomorphism (4) Or X [n] ≃ D * Therefore we have By Verdier duality (Theorem 4.5) with F * = k X the later is also isomorphic to H n−p c (X; k X ) ∨ .
Definition 4.12. Let X be a definably normal, definably locally compact definable manifold of dimension n with an orientation k-sheaf Or X . By a k-orientation we understand an isomorphism k X ≃ Or X of k-sheaves. We shall say that X is k-orientable if a k-orientation exists and k-unorientable in the opposite case. Proposition 4.13. Let X be a definably connected, definably normal, definably locally compact definable manifold of dimension n with an orientation k-sheaf Or X . Then (1) H n c (X; k X ) ≃ k if X is k-orientable.
(2) H n c (X; k X ) ≃ 0 if X is k-unorientable. Proof. Since X is definably normal and definably connected, Proposition 4.1 in [13] implies that H 0 (X; k X ) = k and so (1) follows at once from the Poincaré duality (Theorem 4.11).
For (2), suppose that H n c (X; k X ) = 0. Then by Theorem 4.11 there is a non trivial section s of Or X over X. By our Definition 2.12, the support of s is a closed subset of the o-minimal spectrum of X. Since Or X is locally isomorphic to k X it follows that the support of s is also an open subset of the o-minimal spectrum of X. But since the o-minimal spectrum of X is connected (Theorem 2.5) it follows that the support of s is the o-minimal spectrum of X. Thus Or X ≃ k X . Theorem 4.14 (Alexander duality). Let X be a definably normal, definably locally compact, k-orientable definable manifold of dimension n. For Z a closed definable subset of X there exists an isomorphism Proof. By (4) we have H p Z (X; k X ) ≃ H p−n Z (X; D * ) ≃ H p−n Hom(k Z , D * ). By Verdier duality (Theorem 4.5) with F * = k Z the later is also isomorphic to H n−p c (X; k Z ) ∨ ≃ H n−p c (Z; k X ) ∨ .
4.4.
Duality in o-minimal expansions of fields. In this subsection we assume that the o-minimal structure M is an expansion of an ordered field.
Let X be a Hausdorff definable manifold of dimension n. Then has we saw in Example 4.10 X is affine, definably normal with an orientation k-sheaf.
In o-minimal expansions of fields we have o-minimal singular homology and cohomology theories satisfying the Eilenberg-Steenrod axioms adapted to the ominimal site ( [15], [24]). By [15] the o-minimal singular cohomology theory with coefficients in a field k is isomorphic to the o-minimal sheaf cohomology theory with coefficients in the constant sheaf k X . Because of this isomorphism, below we will use the standard notation from o-minimal singular cohomology and write k for k A and H * (A, B; k) for H * A\B (A; k) where B ⊆ A ⊆ X are definable subsets of X.
O-minimal singular homology theory can be used to obtain an orientation theory for definable manifolds ([1], [2]). (In the papers [1] and [2], orientation is defined by taking homology with coefficients in Z but replacing Z by k and considering homology groups as k-vector spaces one gets the theory of k-orientations.) Our goal here is to show an Alexander duality for homology and to conclude that the two orientation theories agree.
First observe that if B ⊆ A are definably locally closed definable subsets of X, then Let Λ be the directed system of definably locally closed subsets D of A such that B ⊆ D ⊆ A and A \ D ∈ c, directed by reverse inclusion. Since the map that sends D ∈ Λ into D is cofinal (even surjective) in the directed system of definable closed subsets C of A such that B ⊆ C ⊆ A and A \ C ∈ c, directed by reverse inclusion, it follows that to prove (5) | 2010-03-23T14:34:37.000Z | 2010-03-23T00:00:00.000 | {
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267778441 | pes2o/s2orc | v3-fos-license | A systematic review of interventions that impact alcohol and other drug-related harms in licensed entertainment settings and outdoor music festivals
Background Harms associated with the use of alcohol and other drugs (AOD) in licensed entertainment settings (LES) and outdoor music festivals (OMF) are ongoing public health and criminal justice concerns. This systematic review provides a comprehensive, synthesized report on the evidence base of interventions that impact harm in these settings, and how they affect health, behavioral, and criminal justice outcomes. Methods Nine databases were searched for experimental and observational studies published between 2010 and 2021. Studies were included if they were peer-reviewed, published in English, described interventions which could impact AOD-related harms in LES or OMF (and were delivered in these environments), and reported on health, criminal justice and/or behavioral outcomes. Methodological quality was assessed using the Effective Public Health Practice Project’s Quality Assessment Tool for Quantitative Studies and the Critical Appraisal Skills Program for qualitative studies. A narrative synthesis was conducted to synthesize outcomes across studies. The review protocol was registered in PROSPERO (CRD42020140004). Results Of the 48,303 studies screened, 100 met the inclusion criteria. 86 focused solely on reducing alcohol-related harm, 7 on reducing illicit drug-related harm, and 7 on both. Most (n = 88) focused on LES and evaluated changes in laws and regulations (n = 28) and/or multicomponent interventions/policies (n = 41). Multicomponent interventions showed the best results for both health (62% positive) and criminal justice (84% positive) outcomes, with 71% of studies being rated as strong quality. There was also good evidence to support the careful application of trading hour restrictions and limited but promising evidence to support medical services and drug checking. Conclusion The breadth, quality and volume of evidence regarding what works in reducing AOD-related harm in recreational settings have increased in the past decade, particularly regarding LES. Findings support onsite medical services (reducing ambulance transfer rates), multicomponent interventions targeting alcohol accessibility and availability (reducing assaults), and drug checking services, but suggest other interventions such as drug detection dogs may exacerbate harm. Further, higher quality research is required to address identified gaps in the evidence base, particularly on optimal interventions within OMF, around illicit drugs more broadly and in the Global South. Supplementary Information The online version contains supplementary material available at 10.1186/s12954-024-00949-4.
AOD use in LES and OMF has been associated with a diverse array of harms.These harms include excessive intoxication and overdose [15], poor mental health [16,17], offending behaviors such as violence or public disorder offenses [18,19], sexual risk-taking [1], alcohol-and other drug-affected driving [12,20], sexual violence [21,22], risk of victimization [23], and death [24].As such, AOD use in these settings can place a significant burden on acute health services including ambulances and hospitals [25,26].To prevent and/or reduce the prevalence and impact of these harms, there have been growing number of services, programs, campaigns and interventions introduced around the world.
Harm reduction in licensed entertainment settings and festivals
Harm reduction refers to reducing the health, criminal justice or social harms associated with AOD use without necessarily eliminating use [27].A number of harm reduction interventions have been trialled and implemented in LES and OMF specifically, including drug checking [28], transport interventions [29,30], onsite medical services [31], chill out/roaming support services [32], liquor licensing laws and regulations [33,34], licensed venue staff training [35,36], and policing and compliance strategies [37].These interventions vary in their focus regarding the targeted population group, settings, targeted harm(s) and drug type.In addition, changes in drug-related harms may be an unintended consequence for some strategies that primarily target crime and antisocial behavior (e.g., police using drug detection dogs).In recent years, many of these individual measures have been combined to develop more complex, population-wide, multicomponent approaches that encourage change in local policies, structures, systems, and AOD use cultures.A detailed explanation of each intervention described in this study is provided in Table 1.
Given these types of interventions are increasingly implemented across the world, it is crucial for policymakers and service providers to understand the extent to which they are effective in achieving their intended outcomes, the mechanisms that drive outcomes, whether their effectiveness is limited to particular populations or settings, and/or any unintended consequences.There remain several gaps in knowledge that have not been addressed by existing reviews in the field [15,[33][34][35][36][37][38], as summarized in Additional file 1.First, despite the expansion of interventions that impact AOD-related harms, existing reviews are either outdated or limited in their focus to either LES or OMF settings.Second, they do not consider the full array of levers that could influence or affect harm reduction (i.e., laws, policies, services, training, education, policing, governance mechanisms).Third, most do not provide a methodological quality assessment of studies.Fourth, most existing reviews focus on alcohol or drug-related harm, but not both (despite the frequent simultaneous use of these substances in such settings).Finally, there has been significant expansion of interventions aimed at reducing harm in LES and OMF settings in recent years-the evidence from which is yet to be comprehensively synthesized or reviewed.
This review aims to address these critical knowledge gaps, providing an updated synthesis of interventions that impact harms in LES and OMF settings.We chose the term 'impact' rather than 'effectiveness' to encompass the various outcomes of these interventions-negative, null, or positive-and the diverse effects across domains like health, criminal justice, and behavioral outcomes.This choice allows us to evaluate not just how interventions reduce harm, but also their broader impact, including instances where they may increase harm.Further, the review utilizes appropriate tools to assess the quality of the existing evidence across a broad range of health, criminal justice, and behavioral outcomes.To the best of our knowledge, this review has the largest scope to date regarding intervention types and outcomes examined and, as such, provides evidence to inform AOD harm reduction policy and practice for LES and OMF settings.
Laws and regulations Lockout
A "one-way door policy", whereby entry or re-entry to a licensed venue is prohibited after a specified time, but patrons remaining inside the venue past this time can continue purchasing and consuming until the venue closes
Risk-based licensing (RBL)
Policies applied specifically to premises assessed as being of high risk of alcohol-related harms that may include tailored measures and/or liquor license fees that are levied according to assessed venue risks
Alcohol availability restrictions
These policies aim to restrict the availability of alcohol to consumers and include the banning the sale of "rapid intoxication liquor" (e.g., shots) after midnight or prohibiting the sale of spirits, aiming to mitigate alcohol-related harms by controlling the types and timing of alcohol consumption
Trade hour restrictions and extensions
Restrictions or extensions to the permissible hours during which alcohol can be sold or consumed, strategically impacting alcohol-related harms by controlling availability and consumption patterns to promote safer and more controlled recreational environments
Staggered closing times
Varied end-of-service hours for various licensed premises aiming to mitigate alcohol-related harms by reducing the simultaneous dispersal of alcohol-affected patrons onto the streets
Legal purchase age
The legally established minimum age at which individuals are permitted to buy alcohol, that aims to mitigate potential harm by restricting access and consumption among underage patrons.Minimum ages vary across the world
Smoking bans
Regulatory prohibitions on the consumption of tobacco (and sometimes nicotine through electronic nicotine devices).These bans are interrelated with alcohol and other drug-related harm mitigation by reducing exposure to second-hand smoke, risk of drink spiking when patrons left drinks unattended to smoke outside, and risk of overcrowding in smoking areas
Regulatory compliance and enforcement
The rigorous oversight and implementation of responsible service of alcohol (RSA) regulations by law enforcement, liquor licensing authorities, and venue personnel, that aims to mitigate AOD-related harms
Policing strategies
Strategies involving police, such as the use of drug detection dogs, implementation of patron bans, and the application of varying levels of policing enforcement responses
Medical services
Services such as onsite medical personnel, first aid resources, and rapid first response services.These services aim to contribute to enhanced care for attendees and mitigate the strain on local resources by reducing hospitalizations and ambulance transfers Chill/safe space and roaming support services Multi-faceted services that typically encompass drug education booths, peer led AOD care initiatives (e.g., quiet "chill" spaces for those recovering from the effects of substances), and the distribution of harm reduction resources like sunscreen, condoms, water, and earplugs.These initiatives align with onsite medical services by proactively offering assistance, promoting informed decision-making, and cultivating a safer, more supportive event environment
Transport interventions
These interventions encompass 24-h public transportation, designated driver initiatives, ride-share services, and accessible taxi stands, and are offered as strategic measures to curtail alcohol and drug-related harms by mitigating alcohol-and drug-impaired driving, ensuring the safe transportation of affected individuals, and concurrently diminishing incidents of aggression and assault within licensed entertainment areas
Staff and venue intervention
These interventions include staff RSA training, the voluntary use of hand-held breathalyzers to discourage excessive intoxication, in-bar media campaigns promoting water hydration, violence de-escalation training for bar staff, and the strategic deployment of capable guardians such as bouncers, door attendants, and barricades.Staff and venue interventions create safer environments and curb substance-related issues
Drug checking/pill testing
This intervention aims to analyze drug samples directly from the service user, return results to the service user, engage in information exchange between the service user and the service regarding the user and the content and strength/dose of the drug, and provide a tailored intervention
Patron survey and assessment feedback
This intervention includes a street intercept survey with personalized normative feedback on alcohol and other drug-related risks with tips to reduce related harm
Reporting and protocol
The reporting for this systematic review was guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [39] (also see Additional file 2, PRISM Checklist) and the guidelines provided in the Cochrane Consumers and Communication Review Group: Data synthesis and analysis [40].The review protocol was registered in PROSPERO (CRD42020140004).The categories of outcome variables were largely predetermined and listed in the protocol based on prior reviews (e.g., 15,35) and authors' expert knowledge of the field.They were adjusted following the search strategy and data extraction completion to accommodate any unexpected/overlooked outcomes.
Search strategy
The search strategy and syntax were developed, piloted, and refined in consultation with a library expert from the University of New South Wales.Medical subject headings (MeSH) and keywords were utilized and modified for each database as relevant to LES and OMF harm reduction services.These keywords were applied using PICO for quantitative studies-
Multicomponent interventions Sydney "lockout laws"
The "Lockout Laws" in Sydney, Australia, were a set of policy interventions packaged under the official title of the Plan of Management for the Sydney CBD Entertainment Precinct which included: (i) a 1:30am lockout at pubs, bars, registered clubs, nightclubs and karaoke bars; (ii) a 3am cessation of alcohol service in all venues; (iii) a prohibition on the granting of any new liquor licenses; (iv) a ban on takeaway alcohol sales across NSW after 10pm; (v) an extension of 'banning orders' on designated 'trouble-makers' to prevent them entering most licensed premises in the Kings Cross and Sydney's CBD precincts; (vi) a ban on 'shots' and any ready-to-drink beverage with an alcohol by volume content of more than 5%; and (vii) the introduction of a risk-based license fee for all licensed premises based on license type, compliance history and trading hours
Queensland "tackling alcohol-fuelled violence" (TAFV) policy
The TAFV policy in Queensland, Australia, included the following in stages: (i) a restriction on the service of alcohol from 5 to 3am in the state's 15 designated late-night entertainment precincts (Safe Night Precincts; SNPs); (ii) a ban on the sale of shots (or high-alcohol beverages); and (iii) mandatory ID scanners
Newcastle liquor licensing restrictions
The Newcastle Central Business District (CBD) multicomponent intervention included: (i) earlier closing (3am) and lockout (1am), relaxed to 3:30am and 1:30am months later, (ii) the requirement for licensees to adopt management plans, which were subject to compliance audits, and had a dedicated RSA officer from 11pm until closing, (iii) a ban on serving shots after 10pm, and cessation of selling alcohol 30 min before closing, (iv) a ban on drink stockpiling, and (v) requirements to adopt shared radio procedures and notify all staff of the conditions
US multicomponent interventions
Multicomponent interventions trialled in the USA have largely been designed with RSA and law/liquor licensing enforcement measures at their core
New Zealand' s sale and supply of alcohol act
The Sale and Supply of Alcohol Act included trading hour limits for on-licensed and off-licensed premises, new RBL fees, changes to licensing procedures, and the legal enforcement of one-way door policies
UK multicomponent studies
These largely include Cumulative Impact Zones (CIZ)-specific zones within night-time entertainment districts whereby local authorities may apply additional measures to reduce harm and increase public safety
SALUTT and STAD interventions in Northern Europe
The Behavioral outcomes in our study refer to the shifts in individual behaviors in nightlife and festival contexts that are not covered by standard health or criminal justice measures.These outcomes are key to assessing the impact of harm reduction interventions on behaviors and practices.While behavioral outcomes may slightly differ in how they are measured across studies, they must be identified by authors as a behavior that can be changed/ manipulated to potentially reduce harm.Despite most studies defining these outcomes in slightly different ways, the concept of binge drinking in particular has a commonly used definition in research.That is, the consumption of four or more standard drinks in a drinking session for women, and five or more for men [43].
Screening and data extraction
The database searches resulted in the identification of 84,524 potentially relevant articles.Following the removal of 36,221 duplicates, 48,303 titles and abstracts were screened by the lead author (CE) using Covidence software [44].This resulted in 442 potentially eligible articles, with a decision regarding full-text screening on 120 of these articles made in consultation with at least one other author.A total of 337 eligible articles were full text screened by the lead author (CE), with each article being fully screened by a second reviewer (either author PW, MB, or CH).Discrepancies between reviewers existed for approximately 10% of these eligible articles, with disagreements being resolved through one-on-one or team discussion.Data extraction for all articles was conducted by the lead author (CE) with duplicate extractions conducted for each article by either PW, MB or CH.Any differences in extractions were discussed during regular team meetings to resolve discrepancies and develop new exclusion criteria as needed.The following data were extracted from each study: author, year of publication, country, study setting, intervention measures, method of analysis, health, criminal justice, and behavioral outcomes (as listed above), the direction of the effect (i.e., positive, negative, null, or mixed) and size of effect (if reported).
Methodological quality assessment tools
Included quantitative studies were assessed for risk of bias and methodological quality using the Effective Public Health Practice Project (EPHPP) quality assessment tool [45].This tool was chosen due to its high construct validity, content validity and inter-rater reliability [45].Further, it consists of assessment criteria (including risk of bias) that accommodates all types of quantitative studies (not just randomized controlled trials).Included qualitative and mixed methods studies were assessed using the Critical Appraisal Skills Programme (CASP) appraisal tool [46].Using these tools, the methodological rigor of each study was assessed by the lead author (CE) and a second rater (either PW, MB, CH, or DD).Conflicts were resolved by consensus.DD triple-checked data extraction and quality assessment for 10% of studies.
Using the EPHPP tool, each quantitative study was rated as strong, moderate, or weak based on six criteria: selection bias, study design, confounders, blinding, data collection method and withdrawals.Based on the tool guidelines, an overall rating was given for each study.A 'strong' overall rating was applied to studies that had no weak ratings for any criterion.Those with a 'moderate' overall rating had one weak rating, and those with a 'weak' overall rating had two or more weak ratings.In the rare case that all criteria had a moderate rating, we gave an overall rating of moderate (as opposed to assigning a 'strong' rating as recommended by the guidelines).
The CASP tool [46] assesses ten criteria, e.g.appropriateness of research design.Each criterion was scored either "yes" = 2, "no" = 0 and "can't tell" = 1.If ≥ 8 of the criteria on the checklist were met, the study was rated as "good" quality; if 5-7 were met, it was rated as "fair" quality; and if < 5 were met, it was rated as "poor" quality.
There was significant heterogeneity in included studies' interventions, study design, analyses and outcomes, resulting in limited studies that estimated the same population parameter [47].Given this, and the inclusion of both quantitative and qualitative studies in this review, we could not systematically extract included studies' effect sizes, nor conduct a meta-analysis.Instead, a narrative synthesis was employed to systematically identify common themes and findings (see, e.g., 48,49).Herein, this review's results are structured around intervention type, as suggested in the guidelines provided by the Cochrane Consumers and Communication Review Group [40].
Search results
The literature search located 84,524 publications, with 100 included in the systematic review.The article identification and selection process are detailed in Fig. 1, following PRISMA guidelines [39].
Key characteristics of the 100 included studies are summarized in Additional file 4. A detailed overview by study is available in Additional file 5. Most studies were conducted in Australia (41%), USA (18%) or the United Kingdom (UK;18%) and focused on harm reduction interventions in LES (88%), with only 11% of studies conducted at OMF, and 1% conducted in both LES and OMF.Studies predominantly focused on reducing harms attributable to alcohol specifically (86%).The majority (59%) of studies assessed single interventions (including laws and regulations, medical services, policing strategies, chill out spaces), while the remaining 41% reported on multicomponent interventions.Of the included studies, most (84%) were strictly quantitative, 6% were strictly qualitative, and 9% used mixed methods.Most studies reported the impacts of the interventions on criminal justice outcomes (65%), followed by health-related outcomes (40%) and behavioral outcomes (20%).The methodological quality of 93 quantitative (or mixed methods) studies was assessed using the EPHPP tool (see Table 3).Of those, 26 were rated methodologically weak, 16 moderate, and 51 strong.
There were 13 qualitative and mixed methods studies that were assessed using the CASP tool (see Table 4).Of those, 2 were rated strong quality, 8 moderate quality, and 3 weak quality.A summary of study outcomes regarding the general direction of intervention effects (i.e., positive effect, negative effect, null effect or mixed effect) is provided in Additional file 6.
Alcohol availability studies
Two strong-rated studies investigated the impacts of reduced alcohol availability [91,125].Different outcomes were observed.First, a study by Taylor and colleagues [125] found that a ban on the sale of "rapid intoxication liquor" (e.g., shots) after midnight in Queensland, Australia, did not affect rates of police-recorded serious assaults.Second, a study by Khurana et al. [91] investigated the impact of a ban on hard liquor sales from local bars in Kerala, India, with only five-star hotel bars permitted to sell hard liquor.The authors found that the policy had no effect on verbal insults but reduced sexual assaults significantly (but not rapes, possibly due to increased reporting).Sixteen additional studies examined the effectiveness of reduced alcohol availability as part of a multicomponent intervention in Sydney [51,73,78,96,[100][101][102]140], Newcastle [86,94,95] and Queensland [26,62,66,72,128]; see multicomponent interventions sub-Sects."Sydney "lockout laws"" and "Queensland "tackling alcohol-fuelled violence" policy".
Risk-based licensing (RBL) schemes
Four studies examined the impacts of RBL schemes [64,108,112,141].Two strong Australian studies [64,112] and one strong Welsh study [108] found that RBL had no significant impact on the incidence of police-recorded assaults nor emergency department (ED) presentations for injuries during high alcohol hours (HAH; considered 8 pm-6am Friday and Saturday nights) except for a reduction in ED injury presentations among men aged 20-39 years in Victoria only [64].These findings were generally supported by those of Miller and colleagues' strong-rated qualitative study [141], which reported that while licensees generally supported RBL, they did not perceive RBL to have an impact on the level of alcoholrelated harm and required modifications to be more effective.Twelve additional studies examined the effectiveness of liquor licensing changes as part of a multicomponent intervention [51, 63, 73, 76-78, 96, 100-102, 105, 140] see multicomponent sub-Sects."Sydney "lockout laws"", "New Zealand's sale and supply of alcohol act" and "Other multicomponent interventions".
Change in trading hours
Ten studies investigated the impact of changes in licensed venue trading hours on alcohol-related harm outcomes [53,57,67,81,87,88,92,119,122,129].
Table 4 Methodological quality ratings using CASP criteria
Note.Criteria include: 1) clear statement of aims; 2) appropriateness of qualitative methodology; 3) appropriateness of research design; 4) appropriateness of recruitment strategy; 5) appropriateness of data collection; 6) consideration of relationship between researcher and participants; 7) consideration of ethical issues; 8) rigor of analysis; 9) clear statement of findings; and 10) value of research.Scores included: "yes" = 2; "no" = 0 and "can't tell" = 1.Overall quality ratings included: "good" (≥ 8); "fair" (5-7) or "poor" (< 5) (Casp 2013) Three of these studies examined the effect of 24-h trading licenses [87,88,119].A moderate-rated before-and-after study [119] by Ragnarsdóttir et al. showed that extending trading hours to 24/7 in Reykjavik, Iceland's licensed entertainment precincts increased police-reported violent offenses and ED admissions.In contrast, two studies by Humphreys and colleagues, one strong [87] and one of moderate quality [88], found that extending trading hours from 11 pm to 24/7 in Manchester, England, did not result in changes in violence, though there was some evidence of a shift in weekend violence to later hours [54].Smaller extensions in trading hours were introduced in two strong-rated studies in Amsterdam [67] and England and Wales [81].Findings showed contrasting results, with the extension in Amsterdam being associated with an increase in alcohol-related injuries, while England and Wales experienced a decrease in road traffic accidents, especially on Friday and Saturday nights among younger drivers.
Four other studies across Europe examined the impact of both trading hour restrictions and extensions (when closing times were staggered) following policy changes that allowed licensed venues in Preston [92] and Hartlepool [57] in England, or municipalities in Bavaria, Germany [129] or Norway [122] to determine venue opening and closing hours.Two moderate-rated studies [92,129] and one weak quality study [57] found that when these policies led to a slight increase in trading hours, there was an observed decrease in alcohol-related crimes [92]; violence against the person [57,129] and criminal damage [57].In contrast, Rossow and colleagues' [122] strongrated study reported a statistically significant increase in assaults following closing hour extensions in Norway.Brown and colleagues [57] also reported an increase in the number of ambulance attendances and public order offenses (antisocial behavior) in England.Furthermore, Tesch and colleagues [129] found that restricted trading hours led to an increase in night-time violence in Bavarian towns with higher levels of day-time violence.
A further strong study in Spain found reduced trading hours linked to decreased alcohol-related hospitalizations [53].
Legal purchase age
One moderate-rated study examined the impacts of a legal purchase age policy.A before-and-after study design was used to examine the impact of a reduction in the legal purchase age for alcohol from 20 to 18 years in New Zealand [83].Finding showed that the policy resulted in a threefold increase in rates of problems per drinking occasion at pubs and nightclubs, across all drinking quantities.
Smoking ban
Five studies described various impacts of smoking bans in licensed venues, including four strong-rated quantitative studies from the USA [54,56,93,135] and one fair rated observational study from Scotland [139].Young-Wolff and colleagues [135] reported the only positive health outcome, with higher likelihood of alcohol use disorder remission following the introduction of a ban [135].Two studies reported a decline in violence/assaults following a ban [56,139], while one study reported no impact on the rate of assaults [93].Mixed results were also reported regarding sexual assaults, with Forsyth and colleagues [139] reporting an increase in sexual assaults in Scotland, while no change was reported following the introduction of a ban in Minnesota, USA.[93].Forsyth and colleagues [139] also reported mixed findings for behavioral outcomes, observing an increase in risky alcohol consumption practices, and a decrease in overcrowding in licensed venues following a ban.Null findings were reported in USA-based studies for traffic-related health outcomes (alcohol-related road traffic accidents or car crash fatalities, [54]) and other crime outcomes (general crime, or public order offenses [93]).
Regulatory compliance and enforcement
A moderately rated before-and-after study by Tomedi and colleagues [130] found that enhanced enforcement of laws prohibiting the sale of alcohol to intoxicated patrons in on-premised licensed venues significantly reduced the proportion of adults that engaged in risky consumption practices (i.e., drinking eight or more standard drinks) in these settings during their most recent drinking episode in New Mexico, USA.Six additional studies examined the effectiveness of enhanced regulatory compliance and enforcement as part of a multicomponent intervention [55,71,113,131,134,137]; see Sects."UK multicomponent interventions" and "SALUTT and STAD interventions in Northern Europe".
Drug checking/pill testing
Two cross-sectional weak-rated studies by Measham examined the effectiveness of drug checking at OMFs in the UK [28,99].Findings indicated that drug checking was associated with reduced dose size and increased rates of the disposal of dangerous drugs [99], as well as a 95% reduction in AOD-related hospital presentations [28].One additional study examined the effectiveness of drug checking as part of a multicomponent intervention at a OMF [110]; the results of this study are described in the 'Multicomponent intervention' Sect."Other multicomponent interventions".
Transport interventions
There were six studies that examined the effectiveness of transport interventions [29,30,89,90,106,121].Three studies examined the impact of extending public transport hours, including one strong-rated study that investigated the impacts of a gradual extension of public transport operating hours in Washington DC in the USA from midnight to 3am [89], and two weak-rated studies by Curtis and colleagues [29,30] which investigated the impacts of 24-h public transport in Melbourne, Australia.Findings showed that extending public transport hours had limited impact on a range of outcomes including driving under the influence (DUI) arrests (however areas with bars within walking distance to transit stations noted decreases in DUI arrests), total alcohol-related arrests for public nuisance and disorder [89], alcoholrelated injuries, police-recorded assaults, ambulance attendances, and road traffic accidents on Friday and Saturday nights [29], with some evidence of displacement of road crashes over adjacent hours early Sunday morning rather than an impact of the intervention [29].Three studies based in the USA examined the effects of an intervention that included a designated driver/ride sharing measure.One weak-rated study investigating the effects of designated drivers at a college bar in Tallahassee, and one strong-rated study in Ohio that evaluated a ride sharing service found such measures led to an increase in the proportion of patrons riding with/acting as a designated driver (12-24%; [90]), or stating that they would be less inclined to drive after drinking (71%; [106]).Studies reported mixed results regarding any changes in the number of alcohol-attributable crashes (or single vehicle night-time crashes as a proxy) following the interventions.While Miller and colleagues [106] reported no significant change following a rideshare promotion, Rivara et al. [121] reported a substantial reduction in single vehicle night-time crashes among 21-34-year-old drivers as part of their weak-rated study, conducted in Seattle, USA that consisted of a multi-pronged social marketing campaign promoting taxi use and designated drivers.Two additional studies examined the effectiveness of safe taxi ranks (i.e., ranks manned by security staff ) as part of a multicomponent intervention [63,105].The results of these studies are described in the 'Multicomponent intervention' Sects."UK multicomponent interventions" and "SALUTT and STAD interventions in Northern Europe".
Policing strategies
There were six studies that examined the effectiveness of policing strategies [17,65,82,85,123,127].Studies were limited to examining the effects of strategies on criminal justice and behavioral outcomes (i.e., not health outcomes).Three cross-sectional studies (two rated weak [82,85] and one strong [17]) examined the impacts of drug detection dogs at OMF or LES in Australia.Each showed the presence of drug detection dogs led to increased risky consumption and behaviors, with 10% [82] to 36% [85] of participants reporting they concealed drugs internally, or 'panic' consumed some/all their drugs in response to the presence of drug detection dogs [17].In addition, Hickey and colleagues [85] reported that 13-43% of participants (among different subgroups) had been arrested, cautioned, or fined following positive notifications from drug detection dogs.
Two studies examined the effects of police-imposed bans within licensed entertainment precincts on criminal justice outcomes [65,127].The first study by Curtis et al. [65], which had a moderate rating, reported a significant increase in antisocial behavior charges, specifically public order offenses, following the introduction of 72-h police-imposed bans in Victoria, Australia.In contrast, a strong-rated study investigating the impact of 10-day police-imposed bans, along with mandatory ID scanners, in three licensed entertainment precincts in Queensland found no evidence of changes in serious assaults, common assaults, or offenses related to public order/good order [127].A further moderate-rated study by Rowe et al. [123] found that one of three levels of policing enforcement responses (letters, incident reports or feedback meetings) was associated with significant reductions in police incidents and rates of intoxicated patrons in high-risk licensed venues in regional New South Wales, Australia.
Medical services
There were five weak-rated studies that examined the impact of medical services at OMFs on health outcomes in Australia [75], Canada [97], the UK [50,133] and the USA [31].Four were cross-sectional studies [31,75,97,133] and one was a prospective cohort study [50].Four out of the five studies [31,50,75,97] demonstrated a significant reduction in ambulance transfer rates by 65-78%, due to the availability of medical personnel on site at the OMF.Similar reductions were reported by Wood and colleagues [133] who compared transfer rates from an OMF and the OMF after party event, reported 45% less transfers for AOD toxicity from the after party, where an ambulance treatment tent was present [133].
Chill/safe spaces and roaming support services
There were five studies that examined the effectiveness of chill/safe spaces and roaming support services, finding mixed impacts on criminal justice and health outcomes [32,59,74,79,126].Four were rated as weak [32,59,74,79] and one as strong [126].Two Australian studies showed that the introduction of a chill/safe space [32] or street support services [126] in licensed entertainment precincts had no significant impact on hospital admissions [32,126] or ambulance attendances [126].The impact of support services on assaults in Australian cities was mixed, with some evidence of significant reductions in general and sexual assaults in Sydney [74] and serious assaults during HAH in Cairns, [126], while Ward and colleagues [32] reported an increase in violence in taxi queues in Melbourne.Doran and colleagues [74] also reported a reduction in road traffic accidents (12%) and thefts (40%), while Ward [32] found no significant association between the introduction of a mobile van and chill/safe space and the proportion of policereported incidents (public order, theft, property damage or drug offenses) during HAH.In contrast, an evaluation of a roaming support service in licensed entertainment precincts in two cities in the UK found the introduction of the service was associated with an increase in policerecorded crime levels (including violence and sexual assaults) [79].The only study to examine a safe space initiative at a multiday festival was in Portugal by Carvalho and colleagues [59], which found that patrons presenting to the service with difficult psychedelic and emotional experiences showed a significant positive effect on resolving their mental health episode (p < 0.05), thus confirming crisis resolution.
Staff and venue interventions
There were five studies that examined the effectiveness of staff and venue interventions across a range of late-night entertainment settings [61,84,109,136,138], showing conflicting impacts on a range of outcomes.Two strongrated studies by Ham et al. [84] and Moore et al. [109] examined the impact of staff training in responsible service of alcohol (RSA) and conflict resolution, showing no significant change in the number of assaults [84] and an increase in reported assaults following visits and risk audits by Environmental Promotion Officers [109].Ham et al. [84] also reported no statistically significant changes in the number of reported brawls but did find a decrease (16%) in reports of public disorder, with no evidence of displacement.In a weak-rated study by Charlebois and colleagues [61], the promotion of free water led to a reduction in incidence of hazardous drinking (78%), compared to the control bar participants (87%).
A qualitative moderate-rated study explored the impacts of the voluntary adoption of hand-held breathalyzers on the doors of licensed venues in the UK [138].Mixed findings were reported, with some security staff reporting reduced aggression at the door, while others reported that the intervention was not useful.Finally, a weak-rated observational study conducted by Zawisza et al. [136] [136]examined the impact that the presence of place managers and capable guardians (e.g., police, security staff, bar staff ) in licensed entertainment precincts (Arkansas, USA) had on incidents of aggression.The authors observed a reduction in the number of incidents of aggression during a 6-week period [136].
Patron surveys and assessment feedback
There were two studies that examined the impact of patron surveys and assessment feedback in licensed entertainment precincts on risky consumption practices [52] and driving offenses [107].The moderate-rated RCT study by Baldin and colleagues [52] found that their web-based survey with personalized normative feedback on alcohol-related risks and tips to reduce harms led to a significant reduction in weekly binge drinking among participants at six-month follow-up of 38% (p = 0.026); however no significant effect was observed for the control group.In contrast, the weak-rated study by Monezi and colleagues [107] found that their patron survey (which included a module on how alcohol consumption affects driving capacity) had no significant impact on drink driving behaviors.
Sydney "lockout laws"
Eight studies reported on the effectiveness of the "lockout laws" in Sydney, Australia [51,73,78,96,[100][101][102]140].Of these, six strong-rated studies examined changes in police-reported assaults, all showing statistically significant declines over time that ranged from 22 to 49% [51,73,96,[100][101][102]. Two of the more recent studies showed that the declines in violence in Sydney CBD and Kings Cross were sustained between 2014 and 2021 [51,96].The decline in assaults was also reflected in a decline in hospitalizations, as reported in Fulde and colleagues' [78] weak-rated study.This declining trend in violence was echoed by residents, patrons and music industry stakeholders in the lockout zone during focus groups with Hughes and colleagues [140] as part of their moderate-rated qualitative study.However, participants also reported possible displacement of violence to adjacent suburbs.Evidence of violence displacement was also found in a strong-rated quantitative study by Donnelly et al. [73] with a 12% increase in assaults within the Proximal Displacement Areas, and a 17% increase in assaults within the Distal Displacement Areas.The displacement was significant and increased over time, albeit with an overall net reduction of non-domestic alcohol-related violence across target and adjacent precincts [73].
Queensland "tackling alcohol-fuelled violence" policy
Five studies evaluated the effectiveness of the TAFV Policy implemented in SNPs in Queensland, Australia, in reducing alcohol-related harms [26,62,66,72,128].Four strong studies indicated a significant reduction in the number of assaults [62,128], hospitalizations [26], and ambulance callouts [66].The moderate quality study by Devilly and colleagues [72] reported no meaningful change in public order offenses and an increase in drug offenses.
Newcastle liquor licensing restrictions
Five studies evaluated the effectiveness of legislative changes in Newcastle, Australia [86,94,95,105,132].Four were rated strong [86,94,95,105] and one moderate-rated study [132] assessed community perceptions and experiences of crime, as well as support for restrictions.Miller et al. [105] compared the Newcastle CBD intervention with Geelong's own set of interventions which included: Geelong-(I) ID scanners, (ii) increased policing and CCTV, (iii) taxi ranks, and (iv) risk-based licensing.Similarly, Kypri et al. [95] compared the Newcastle intervention with Hamilton.The legislative changes led to significant long-term declines in police-recorded assaults [94,95] and alcohol-related injury presentations to hospital [86,105].Comparison to a control site found that reduced trading hours rather than the lockout was the key mechanism to change [95].Results of a survey of Newcastle residents further supports these findings, highlighting a significant decrease in general crime, risky alcohol consumption and witnessing or being involved in physical violence [132].
US multicomponent interventions
Six studies reported on multicomponent interventions in the USA [58,60,76,80,134,137].Multicomponent interventions trialled in the USA have largely been designed with RSA and law/liquor licensing enforcement measures at their core.In Zhang and colleagues' strong study [137] they reported a reduction in violent crime in Atlanta, USA, following the introduction of restricted trading hours combined with a reduced density of alcohol outlets, and the enforcement of laws prohibiting alcohol sales to minors.In contrast, two strong studies examined the impact of increased trading hours combined with enhanced law enforcement in licensed entertainment precincts in San Marcos, Texas, USA [60] and Little Rock, Arkansas, USA [58].While Chamlin et al. [60] found the intervention to have no effect on public order offenses or complaints of DUI, Burgason et al. [58] reported a 23% reduction in reported violent crime with their intervention also including improved lighting and video surveillance and increased penalties.
Three studies based in the USA examined road traffic accidents and DUI related arrests and accidents following interventions that focused on improving RSA training and increasing onsite/liquor licensing law enforcement.In Fell and colleagues' [76] strong-rated before-and-after controlled study targeting problem bars in New York and Ohio, they found no statistically significant changes in road traffic accidents pre-post study or between intervention and control bars, However, findings showed a decline in reported DUI arrests post-intervention and a significant short-term decline in self-reports of drink driving in New York.Similarly in South Carolina, USA, a moderate-rated non-randomized controlled trial which comprised of a RSA practices toolkit, onsite law enforcement and a media campaign in licensed entertainment precincts, resulted in a significant reduction in alcoholinvolved crashes and DUI arrests in the year following the intervention [80].Similar to Fell and colleagues' [76] study, Xu et al. 's [134] strong-rated study targeted problem alcohol outlets in New Orleans.Additional enforcement staff combined with increased license fee and expanded powers for the alcohol license board were found to have no impact on the crime rate over two years post-intervention [134].
New Zealand's sale and supply of alcohol act
One weak study [77] and one moderate study [120] examined the impact of the Act on ED attendances [77] and drink driving [120].The cross-sectional study by Ford et al. [77] reported a non-significant reduction in ED attendees, while Randerson et al. [120] found a 24% reduction in apprehension of drivers with a BAC of 0.008, and 35% drivers reported drinking less before driving after the law change.
UK multicomponent interventions
Five strongly rated studies, all conducted in UK, examined the impact of introducing cumulative impact zones (CIZs) and the impact of implementing new local licensing guidelines and increased venue inspections [68][69][70][71]118].The study designs included data linkage methods [68], a quasi-randomized control trial [71], a before-andafter study [69] and two interrupted time series (ITS) analyses [70,118].CIZs were found to have a positive impact on alcohol-related hospital admissions and crime, particularly alcohol-related violent crimes and sexual assaults (but not public order offenses).For example, one strong before-and-after study by De Vocht et al. [69] examined the impact on alcohol-related hospital admissions, reporting reductions following the introduction of CIZs and intense liquor license scrutiny, measured as the number of refusals of new liquor license applications.In Pliakas and colleagues' [118] strong-rated study, they found no impact on alcohol-related ambulance callouts.The quasi-randomized control trial [71] found that closing a nightclub and improvements in liquor licensing guidance and inspections were collectively associated with a reduction in antisocial behavior/public order incidents, but not other crime (including assaults) or acute health service attendance.
SALUTT and STAD interventions in Northern Europe
Five studies evaluated the STAD and modified versions such as SALUTT, aimed at reducing alcohol-related harms in LES [55,113,114,124,131].The moderate-and strong-rated ITS studies evaluating STAD in Stockholm and Visby in Sweden both reported significant reductions in police-recorded violence in the entertainment precincts of 29% and 70%, respectively [113,114].This was despite the closing time in Visby being extended from 2 to 3am.These findings were supported by the work of Trolldal et al. [131] and Brännström et al. [55], who conducted strong-and moderate-rated studies examining the impact of STAD (and modified versions) much larger areas of Sweden (237 and 288 municipalities, respectively).Both studies found STAD led to significant reductions in police-recorded assault.However, Trolldal and colleagues [131] identified the implementation of the community coalition steering group as the key driver of the reduction (rather than the RSA training or supervision components of the intervention).In contrast to findings of the STAD studies, a strong-rated evaluation found the SALUTT intervention to have no statistically significant effect on violence in and around licensed premises [124].
Other multicomponent interventions
Two strong-rated studies in Victoria, Australia, examined the impact of multi-pronged community-based interventions on ED presentations [63,103] and policerecorded assaults [63].While the Fremantle liquor accord regulations included a broad range of measures including patron bans, RSA guidelines, one-way door policy, awareness campaigns, liquor license freeze, safe taxi rank, policing operations, night watch radio program, ID scanners, and RBL [63], the Geelong-based intervention included measures more heavily focused on increasing licensed venue and police collaboration and enforcement.ITS analyses showed that neither intervention had a significant effect on the count of assaults, or rate of ED presentations, at any research sites during peak weekend times.While findings by Miller et al. [103] indicated that ID scanners and a safe drinking awareness campaign were associated with an increase in ED attendances, authors suggest these findings to be coincidental rather than correlational.
A weak-rated study by Navarro et al. [111] examining a multi-stakeholder/multicomponent intervention in regional NSW that included a letter from the mayor to licensees asking to brief security staff and ensure RSA, local media briefings, increased police visibility, incident feedback through media and stakeholder meetings.The intervention had no effect of the intervention on assaults but a small statistically significant effect on alcoholrelated sexual assaults.
A weak-rated study in Mexico found reduced trading hours linked to decreased alcohol-related assaults [117].The intervention involved a combination of restricted bar opening hours and the introduction of a 10PM cut-off for alcohol sales by off-premise stores.
One weak study examined the effect of a multicomponent intervention at a festival, on both alcohol and illicit drug harms, reporting a reduction in ambulance attendances and acuity, and a decrease in the utilization for local, community-based health services [110].It is important to note that isolating specific outcomes for individual interventions within multicomponent studies, such as Munn and colleagues [110], can be challenging due to their inherent design focus on collective effectiveness rather than singular components.
Discussion
This systematic review synthesized the impact of interventions on AOD-related harms in LES and OMF alongside their impacts on health, criminal justice, and behavioral outcomes.A dominant majority of studies focused on interventions in LES (88%) when compared to OMF settings (11%), or both (1%).A similar majority of studies examined reducing harms associated with alcohol consumption compared to illicit drug consumption (7%) or both (7%).Further, multicomponent interventions (39%) were common compared with stand-alone interventions such as policing strategies (6%), medical service provision (5%), chill spaces and roaming support services (5%), and drug checking services (2%), and there was a considerably stronger emphasis on evaluating criminal justice outcomes (65%) than health outcomes (40%) and behavioral outcomes (18%).
This review provides promising evidence into the effectiveness of different interventions that may impact AODrelated harms, including highlighting what works and what does not.Multicomponent interventions showed the best results for both health and criminal justice outcomes, with 28/39 (72%) of studies being of strong quality, and 33/39 (85%) of studies reporting at least one positive outcome (e.g., decreased hospitalizations and assaults).Further, the evidence available regarding medical services and drug checking at OMF, while limited, suggests that they are promising approaches to decrease hospitalizations, ambulance attendances and the overall burden on local healthcare infrastructure.There was also good evidence to support the careful application of trading hour restrictions [53,[115][116][117]122], but less so for staggered closing times [88,92], mandatory ID scanners and police-imposed patron bans [65,127].
When applied as standalone measures, laws and regulations interventions showed mixed effects (see Additional file 6), with some unintended consequences.There was no supporting evidence for the use of police-issued patron bans [65,127], however those examined were short-term and the examination of longer-term bans is required.There was also no supporting evidence for the use of drug detection dogs [17,82,85]: a significant finding given their increased use across many countries with the aim of managing public safety.The literature canvassed in this review suggests that LES patrons and festivalgoers often engage in risky drug consumption practices when police and dogs are present, due to fear of criminal justice repercussions, with their presence also acting as a barrier to medical help-seeking [17,82,85].
This review builds on the findings of prior reviews in the field.Reviews from over a decade ago (for example see Akbar et al. [15]) identified this topic as one that is under-researched.However, the large number of studies identified through this review highlight a period of intense research investment since the review by Akbar and colleagues [15], which only included 14 studies, provides promising evidence on some interventions and services, and consolidates the evidence on several interventions previously tagged as "promising, " such as multicomponent interventions.This review also highlights the significant increase in high-quality research that has been conducted in recent years in entertainment precincts, and the continued lack of research (including high-quality research) being conducted in other high-risk settings such as OMF.
This review has highlighted continued gaps in the existing evidence base.Of note is the heavy focus on: 1) harm associated with LES (with few in OMF settings), 2) alcohol-related harm (rather than other drug-related harm), and 3) criminal justice outcomes (46%), specifically assault and violence.This focus is problematic as it reduces the capacity to assess impacts on health or across multiple outcome domains.More broadly, we see considerable variability in outcomes used by different studies, reducing the capacity to conclusively identify whether the interventions reduce harm, in what ways and if there are any unintended effects.Improving the study designs such that future harm reduction evaluations assess impacts across multiple outcome domains (health, criminal justice and behavioral) and using consistent and highquality research methods is recommended.Furthermore, while studies focused on the evaluation of medical service provision at OMF demonstrated promising findings, there is a clear need for more high-quality research to be conducted in this domain.
In addition, this review highlights the volume of highquality evaluations of multicomponent interventions implemented in LES.While these interventions are largely proving successful, there is heterogeneity regarding the measures included in multicomponent interventions internationally.This heterogeneity, and the implementation design, results in an inability to decipher the key drivers of the success.Adding to this ambiguity is the conflicting evidence regarding some of the measures included in multicomponent interventions when delivered as stand-alone interventions.This highlights the need for more comprehensive evaluations of multicomponent interventions to identify what works to improve outcomes, how and for whom.
Limitations
This review has four main limitations.First, we acknowledge the extensive heterogeneity in study designs, strategies, sample sizes, and outcomes, which has been a significant factor in our methodology and analysis.This diversity, encompassing both quantitative and qualitative research methodologies, presented unique challenges in synthesizing the data, consequently preventing a metaanalysis from being conducted.We have intentionally included a broad range of interventions, study designs, and impacts to capture the comprehensive scope of the subject matter.However, this inclusivity also brought with it challenges of balancing and weighing the contributions of diverse study types.This aspect, in particular, should be considered when interpreting the findings of our review.We refer the reader to our earlier discussion where we justify the inclusion of this wide spectrum of studies and acknowledge the implications of their heterogeneity on our results.Second, the study was limited by design to English language publications only.Third, an overwhelming majority of studies were conducted in developed countries (n = 91) such as Australia, the USA or the UK which have more similar drinking cultures.This means that we are unable to explore differences in cultural norms that may affect AOD use and harms in different milieus particularly beyond the Global North.Moreover, including publications only in English and largely from developed countries impacts the generalizability and translatability of the research findings.Fourth, this review did not include grey literature, potentially increasing publication bias given researchers may be less inclined to publish null or negative findings in academic journals [142].However, approximately half of all health, criminal justice and behavioral outcomes reported in studies in this review found null and negative effects (see Additional file 6), suggesting that publication bias is likely to be minimal.
Implications for policy and practice
This review provides an up-to-date summary for policymakers of what works in LES and OMF to reduce AOD-related harms.Firstly, it highlights that there are interventions that can be deployed in LES and OMF to improve health, criminal justice and behavioral outcomes-whether they be overdose, injuries, mental health conditions, hospitalizations, assaults, or road traffic accidents.Secondly, it highlights the benefits of maintaining and expanding investment in key harm reduction approaches, namely onsite medical services and multicomponent interventions.Based on results presented in this review, each of these intervention types can be deemed as effective tools for harm reduction.Thirdly, findings of this review highlight a number of areas where caution is recommended.These include the deployment of drug detection dogs at OMF, whereby their presence was found to be related to increase risky consumption practices and potential harms.Finally, this review clearly demonstrates the importance of continuing to invest in high quality study design and evaluations to ensure that harm reduction interventions are effective.
Conclusion
To the best of our knowledge, this is the most up-todate and comprehensive review of strategies that aim to impact AOD-related harm in LES and OMF around the world.Findings provide consistent support for onsite medical services for reducing ambulance transfer rates, multicomponent interventions targeting alcohol accessibility and availability for reducing assaults, and promising behavioral outcomes for patrons who use drug checking services.Furthermore, findings of this review suggest that policing strategies rarely reduce criminal justice outcomes and, in some instances, lead to an exacerbation of negative health outcomes.This review thus holds clear implications for policy and practice in these settings, and highlights the need for studies that address identified evidence gaps in OMF settings, around illicit drugs more broadly and in the Global South.Continuing to grow this evidence is important as harm reduction measures become increasingly prevalent across the world.
Fig. 1
Fig. 1 PRISMA flow diagram of study retrieval process.Note.LES: Licensed entertainment settings; OMF: Outdoor music festivals; AOD: Alcohol and other drugs
Table 2
Search string (PubMed) 13. 6 OR 7 OR 8 OR 9 OR 10 OR 11 OR 12 14.Nightlife settings OR safer nightlife OR licensed venue* OR recreational setting* or licensed premise* OR entertainment precincts OR licensure OR pub OR club OR patron OR attendee* OR nightclub* OR disco* OR bar OR lounge OR festival* OR rave OR music 15. 5 AND 13 AND 14 16.Limit 15 to: language: English; publication date: 2010-2021
Table 3
Methodological quality ratings using EPHPP criteria
Table 3
(continued) Note.S: Strong; M: Moderate; W: Weak; N/A: Not applicable *Ratings for blinding and withdrawals were only applicable where the study was an RCT design | 2024-02-22T12:03:30.575Z | 2024-02-21T00:00:00.000 | {
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251876684 | pes2o/s2orc | v3-fos-license | Functionalization of Graphite with Oxidative Plasma
Surface-modified graphite is studied as an electrode material, an adsorbent, and a membrane component, among other applications. Modifying the graphite with plasma can be used to create relevant surface functionalities, in particular, various oxygen groups. The application of surface-oxidized graphite often requires its use in an aqueous environment. The application in an aqueous environment is not an issue for acid-oxidized carbons, but a discrepancy in the structure–activity relationship may arise because plasma-oxidized carbons show a time-dependent decrease in the degree of functionalization and related properties. Moreover, plasma-oxidized materials are often characterized in terms of their chemical and physical properties, most notably their degree of functionalization after plasma treatment, without contact with water. In this study, we used low-temperature plasma oxidation with pure oxygen and carbon dioxide and sample-washing with concentrated nitric and sulfuric acids. To evaluate the electronic properties of modified graphite, the work function changes and surface oxygen content were measured just after plasma modification and after water immersion. We show that water immersion drastically decreases the work function of plasma-treated samples, which is accompanied by a decrease in the number of radicals introduced by plasma. Our results demonstrate that the increase in stable work function as a result of plasma treatment, brought about by an increase in the surface oxygen species concentration, can be realized most effectively for the acid-washed graphite.
Introduction
Natural graphite can be classified into three principal types: crystalline small flake graphite (or flake graphite), crystalline vein or lump graphite, and amorphous graphite (very fine flake graphite). Each natural graphite has different physical properties, appearance, chemical composition, and impurities [1]. Recently, much attention has been focused on the use of economically attractive electrodes based on natural graphite. It is relatively abundant and inexpensive, has good durability and relatively low mass, and has excellent porosity and conductivity [2,3]. Moreover, an improvement in the electrochemical performance of graphite can be achieved through several methods such as mild oxidation, surface deposition of metals/metal oxides, or polymer coating [4]. Such actions may lead to significant changes in the surface of the material, such as the removal of some reactive sites and/or defects, the creation of an oxide layer, the formation of a porous structure, the change in electronic conductivity, and the reduction in the thickness of the solid electrolyte interface layer [5]. To obtain the best properties, the carbon content in graphite must be maximized. It can be achieved by purification with acids such as HCl, HNO 3 , H 2 SO 4 , or HF. According to the authors [6], a maximum carbon content of 98.4% was obtained for acid treatment of flake graphite. The combined acid-alkali-acid (H 2 SO 4 /H 2 O 2 -NaOH-HCl) treatment improved this score to 99.68% or even 99.72% if HF was also employed. Nitric acid treatment is also a very popular method for introducing surface oxygen functional groups (OFG), mainly carboxylic, onto various carbon materials, such as amorphous car-improved by using plasma. A clear correlation between their electrocatalytic activity and work function indicates that the latter can be a common descriptor of activity [24]. We recently reported that the degree of functionalization of the plasma-modified graphene paper is controlled by the post-plasmatic reactions in water. Immersion in water of plasmatreated graphene paper resulted in stable surface functionalization, confirmed by the constant value of the work function [25].
The need for preliminary purification of natural graphite before its application and possible surface functionalization with oxygen groups motivated us to investigate the effect of acid washing on plasma oxidation. We aimed to evaluate the effect of plasma treatment on the electronic properties and surface composition of the off-the-shelf graphite material washed with different acids. We used oxygen and carbon dioxide plasmas to evaluate the extent of possible WF changes and confronted the results for two approaches: plasma oxidation of the off-the-shelf sample and the same treatment of graphite, which underwent acid purification.
Results
The most spectacular changes are observed by following the contact potential difference (CPD) with the Kelvin probe for the samples before and after plasma treatment, Figure 1. The initial increase in the CPD is of the order of 1 V, equivalent to an increase of 1 eV of the work function of the material. The observed modification of the electronic properties of the surface is not stable over time and decreases substantially, with the magnitude of the effect depending on the type of plasma (O 2 , CO 2 ) and the time of treatment. As presented in Figure 1 CO 2 plasma treatment results in a higher decrease in CPD than O 2 plasma. The graphs of the short-term evolution of CPD for different plasma treatments are presented in Figures S1 and S2 in Supplementary Materials. Acid-washed samples exhibit a similar pattern of high initial increase in CPD followed by substantial decrease over time. reactivity of carbon-based electrocatalysts, for example, for the oxygen reduction reaction, is correlated with the functional oxygen groups present in the carbon materials [23] and can be successfully improved by using plasma. A clear correlation between their electrocatalytic activity and work function indicates that the latter can be a common descriptor of activity [24]. We recently reported that the degree of functionalization of the plasmamodified graphene paper is controlled by the post-plasmatic reactions in water. Immersion in water of plasma-treated graphene paper resulted in stable surface functionalization, confirmed by the constant value of the work function [25]. The need for preliminary purification of natural graphite before its application and possible surface functionalization with oxygen groups motivated us to investigate the effect of acid washing on plasma oxidation. We aimed to evaluate the effect of plasma treatment on the electronic properties and surface composition of the off-the-shelf graphite material washed with different acids. We used oxygen and carbon dioxide plasmas to evaluate the extent of possible WF changes and confronted the results for two approaches: plasma oxidation of the off-the-shelf sample and the same treatment of graphite, which underwent acid purification.
Results
The most spectacular changes are observed by following the contact potential difference (CPD) with the Kelvin probe for the samples before and after plasma treatment, Figure 1. The initial increase in the CPD is of the order of 1 V, equivalent to an increase of 1 eV of the work function of the material. The observed modification of the electronic properties of the surface is not stable over time and decreases substantially, with the magnitude of the effect depending on the type of plasma (O2, CO2) and the time of treatment. As presented in Figure 1 CO2 plasma treatment results in a higher decrease in CPD than O2 plasma. The graphs of the short-term evolution of CPD for different plasma treatments are presented in Figures S1 and S2 in Supplementary Materials. Acid-washed samples exhibit a similar pattern of high initial increase in CPD followed by substantial decrease over time. To obtain a stable surface of the plasma-modified graphites, the samples were washed with deionized water. Such a treatment was chosen to remove the static electricity charging of the samples created by plasma treatment and to promote surface reactions that lead to changes in surface composition. This treatment allowed the material to have stable surface properties, as evidenced by a constant value of the change in work function (change in CPD) relative to the starting material ( Figure 2). To obtain a stable surface of the plasma-modified graphites, the samples were washed with deionized water. Such a treatment was chosen to remove the static electricity charging of the samples created by plasma treatment and to promote surface reactions that lead to changes in surface composition. This treatment allowed the material to have stable surface properties, as evidenced by a constant value of the change in work function (change in CPD) relative to the starting material ( Figure 2). The effect of water for different plasma treatment times for O2 and CO2 gases is presented in Figure 3. An immediate decrease in work function is easily observed, and the final CPD values vary depending on the type of plasma and the treatment time. The increase in final work function due to plasma and water treatment is more pronounced for O2 plasma than for CO2 plasma. In the case of oxygen plasma, the maximum changes of surface electronic properties (ΔCPD) are observed for 10 min of plasma treatment, while for carbon dioxide plasma the maximum is much less pronounced and is located at 5 min of plasma treatment. XRD characterization was performed to verify the structural integrity of the modified graphite samples; Figure 4. The diffraction patterns exhibit typical two maxima assigned to the crystal planes (002) and (004). Notably, no other phases are detected. Neither plasma nor acid treatments induce noticeable structural changes. To further analyze the data, the crystallite sizes were determined with the Scherrer formula using (002) diffraction maximum [26]. For the unmodified graphite sample, it is equal to about 80 nm, whereas plasma modification results in a slight decrease to 65 nm. Both acid washing treatments result in a decrease in the average crystallite size of graphite to 50 nm, which is not further influenced by plasma oxidation. The effect of water for different plasma treatment times for O 2 and CO 2 gases is presented in Figure 3. An immediate decrease in work function is easily observed, and the final CPD values vary depending on the type of plasma and the treatment time. The increase in final work function due to plasma and water treatment is more pronounced for O 2 plasma than for CO 2 plasma. In the case of oxygen plasma, the maximum changes of surface electronic properties (∆CPD) are observed for 10 min of plasma treatment, while for carbon dioxide plasma the maximum is much less pronounced and is located at 5 min of plasma treatment. The effect of water for different plasma treatment times for O2 and CO2 gases is presented in Figure 3. An immediate decrease in work function is easily observed, and the final CPD values vary depending on the type of plasma and the treatment time. The increase in final work function due to plasma and water treatment is more pronounced for O2 plasma than for CO2 plasma. In the case of oxygen plasma, the maximum changes of surface electronic properties (ΔCPD) are observed for 10 min of plasma treatment, while for carbon dioxide plasma the maximum is much less pronounced and is located at 5 min of plasma treatment. XRD characterization was performed to verify the structural integrity of the modified graphite samples; Figure 4. The diffraction patterns exhibit typical two maxima assigned to the crystal planes (002) and (004). Notably, no other phases are detected. Neither plasma nor acid treatments induce noticeable structural changes. To further analyze the data, the crystallite sizes were determined with the Scherrer formula using (002) diffraction maximum [26]. For the unmodified graphite sample, it is equal to about 80 nm, whereas plasma modification results in a slight decrease to 65 nm. Both acid washing treatments result in a decrease in the average crystallite size of graphite to 50 nm, which is not further influenced by plasma oxidation. XRD characterization was performed to verify the structural integrity of the modified graphite samples; Figure 4. The diffraction patterns exhibit typical two maxima assigned to the crystal planes (002) and (004). Notably, no other phases are detected. Neither plasma nor acid treatments induce noticeable structural changes. To further analyze the data, the crystallite sizes were determined with the Scherrer formula using (002) diffraction maximum [26]. For the unmodified graphite sample, it is equal to about 80 nm, whereas plasma modification results in a slight decrease to 65 nm. Both acid washing treatments result in a decrease in the average crystallite size of graphite to 50 nm, which is not further influenced by plasma oxidation. The structural characterization by XRD was supported by Raman spectrosco ies. The Raman spectra of the reference (GREF) and modified graphite samples, with oxygen (G(O2)) or carbon dioxide (G(CO2)) plasmas using 1, 5, 10, and 20 exposure time, and with nitric or sulfuric acid are presented in Figure 5A. In a intense bands are present at ~1580 (G) and ~2725 cm −1 (2D), which are distinc graphite. The G band is related to the vibrational mode of graphitic carbon, whe 2D band, with typical graphite asymmetry, is the result of the two-phonon lattic tional process [27]. In the spectra of the modified samples, the disorder-induced at ~1355 cm −1 appears with its intensity slightly decreasing with plasma treatme while the band associated with the carbon amorphization process at ~1620 cm − almost unnoticed. To assess the degree of disorder of the graphite structure, the I I2D/IG ratios were calculated ( Figure 5B). The low intensity of the D band, togeth the irrelevant variations in the I2D/IG and ID/IG values, indicates that the extent of d of the bulk graphite structure is negligible, regardless of the modification pr (plasma versus acid treatment, plasma exposure time). This assertion, reinforced highly ordered structure inferred from the XRD results ( Figure 3), is in line with p research [17]. The slight decrease in the ID/IG ratio within the G(O2) and G(CO2) se be attributed to the removal of disordered graphitic layers with a longer plasma e time [17]. Similarly, the specific surface area is not influenced by 1 min of oxygen (the same 12 m 2 •g −1 before and after) and only slightly decreases after prolonged treatment (8 m 2 •g −1 after 20 min). The morphology of the oxidized graphite sam not differ appreciably from that of the starting material even after a harsh sulfu treatment (scanning electron microscopy, SEM, pictures in Figure S3). The structural characterization by XRD was supported by Raman spectroscopy studies. The Raman spectra of the reference (GREF) and modified graphite samples, treated with oxygen (G(O2)) or carbon dioxide (G(CO2)) plasmas using 1, 5, 10, and 20 min of exposure time, and with nitric or sulfuric acid are presented in Figure 5A. In all cases, intense bands are present at~1580 (G) and~2725 cm −1 (2D), which are distinctive for graphite. The G band is related to the vibrational mode of graphitic carbon, whereas the 2D band, with typical graphite asymmetry, is the result of the two-phonon lattice vibrational process [27]. In the spectra of the modified samples, the disorder-induced D band at~1355 cm −1 appears with its intensity slightly decreasing with plasma treatment time, while the band associated with the carbon amorphization process at~1620 cm −1 (D') is almost unnoticed. To assess the degree of disorder of the graphite structure, the I D /I G and I 2D /I G ratios were calculated ( Figure 5B). The low intensity of the D band, together with the irrelevant variations in the I 2D /I G and I D /I G values, indicates that the extent of disorder of the bulk graphite structure is negligible, regardless of the modification procedure (plasma versus acid treatment, plasma exposure time). This assertion, reinforced by the highly ordered structure inferred from the XRD results ( Figure 3), is in line with previous research [17]. The slight decrease in the I D /I G ratio within the G(O2) and G(CO2) series can be attributed to the removal of disordered graphitic layers with a longer plasma exposure time [17]. Similarly, the specific surface area is not influenced by 1 min of oxygen plasma (the same 12 m 2 ·g −1 before and after) and only slightly decreases after prolonged plasma treatment (8 m 2 ·g −1 after 20 min). The morphology of the oxidized graphite samples did not differ appreciably from that of the starting material even after a harsh sulfuric acid treatment (scanning electron microscopy, SEM, pictures in Figure S3). A complementary characterisation of the materials was performed by thermogravimetric studies, Figure 6. Plasma treatment was found to have no influence on the thermal stability of graphite. However, treatment with acids, especially nitric acid, increases thermal stability. The effect is due to the removal of iron oxide impurity, which acts as an oxidation catalyst [28]. The oxygen plasma treatment of the acid-washed samples did not change their thermal stability appreciably, similarly to that of the unwashed graphite. Normalized XPS C 1 s and O 1 s spectra of plasma and acid-treated graphites are collected in Figure 7. The full sets of narrow scan XPS spectra for the investigated samples can be found in Supplementary Materials, Figures S4-S14. A comparison of the normalized spectra for O2 and CO2 plasma modified samples indicates that speciation of the oxygen functional groups does not change. Although the C 1 s spectra of acid-washed graphites (nitric acid-washed, GN, sulfuric acid-washed, GS) do not differ noticeably, plasma treatment in these cases results in an increased intensity of around 286.1 eV, characteristic A complementary characterisation of the materials was performed by thermogravimetric studies, Figure 6. Plasma treatment was found to have no influence on the thermal stability of graphite. However, treatment with acids, especially nitric acid, increases thermal stability. The effect is due to the removal of iron oxide impurity, which acts as an oxidation catalyst [28]. The oxygen plasma treatment of the acid-washed samples did not change their thermal stability appreciably, similarly to that of the unwashed graphite. A complementary characterisation of the materials was performed by thermogravimetric studies, Figure 6. Plasma treatment was found to have no influence on the thermal stability of graphite. However, treatment with acids, especially nitric acid, increases thermal stability. The effect is due to the removal of iron oxide impurity, which acts as an oxidation catalyst [28]. The oxygen plasma treatment of the acid-washed samples did not change their thermal stability appreciably, similarly to that of the unwashed graphite. Normalized XPS C 1 s and O 1 s spectra of plasma and acid-treated graphites are collected in Figure 7. The full sets of narrow scan XPS spectra for the investigated samples can be found in Supplementary Materials, Figures S4-S14. A comparison of the normalized spectra for O2 and CO2 plasma modified samples indicates that speciation of the oxygen functional groups does not change. Although the C 1 s spectra of acid-washed graphites (nitric acid-washed, GN, sulfuric acid-washed, GS) do not differ noticeably, plasma treatment in these cases results in an increased intensity of around 286.1 eV, characteristic Normalized XPS C 1 s and O 1 s spectra of plasma and acid-treated graphites are collected in Figure 7. The full sets of narrow scan XPS spectra for the investigated samples can be found in Supplementary Materials, Figures S4-S14. A comparison of the normalized spectra for O 2 and CO 2 plasma modified samples indicates that speciation of the oxygen functional groups does not change. Although the C 1 s spectra of acid-washed graphites (nitric acid-washed, GN, sulfuric acid-washed, GS) do not differ noticeably, plasma treatment in these cases results in an increased intensity of around 286.1 eV, characteristic for C-O-type functional groups [29]. The O 1 s spectra of acid-washed samples differ from those of the starting material, probably due to the removal of inorganic impurities, such as Fe 2 O 3 . Following Table S1, it can be concluded that acid washing removes iron oxide impurities and uncovers SiO 2 , changing the oxygen balance in the sample. To account for the changes in the chemical composition of graphite due to the removal of Fe 2 O 3 and the uncovering of SiO 2 , the corrected oxygen atomic composition bound in the functional carbon-oxygen groups was obtained by subtracting the oxygen from Fe 2 O 3 (Fe at.% × 1.5), SiO 2 (Si at.% × 2), and SO 3 where adequate (S at.% × 3). The summary of surface quantification with XPS is collected in for C-O-type functional groups [29]. The O 1 s spectra of acid-washed samples differ from those of the starting material, probably due to the removal of inorganic impurities, such as Fe2O3. Following Table S1, it can be concluded that acid washing removes iron oxide impurities and uncovers SiO2, changing the oxygen balance in the sample. To account for the changes in the chemical composition of graphite due to the removal of Fe2O3 and the uncovering of SiO2, the corrected oxygen atomic composition bound in the functional carbon-oxygen groups was obtained by subtracting the oxygen from Fe2O3 (Fe at.% × 1.5), SiO2 (Si at.% × 2), and SO3 where adequate (S at.% × 3). The summary of surface quantification with XPS is collected in Table S1, Supplementary Materials. The EPR spectra of the investigated samples registered at room temperature are shown in Figure 8. The composite EPR spectrum for the reference sample (GREF) was simulated as a superposition of two components characterized by g∥ = 2.0459 and g⏊ = 2.0028 for the first one (GREFC-1), while the second center was characterized by g∥ = 2.0198 and g⏊ = 2.0002 (GREFC-2). The simulation represented a very good fit; however, a small discrepancy can be observed around g~2 which might be attributed to an additional sharp signal. Due to the very small intensity, this line was not included in the simulation. The signal denoted GREFC-1 was attributed to electrons that can travel through a large number of crystallites before a change in spin orientation takes place [30]. The observed shape of the EPR spectrum is due to a time averaging of the anisotropy owing to electron motion. The second signal, GREFC-2, originates from the electron whose spin flipped after only crossing a few crystallites. The signal from plasma treated graphite shows a similar shape and also consists of two components (g∥ = 2.0459 g⏊ = 2.0027 and SG-2 g∥ = 2.0199 g⏊ = The EPR spectra of the investigated samples registered at room temperature are shown in Figure 8. The composite EPR spectrum for the reference sample (GREF) was simulated as a superposition of two components characterized by g = 2.0459 and 2.0039), though, its intensity increased significantly. After immersion of the sample in water, the shape and intensity of the EPR signal returned to that observed for the native graphite (g∥ = 2.0459 g⏊ = 2.0028, and g∥ = 2.0198 g⏊ = 2.0002). = 2.0002 (GREFC-2). The simulation represented a very good fit; however, a small discrepancy can be observed around g~2 which might be attributed to an additional sharp signal. Due to the very small intensity, this line was not included in the simulation. The signal denoted GREFC-1 was attributed to electrons that can travel through a large number of crystallites before a change in spin orientation takes place [30]. The observed shape of the EPR spectrum is due to a time averaging of the anisotropy owing to electron motion. The second signal, GREFC-2, originates from the electron whose spin flipped after only crossing a few crystallites. The signal from plasma treated graphite shows a similar shape and also consists of two components (g = 2.0459 2.0039), though, its intensity increased significantly. After immersion of the sample in water, the shape and intensity of the EPR signal returned to that observed for the native graphite (g∥ = 2.0459 g⏊ = 2.0028, and g∥ = 2.0198 g⏊ = 2.0002).
Discussion
The physicochemical characterization of modified graphites shows that the applied treatments influence neither the structure nor the morphology of the commercial graphite used. This is expected for plasma treatment, since oxidation is restricted to the near-surface region (Tables S1-S3). The increase in the Si content of SiO2 in the plasma-treated samples is a sign of total oxidation of some of the carbon components that reveal the silica impurity. Nevertheless, plasma treatment does not influence the thermal stability of graphite, most likely due to a negligible effect on the structure of the material and the introduction only of unstable oxygen species. In contrast, acid washing increases the thermal stability of the graphite material as a result of the removal of iron impurity, which acts as an oxidation catalyst.
The surface changes of graphite result in an increase in work function due to the mild oxidation. However, the changes are unstable over time and a gradual decrease in the work function is observed. This effect can be explained by considering two components of the modifications: electrostatic and chemical. The former is related to the effect of graphite powder charging with static electricity during plasma treatment and a discharge during measurement with the Kelvin probe, where the sample is grounded. The latter component is responsible for more stable surface modification-the introduction of oxygen functional groups. To equilibrate the samples and remove the charging, graphite powders were immersed in water after plasma treatment. This procedure also decreases the number of unpaired electrons as shown by EPR measurements. The additional effect of such treatment is related to the surface reactivity upon activation in plasma. As expected, for each plasma treatment, the values of the work function (CPD) decreased substantially but were higher than for the original samples (ΔCPD > 0, Figure 9). 2.0039), though, its intensity increased significantly. After immersion of the sam ter, the shape and intensity of the EPR signal returned to that observed for graphite (g∥ = 2.0459 g⏊ = 2.0028, and g∥ = 2.0198 g⏊ = 2.0002).
Discussion
The physicochemical characterization of modified graphites shows that th treatments influence neither the structure nor the morphology of the commercia used. This is expected for plasma treatment, since oxidation is restricted to the face region (Tables S1-S3). The increase in the Si content of SiO2 in the plasm samples is a sign of total oxidation of some of the carbon components that revea impurity. Nevertheless, plasma treatment does not influence the thermal s graphite, most likely due to a negligible effect on the structure of the materia introduction only of unstable oxygen species. In contrast, acid washing increase mal stability of the graphite material as a result of the removal of iron impur acts as an oxidation catalyst.
The surface changes of graphite result in an increase in work function due t oxidation. However, the changes are unstable over time and a gradual decre work function is observed. This effect can be explained by considering two co of the modifications: electrostatic and chemical. The former is related to the graphite powder charging with static electricity during plasma treatment and a during measurement with the Kelvin probe, where the sample is grounded. component is responsible for more stable surface modification-the introduction functional groups. To equilibrate the samples and remove the charging, graphite were immersed in water after plasma treatment. This procedure also decreases ber of unpaired electrons as shown by EPR measurements. The additional effe treatment is related to the surface reactivity upon activation in plasma. As exp each plasma treatment, the values of the work function (CPD) decreased substa were higher than for the original samples (ΔCPD > 0, Figure 9). 2.0039), though, its intensity increased significantly. After immersion of the sample in water, the shape and intensity of the EPR signal returned to that observed for the native graphite (g∥ = 2.0459 g⏊= 2.0028, and g∥ = 2.0198 g⏊ = 2.0002).
Discussion
The physicochemical characterization of modified graphites shows that the applied treatments influence neither the structure nor the morphology of the commercial graphite used. This is expected for plasma treatment, since oxidation is restricted to the near-surface region (Tables S1-S3). The increase in the Si content of SiO2 in the plasma-treated samples is a sign of total oxidation of some of the carbon components that reveal the silica impurity. Nevertheless, plasma treatment does not influence the thermal stability of graphite, most likely due to a negligible effect on the structure of the material and the introduction only of unstable oxygen species. In contrast, acid washing increases the thermal stability of the graphite material as a result of the removal of iron impurity, which acts as an oxidation catalyst.
The surface changes of graphite result in an increase in work function due to the mild oxidation. However, the changes are unstable over time and a gradual decrease in the work function is observed. This effect can be explained by considering two components of the modifications: electrostatic and chemical. The former is related to the effect of graphite powder charging with static electricity during plasma treatment and a discharge during measurement with the Kelvin probe, where the sample is grounded. The latter component is responsible for more stable surface modification-the introduction of oxygen functional groups. To equilibrate the samples and remove the charging, graphite powders were immersed in water after plasma treatment. This procedure also decreases the number of unpaired electrons as shown by EPR measurements. The additional effect of such treatment is related to the surface reactivity upon activation in plasma. As expected, for each plasma treatment, the values of the work function (CPD) decreased substantially but were higher than for the original samples (ΔCPD > 0, Figure 9).
Discussion
The physicochemical characterization of modified graphites shows that the applied treatments influence neither the structure nor the morphology of the commercial graphite used. This is expected for plasma treatment, since oxidation is restricted to the nearsurface region (Tables S1-S3). The increase in the Si content of SiO 2 in the plasma-treated samples is a sign of total oxidation of some of the carbon components that reveal the silica impurity. Nevertheless, plasma treatment does not influence the thermal stability of graphite, most likely due to a negligible effect on the structure of the material and the introduction only of unstable oxygen species. In contrast, acid washing increases the thermal stability of the graphite material as a result of the removal of iron impurity, which acts as an oxidation catalyst.
The surface changes of graphite result in an increase in work function due to the mild oxidation. However, the changes are unstable over time and a gradual decrease in the work function is observed. This effect can be explained by considering two components of the modifications: electrostatic and chemical. The former is related to the effect of graphite powder charging with static electricity during plasma treatment and a discharge during measurement with the Kelvin probe, where the sample is grounded. The latter component is responsible for more stable surface modification-the introduction of oxygen functional groups. To equilibrate the samples and remove the charging, graphite powders were immersed in water after plasma treatment. This procedure also decreases the number of unpaired electrons as shown by EPR measurements. The additional effect of such treatment is related to the surface reactivity upon activation in plasma. As expected, for each plasma treatment, the values of the work function (CPD) decreased substantially but were higher than for the original samples (∆CPD > 0, Figure 9). Quantification of oxygen functional groups in graphite samples is shown in Figure 10. The oxygen content (based on Table S1) in the O2 and CO2 plasma and water-treated samples is higher than in the original graphite, which explains the increase in work function. Interestingly, CO2 plasma is more effective in introducing OFGs, but water treatment leads to their decrease. In contrast, oxygen plasma appears to also activate the materials, similarly to the effect reported for graphene paper [25]. Quantification of oxygen functional groups in graphite samples is shown in Figure 10. The oxygen content (based on Table S1) in the O 2 and CO 2 plasma and water-treated samples is higher than in the original graphite, which explains the increase in work function. Interestingly, CO 2 plasma is more effective in introducing OFGs, but water treatment leads to their decrease. In contrast, oxygen plasma appears to also activate the materials, similarly to the effect reported for graphene paper [25]. Quantification of oxygen functional groups in graphite samples is shown in Figure 10. The oxygen content (based on Table S1) in the O2 and CO2 plasma and water-treated samples is higher than in the original graphite, which explains the increase in work function. Interestingly, CO2 plasma is more effective in introducing OFGs, but water treatment leads to their decrease. In contrast, oxygen plasma appears to also activate the materials, similarly to the effect reported for graphene paper [25]. The investigated, off-the-shelf graphite contained impurities in the form of SiO 2 and Fe 2 O 3 . To purify the material, nitric and sulfuric acids were used. Acid treatment resulted in substantial removal of iron oxide impurity. At the same time, the work function of the graphite increased, despite the substantial decrease in the content of the oxygen functional groups. However, as evidenced in Figure 7G,H, the shift in the O 1s spectra may indicate a change in OFG speciation, leading to the modification of the work function. In the case of the sulfuric acid-washed sample, sulfate groups are present on the surface (Figures S12 and S13) possibly adding to the observed increase in work function. However, these groups disappear after oxygen plasma treatment followed by water washing ( Figure S14). Moreover, intimate interaction between the graphite and iron impurities may result in a decreased work function of the of-the-shelf graphite. Nevertheless, plasma treatment of the acid-washed samples resulted in the incorporation of stable oxygen functional groups, which further increased the work function graphite.
Plasma, consisting of radicals and free electrons, can transfer these species and charges to the materials in contact. The studied graphite already contains radicals in the form of free electrons, but plasma treatment increases their concentration, as shown in Figure 8. Following the adopted experimental procedure, immersion in water results in the removal of these additional radicals. Electron paramagnetic resonance studies allow for some insight into the state of the graphite samples upon plasma treatment. The increase in the radical signal coincides with the increase in the work function and the surface oxygen. Similarly, the decrease in the radical signal for the water-treated graphite follows the decrease in work function. Changes in the surface oxygen content do not increase uniformly after the washing of the plasma-oxidized samples. Therefore, the reactivity in water-increase or decrease in the number of functional oxygen groups-will depend on the interplay between surface disordering due to ion bombardment, the formation of radicals, as well as the number and type of OFG already present.
Materials and Methods
Graphite powder (Polskie Odczynniki Chemiczne, POCH) was modified in this work using both acid immersion and plasma treatment. The wet acid modification was carried out by placing 2 g of graphite in a round bottom flask and adding 80 mL of concentrated acid. Sulfuric acid (VI) (Chempur) with a concentration of 95% and nitric acid (V) (POCH Basic) with a concentration of 65% were applied. The mixtures were then heated to a temperature of about 96 • C for 16 h. The product was then filtered on a Buchner funnel and washed with deionized water until pH was close to neutral. The obtained powders were dried and ground in a mortar.
Plasma treatment was carried out using the commercial cold plasma system (Femto-Diener Electronic GmbH, Nagold, Germany) with a generator frequency of 40 kHz, employing pure oxygen (Air Products, 99.9998% O 2 ) or CO 2 (Air Products, X50S 37.5 K, ultra-pure) as feed gases for plasma generation. The optimization of the plasma modification parameters was performed with the use of 100% power and 0.2 mbar pressure for different plasma treatment times: for oxygen plasma, the times were: 1, 5, 10, and 20 min (samples designation: G1(O2), G5(O2), G10(O2), G20(O2)), and for CO 2 plasma, 1, 5, 10, and 20 min (samples designation: G1(CO2), G5(CO2), G10(CO2), G20(CO2)). The plasma treatment was applied to unmodified graphite and samples already treated with concentrated acids. After plasma treatment, the samples were immersed in deionized water (DI) for surface stabilization and dried at 60 • C. Based on the relative changes in work function before and after water immersion, 5 min of plasma treatment was selected to modify acid-treated samples, as well as samples for electron paramagnetic resonance (EPR) and X-ray photoelectron spectroscopy (XPS) studies.
To determine the effect of applied modifications on the electronic properties of graphite, the work function changes were followed. Work function studies for plasma treatment were performed in a series of three measurements: untreated sample, sample just after plasma treatment, and sample immersed in DI water just after plasma treatment.
Changes in the work function of graphite samples were investigated by measuring contact potential difference (∆CPD) measurements. The experiments were carried out using the Kelvin probe method with a KP6500 device (McAllister Technical Services, Coeur d'Alene, ID, USA). The stainless steel plate (d = 3 mm) was used as an electrode (WF ref = 4.3 eV). The measurements were carried out under ambient conditions (room temperature, atmospheric pressure), with vibration frequency at 114 Hz and amplitude at 40 a.u.
The structural analysis of the reference and functionalized samples was carried out using a Rigaku Multiflex diffractometer, using Cu Ka radiation (40 kV, 40 mA). Powder X-ray diffractograms were collected in the 2theta range of 10-70 • (step size of 0.02 • and accumulation time of 3 s). µRaman spectra of the samples were collected using a Renishaw InVia spectrometer equipped with a 514 nm laser. Measurements were carried out in the spectral range of 1000-3000 cm −1 with a resolution of 1 cm −1 . The accumulation of ten scans was applied for each spectrum.
Thermogravimetric analysis of reference and functionalized samples was performed using TGA/DSC 1 equipment (Mettler Toledo). Approximately 10 mg of the sample were heated in the flow of synthetic air 40 mL·min −1 (mixed with Ar 20 mL·min −1 ) in the temperature range of 25-1100 • C with a heating rate of 20 degrees per minute.
The surface composition was examined using XPS with a SESR4000 analyzer (Gammadata Scienta, Uppsala, Sweden, the XPS setup was provided by Prevac, Rogów, Poland). The base pressure in a vacuum chamber was below 5 × 10 −9 mbar, and monochromatized Al-Kα source with the 250 W at 1486.6 eV emission energy was used with the pass energy for selected narrow-range binding energy scans of 100 eV. To process the raw data, CasaXPS Version 2.3.24PR1.0 (Casa Software Ltd., Teignmouth, UK) was used [31].
Local structural changes of modified graphites were investigated using a Renishaw InVia spectrometer equipped with a 514 nm laser. µRaman spectra were collected in the range of 1000-3000 cm −1 with a resolution of 1 cm −1 , by accumulating ten scans for each spectrum.
The number and type of paramagnetic species in the graphite samples were investigated using EPR spectroscopy. Continuous wave EPR spectra were measured with a Bruker Elexsys E500 X-band spectrometer equipped with the Xepr data system for spectra acquisition and manipulation and the super high-sensitivity cavity ER 4122 SHQE. Spectra were recorded with a 100 kHz magnetic field modulation. In standard experiments, the microwave power was 2 mW, sweep width 20 mT, modulation amplitude 0.2 mT, number of points 1024, and 4 scans were acquired. The EPR spectra were simulated using the EPRsim32 program written in Microsoft Visual C ++ 6.0 using the Microsoft Foundation Class library [32].
Conclusions
Graphite is being investigated for potential and improved applications in many areas where tuning the surface properties is paramount to its performance. In this study, we evaluated the tuning of the surface electronic properties and surface oxygen content by oxidative plasma modification of the sample of commercial graphite. Since the surface of the plasma-oxidized graphite is unstable over time, simple washing with water was chosen to obtain a stable material. Additionally, because the various graphite samples may differ according to their origin, the effect of acid washing was also evaluated. The main finding of this research is that the combination of acid and plasma treatment is the most effective way to modify the electronic properties and surface functionalization of microcrystalline graphite material.
The nitric and sulfuric acid washing results in a decrease in iron oxide impurity, which increases the thermal stability of graphite. For the investigated graphite samples, the increase in the stable work function due to plasma treatment is caused by (1) an increase in the concentration of surface oxygen species and (2) a change in the speciation of the surface oxygen species. Stable plasma functionalization with oxygen functional groups is most evident in nitric and sulfuric acid washed samples, which is reflected in the highest observed increase in work function for acid-treated samples and a visible increase in the relative content of C-O functional groups. | 2022-08-28T15:18:32.235Z | 2022-08-25T00:00:00.000 | {
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266982757 | pes2o/s2orc | v3-fos-license | The First Case of a Homozygous CCNO NM 021147.4 Mutation Associated With Primary Ciliary Dyskinesia in Two Indian Siblings
Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease marked by organ lateralization in 50% of patients, chronic sinopulmonary disease, infertility in men, and neonatal respiratory distress. Respiratory control cells contain CCNO in their apical cytoplasm, which is necessary for the development of multiciliate cells, basal body amplification, and migration. Reduced generation of multiple motile cilia, a rare form of PCD, has been linked to CCNO gene abnormalities. Individuals with CCNO mutations have been reported to suffer from severe lower respiratory infections that cause progressive impairment of lung function. For the first time, we describe the CCNO NM 021147.4 (c.258 262dup.p, Gln88argfs*8 Homozygous) gene mutation in an Indian consanguineous family that resulted in severe PCD.
Introduction
Primary ciliary dyskinesia (PCD), an autosomal recessive condition characterized by motile cilia malfunction, displays clinical and genetic variation.Some of the clinical symptoms of PCD include left-right lateralization, infertility, and chronic upper and lower respiratory illness [1][2][3][4][5].There is no one best way to diagnose PCD; instead, a variety of methods can be used, such as a combination of nasal nitric oxide (nNO), high-speed genetic analysis, immunological fluorescence of ciliated cells, transmission electron microscopy (TEM), high-speed video microscopy analysis, immune fluorescence of ciliated cells, and genetic analysis (gene panel analysis or extensive genetic analysis) [6,7].
There is a significant correlation between phenotype and specific genetic changes.Reduced generation of multiple motile cilia (RGMC) has been linked to CCNO gene mutations, which are also more likely to have a more severe respiratory disease phenotype with pulmonary failure at a younger age [8].
For the first time, we describe the CCNO NM 021147.4(c.258 262dup.p,Gln88argfs*8 homozygous) gene mutation that caused severe PCD in a consanguineous Indian family.
This article was previously presented as a meeting abstract at the 2022 American Thoracic Society International Conference Meeting on May 13-18, 2022.
Case Presentation
Our case study focuses on two consanguineous sisters from India who visited the Pediatric Pulmonary Department at Sidra Hospital in Qatar when they were 17 and 15 years old.They presented with chronic wet cough and were found to have progressive loss of lung function that eventually led to end-stage lung disease and the requirement for lung transplantation in the case of the elder sister.Since infancy, both siblings had suffered significant lower respiratory infections, chronic rhinorrhea, and recurrent ear infections.
Bronchiectasis was detected by computed tomography (CT) of the chest when the elder sister was 14 years old (Figure 1) and the younger sister was seven years old (Figures 2, 3).A: CT chest, axial view, lung window.B: CT chest, coronal view, lung window.
CT of the chest shows decreased volume of the left upper lobe and lingula with diffuse cystic bronchiectasis.
There is also bronchiectasis within the left lower lobe with a mucus plug and hyperinflation of the right lung with diffuse tree-in-bud changes of the right middle lobe likely secondary to infection.
Physical examination was pertinent for bilateral crackles and clubbing in both sisters.The elder sister was hypoxic requiring 1-2 L of oxygen per minute via a nasal cannula.Expiratory flow volume (spirometry) revealed significant mixed restrictive and obstructive airway disease, which was worse in the elder sibling.The forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) in the elder sister were 25% and 40% predicted, respectively (Figure 4).Whereas the FEV1 and FVC in the younger sister were 39% and 57% predicted, respectively (Figure 5).
Discussion
We described two Indian sisters who experienced early-onset respiratory symptoms, obstructive ventilatory dysfunction, and situs solitus.They were identified as having PCD based on the full exon of the CCNO gene and low nNO levels.
Patients with ciliogenesis-related mutations in the genes CCNO and MCIDAS have been reported to have a worse phenotype than those with other kinds of PCD, which is consistent with the current cases [8].There are currently 50 known PCD genes that cause a variety of functional defects ranging from abnormal beat patterns through impairment of dynein arms to a complete absence of cilia [9,10].Although nNO showed good diagnostic accuracy as a PCD diagnostic test when compared to the extended reference standard of TEM and/or genetic testing [11], it is crucial to confirm the genetic diagnosis, given the present evidence of links between genotype and phenotype [12].
Patients with PCD, which is secondary to CCNO mutation, have early-onset respiratory symptoms, recurrent lower respiratory tract infections, and progressive loss of lung function, which is consistent with our cases.Situs solitus, like in the cases presented, has only been documented in people with CCNO mutations [8].The other documented symptoms are recurrent pneumonia, sinusitis, and otitis media (Table 1).
TABLE 1: CCNO gene mutation studies.
There are no reports of genetic PCD in India, and only one publication stated that patients with recurrent sinusitis, otitis, and pneumonia in India who also had a fractional exhaled NO level under 10 ppb and who had not undergone genetic testing were likely to have PCD [13].To our knowledge, this is India's first report on PCD with CCNO which accounts for two out of 318 cases that have been published in the literature (Table 1).
In 16 people who had the first CCNO mutation in 2014, a malfunction in the mother centriole formation and migration at a late stage of multiple motile cilia (MMC) differentiation led to a significantly decreased number of MMCs.Congenital mucociliary clearance disorder with RGMC is used to denote this hereditary condition [8].Following the study, there have since been several CCNO reports (Table 1).In a study of five PCD patients from three different Irish traveler families, it was discovered that a sibling pair in Irish family B had the CCNO gene [14].In another investigation, researchers looked for CCNO mutations in 170 Israeli families with mucociliary clearance disorders and identified two novel variations (p.Gly56Alafs38; c.165delC, c.638T>C, p.Leu213Pro), and two known mutations were found in 15 individuals from 10 families (6% prevalence) [15].In Lisbon, Portugal, 12 patients underwent PCD genetic testing confirming the diagnosis, with three presenting CCNO mutations [16].In Turkey, out of a total of 265 patients with PCD during a fiveyear period, 46 had genetically determined PCD using whole-exome sequencing at a single facility, and four had CCNO [17].There have been multiple publications from China.One of these described 58 individuals with PCD, 51 with hereditary PCD, and three with CCNO [18].This presenting case study is not a metaanalysis, and its limitation arises from the small number of patients from India, which has not been previously explored in the current literature.
Conclusions
PCD with CCNO mutations is a relatively rare disease.Our findings highlight the significance of considering PCD based on CCNO mutations in people with situs solitus to have a more severe respiratory disease phenotype with lung failure at earlier ages.It is also critical to include individuals from various racial and ethnic origins in PCD-associated genetic CCNO mutation.
1 1 2 FIGURE 1 :
FIGURE 1: Chest X-ray and CT chest findings of the elder sister.A: Anteroposterior chest X-ray.B: CT chest, axial view, lung window.Chest radiographical imaging of the elder sister showing bilateral varicose bronchiectasis worse on the right side.
FIGURE 2 :
FIGURE 2: Anteroposterior chest X-ray of the younger sister.Chest radiography showing left lower lobe consolidation.
FIGURE 3 :
FIGURE 3: CT chest findings of the younger sister.
FIGURE 4 :
FIGURE 4: Spirometry findings of the elder sister.Spirometric measurement of the elder sister showed mixed restrictive and obstructive airway disease.FVC = forced vital capacity; FEV = forced expiratory volume; PEF = peak expiratory flow; PIF = peak inspiratory flow; FET = forced expiratory time
FIGURE 5 :
FIGURE 5: Spirometry findings of the younger sister.Spirometric measurement of the younger sister showed mixed restrictive and obstructive airway disease.FVC = forced vital capacity; FEV = forced expiratory volume; PEF = peak expiratory flow; PIF = peak inspiratory flow; FET = forced expiratory time
5 .
Gly85Cysfs*10) in CCNO using whole-exome sequencing After birth, 12 out of 16 babies experienced respiratory distress.One of the women was infertile.Everyone exhibited situs solitus, bronchitis, and recurrent infections of the upper and lower respiratory tracts.At the age of 34, two people who had terminal respiratory mean age of presentation was 9.6 (range = 2-15) years.Overall, 62.5% of the population was younger than 5 years old.Clubbing was present in 58 (72.5%)Bronchiectasis on CT of the thorax: 5/5.Hearing loss in 2/5 patients.2/5 of the patients had early-onset severe cardiomyopathy, type III glycogen storage disease, and developmental delay.Neonatal pneumonia affected 1/5 of patients.Recurrent otitis media in 254 years.At a median age of 20 years, the initial genetic diagnosis was made.All situs solitus 11/14 cases (85%) of neonatal respiratory distress syndrome.Otitis media recurrent in 10/15 (67%) people, and 13/14 (93%) people had sinusitis.Documentation of bronchiectasis by radiographic imaging in 13/14 (93%).At the age of 43, lung transplantation was done.The composition of the situs was symptoms that started early.Situs solitus.Due to lung collapse, one required a lobectomy.One patient had two tympanostomy tube insertions due to recurrent otitis media with effusion and at diagnosis (median: 3 years; range, 6 months to 4 years).44 patients had rhinitis, whereas 41 had newborn respiratory distress.The sinusitis returned in 36.14 people had recurrent otitis.Six were hard of hearing.Clubbing was seen in seven.Fourteen had a total inversus situation.Six (atrial septal defect, patent ductus arteriosus, and mitral valve prolapse) had congenital cardiac defects.Four patients had Lobectomy.Four patients had undergone ear, nose, diagnosis was 7.0 years (range = 2 months to 14 years).A chronic wet cough affected 66 out of 75 people.58/75 people had recurrent sinusitis.57/75 people had bronchiectasis.Respiratory distress in newborns was present in 30% of the cases.There were 6/75 cases of postinfectious bronchiolitis obliterans as the first presentation, while 21/ | 2024-01-16T05:10:53.214Z | 2024-01-01T00:00:00.000 | {
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8106246 | pes2o/s2orc | v3-fos-license | Adjusting Phenotypes by Noise Control
Genetically identical cells can show phenotypic variability. This is often caused by stochastic events that originate from randomness in biochemical processes involving in gene expression and other extrinsic cellular processes. From an engineering perspective, there have been efforts focused on theory and experiments to control noise levels by perturbing and replacing gene network components. However, systematic methods for noise control are lacking mainly due to the intractable mathematical structure of noise propagation through reaction networks. Here, we provide a numerical analysis method by quantifying the parametric sensitivity of noise characteristics at the level of the linear noise approximation. Our analysis is readily applicable to various types of noise control and to different types of system; for example, we can orthogonally control the mean and noise levels and can control system dynamics such as noisy oscillations. As an illustration we applied our method to HIV and yeast gene expression systems and metabolic networks. The oscillatory signal control was applied to p53 oscillations from DNA damage. Furthermore, we showed that the efficiency of orthogonal control can be enhanced by applying extrinsic noise and feedback. Our noise control analysis can be applied to any stochastic model belonging to continuous time Markovian systems such as biological and chemical reaction systems, and even computer and social networks. We anticipate the proposed analysis to be a useful tool for designing and controlling synthetic gene networks.
For the case of control that the mean level is changed with the noise level fixed, the tolerance level (tol) is applied to the noise level. The control schemes related to α m , γ m , and α p were significantly dependent on the tolerance level for the wild-type. This is because the control coefficients for the noise level with respect to these parameters were all small (C V s αm = −0.19, C V s γm = 0.13, and C V s αp = −0.01).
Model for ATM-p53-mdm2 oscillations ATM-p53-mdm2 systems show sustained noisy oscillations, which were shown to be successfully modeled by the following linear Langevin equations [1]: where a, p, and m denote the oscillation components of the concentrations of ATM, p53, and mdm2, respectively, and ξ a , ξ p , and ξ m are the independent Gaussian white noise, satisfying ξ a (t)ξ a (t ) = W a δ(t − t ) and the same relationships for the other noise terms ξ p and ξ m with different noise strength W p and W m .
The parameter values are shown in Table S1.
Summation theorem for auto-correlation functions
We derived summation theorems among control coefficients for concentration mean and noise levels (coefficients of variation and even higher order moments) [2] that is analogous to those found in the metabolic control analysis. In this section, we derive another summation theorem regarding to control coefficients for auto-correlation functions.
We consider a system that is described by the master equation. The reaction rate functions can be arbitrary and the global proportionality constants are chosen for perturbations. For example, in the Michaelis-Menten-type rate law: v max is our choice of parameter but the Michaelis-Menten constant K M is not. For more detailed discussion, γ a k a→p k p→a γ p k m→p k p→m γ m W a W p W m 60 ln(2)/100 -0.65 1 60 ln(2)/75 -0.55 0.29 60 ln(2)/144 1400 5000 10000 Table S1. Parameter values for the ATM-p53-mdm2 system. The unit of all the parameters is hour −1 .
The values are taken from [1].
we refer to [2]. The set of the parameters will be denoted by p. If there are n reactions, the number of the parameters (the dimension of p) is n.
Before the derivation of the summation theorem, we note that the rescaling all the parameter p to αp means that all the reactions are increased by the factor of α. This can be alternatively viewed as the unit of time is decreased by the factor of α [2][3][4][5]. This equivalence leads to the following relationship: Specifically, we can obtain the above relationship as follows. The function G(τ, αp) is defined as where the angle bracket means the average over time at the stationary state. With the rescaling in p, the above equation becomes G(τ, αp) = X(α(t + τ ))X(αt) − X 2 = X(αt + ατ )X(αt) − X 2 = G(ατ, p).
By using this relationship, we can obtain the summation theorem as follows. First, an infinitesimal change in the autocorrelation function can be described by the sum of the change due to each individual parameter change by applying the chain rule: Second, the infinitesimal change in the autocorrelation can also be described, by using Eq. (S2), as where δτ = ατ − τ = (α − 1)τ . From Eqs. (S3) and (S4), we obtain the summation theorem:
Jacobian and diffusion matrices
Our model system -a continuous time Markov process -is assumed to show stochastic fluctuations in state variables (here, concentrations) small enough that the fluctuations occur in linear portions of nonlinear reaction rates. Under this assumption, the fluctuation statistics can be approximately described by linearizing the nonlinear reaction rates. This approximation is called the linear noise approximation [6]. The noise strength (concentration covariance) at the stationary state was shown to be computed by solving the Lyapunov equation [6] (also known as the fluctuation dissipation relationship [7,8]): where σ the covariance matrix, J the Jacobian matrix, and D the diffusion matrix.
When a system is governed by the master equation, J and D can be computed from the system's linearized reaction rates. However, when the master equation is not known, e.g., due to unknown source of noise, the system is often described by a phenomenological Langevin equation with Gaussian white noise, where the strength of noise is estimated via parameter fitting procedures. When fluctuations in concentrations are small enough that the Langevin equation can be linearized, the concentration covariance is described by the same (in mathematical structure) ordinary differentical equation that was derived from the master equation [9].
At the stationary state, the equation becomes the Lyapunov equation Eq. (S5) and the diffusion matrix is given by the estimated noise strength.
Consider the two-state gene expression model shown in Fig. 2b. This model system is fully described by the master equation. Under the linear noise approximation (the approximation becomes exact actually, since the model is described by linear reaction rate laws), J and D are given by ) is defined as i, j = 1 corresponds to P a ; i, j = 2 to mRN A; and i, j = 3 to P rotein. For example, both σ 12 and σ 21 represent covariance between P a and mRN A.
For the p53 oscillation model shown in Fig. 6a, the linear phenomenological Langevin equation (S1) was used. J and D were given by with the matrix index (i, j) is defined as i, j = 1 corresponds to ATM; i, j = 2 to P53; and i, j = 3 to mdm2.
The noise strength, W a , W p , and W m were estimated by parameter-fitting [1].
Network C in Fig. 7 is not weakly reversible.
The network can be converted as follows: where S is a constant. This system is not weakly reversible; there is no directed arrow path, e.g., from the complex X 1 +E to the complex E, from X 2 to X 1 , and so on. The deficiency is, however, zero since there are six complexes {ø, E, X 1 + E, X 1 , X 2 , P }, two linkage classes [10], and the rank of the network (the column rank of the stoichiometric matrix [10]) is 4.
Relationship between infinitesimal and finite perturbations
Consider an infinitesimal perturbation in a parameter p such as p → p(1 + ), with 1. The relative change in p can be expressed as The infinitesimal perturbation is applied iteratively in N times to perturb the parameter in a finite amount: where p 0 and p N are the values of the parameter before any iteration performed and after the N -th iteration, respectively. Therefore, we obtain p N = p 0 exp(N ).
When two parameters, p and q, are perturbed infinitesimally with the ratio of 1:2: p → p(1 + ), and q → q(1 + 2 ), with 1. After the N -th iteration of this perturbation, the final parameter values become: p N = p 0 exp(N ), and q N = q 0 exp(2N ).
Therefore, we obtain q N q 0 = p N p 0 2 . | 2014-10-01T00:00:00.000Z | 2012-01-01T00:00:00.000 | {
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264118313 | pes2o/s2orc | v3-fos-license | Recent Advances and Challenges in Management of Colletotrichum orbiculare , the Causal Agent of Watermelon Anthracnose
: The fungus Colletotrichum orbiculare causes watermelon anthracnose and is an important pathogen of watermelon in the United States, causing a significant impact on yield and quality of the produce. The application of fungicides as preventative and post-occurrence control measures is currently being deployed by growers. Further study of the genetic and molecular basis of anthracnose resistance will help in guiding future watermelon breeding strategies. Several conserved virulence factors (effectors) in C. orbiculare have been reported to interact with the host, at times impairing the host immune machinery. A single dominant gene conferring race 1 anthracnose resistance was reported independently on two watermelon germplasm. The recent advances in genomics, transcriptomics, proteomics, and metabolomics could facilitate a better understanding of the interaction between C. orbiculare effectors and host resistance genes in the already sequenced watermelon genome. In this review, we encompass and discuss (i) the history of watermelon anthracnose, taxonomy, morphology, and diversity in races of C. orbiculare ; (ii) the epidemiology of the anthracnose disease and host resistance; (iii) the genetics behind the pathogenesis; and (iv) the current advances in breeding and molecular efforts to elucidate anthracnose resistance.
Introduction
Watermelon is an important crop grown worldwide with 102 Megatons produced in 2021 [1].In the United States (U.S.), the crop had a total economic value of USD 748 million in 2022 [2].Watermelons are grown in most U.S. states, but the majority of the production occurs in Florida, Georgia, California, and Texas because of higher temperatures and a longer growing season [3].The introduction of seedless cultivars has increased the per capita consumption of watermelon by 37% since 1980 [3].As the watermelon industry grew, challenges related to fruit quality, yield, and production methods emerged.As is the case with many crops, disease and pest management is a significant limitation to watermelon production.Diseases are a major priority for watermelon producers since new races of pathogens continue to break down host resistance and develop insensitivity to fungicides.The major diseases of watermelons are Fusarium wilt, anthracnose, gummy stem blight, powdery mildew, Phytophthora, downy mildew, bacterial fruit blotch, and viruses.While Fusarium wilt and Phytophthora are considered the most devastating The genus Colletotrichum is economically and scientifically important because it contains many plant-pathogenic fungi infecting a wide range of crops; those crops include row crops, fruits, flowers, and vegetables.Almost every domesticated crop is a host of a species belonging to the genus Colletotrichum [9].In many hosts, Colletotrichum spp.infect all the aerial parts of the plant, including stems, leaves, fruits, and flowers.Colletotrichum spp.are also significant postharvest pathogens since spores from foliar infections during field growth can cause an infection that progresses during transport or on the market shelves, resulting in complete loss of the crop [10].When the plant pathogens were defined based on host specificity, Colletotrichum consisted of almost 700 species.Later, von Arx reclassified those species in 1957 to 11 taxa based on morphological traits [11,12].Colletotrichum has been used as a model system for biochemical, physiological, and genetic studies.The concept of pathogen races was initially recognized in Colletotrichum lindemuthianum [13].Many of the studies that laid the foundation for the concept of systemic acquired resistance were performed using the cucumber model of Colletotrichum orbiculare [14].
Colletotrichum orbiculare ((Berk.& Mont.)Arx) is an important pathogen of cucurbits including cucumber, muskmelon, watermelon, squash, gourd, pumpkin, cantaloupe, honeydew, and Luffa spp.[15].C. orbiculare can also infect tobacco [16].Anthracnose can cause severe damage, both in the field and postharvest, and is one of the top research priorities for watermelon in the U.S. [17].In the field, C. orbiculare infects all the above-ground parts of plants, including leaves, stems, flowers, and fruits [3,5,8].Infections on any of these plant parts have direct effects on yield.The pathogen also infects all growth stages of the plant, from the seedling stage to mature fruit-bearing plants.Defoliation will leave the plant with poor photosynthetic capacity, stunt fruit development, and expose the mature fruit to direct sunlight, leading to sunburn.Infection on both growing and mature plants leads to unmarketable produce.
Earlier when the taxonomic classification of plant pathogenic fungi was based on plant disease specificity, C. orbiculare was named and identified multiple times by different researchers around the world [12].Cucurbit and bean anthracnose were assumed to be caused by the same fungus, which was named Colletotrichum lagenarium [8].This assumption was discarded in a comparative study of anthracnose fungi, in which bean anthracnose was named Volutella citrulli.Based on modern molecular tests, C. orbiculare is recognized as a species complex, with C. lindemuthianum, C. malvarum, C. orbiculare, and C. trifolii as distinct species [20].Currently, the isolates causing watermelon anthracnose are classified as a subspecies in the C. orbiculare species group.Researchers still differ in classifying this pathogen [18].
Fungal Morphology
The mycelium at first is colorless, thin-walled, septate, and uniformly cylindrical.Many of the cells later increase in diameter up to threefold, becoming thick-walled and dark brown in color [8,18].On culture media, mycelium is first colorless and then pink and black at the end.Pink coloration is sometimes observed in host tissues, with blackening of mycelium being common in fruit lesions.Acervuli, anthracnose mycelium aggregates, branch and become intertwined to send out a layer of short colorless conidiophores [8].From the tip of the conidiophores, spores bud off apically one at a time, piling up to form a pink slimy cluster on top of acervuli.Spores are surrounded by a sticky water-soluble matrix and are single-celled, clear, oblong or ovate, and vaguely pointed at one end [8].Spore size varies from 13 to 19 µm by 4 to 6 µm, and masses are pink in color.Acervuli have two to three long setae scattered among the conidiophores, which are brown, thick-walled bristles 90 to 120 µm in length [8,18].The number of setae in a single acervulus varies and can be up to 36.Spores form heaps as high as setae, with setae supporting the spore mass.Sclerotial bodies are typically observed more on media and fruit lesions and are formed due to the further development of the bases of stromata or acervuli, where the whole mass is enlarged and black in color [8].On media, the spore mass may dry and remain as a part of sclerotial bodies, whereas in fruit lesions, the spores are washed away, and the black stroma that forms the black spots on fruits remains [8].
Germinating spores form an appressorium at the tip of each of the germ tubes, which are brown, melanized, thick-walled, and ovoid to spherical in shape.Appressoria are slightly tapered at one end and flattened on the side of contact with the host [8].Melanization of appressoria is important for pathogenicity.Melanin-deficient mutants have reduced pathogenicity, and melanization is supposed to resist the high turgor pressure within the appressorium and direct the force on the leaf epidermis for penetration [21].Melanization in fungal spores is also assumed to provide protection under adverse conditions like oxygen radicals, high temperatures, irradiation, or lysis by other microbes [22].
Life and Disease Cycle
Colletotrichum orbiculare is the asexual form of the cucurbit anthracnose pathogen and propagates through conidia.It normally exits in the asexual stage, and it rarely undergoes the sexual stage [18,23].There has been no defined complete life cycle for C. orbiculare and only a few reports of the sexual stage of C. orbiculare.The sexual stage of C. orbiculare was reported as a species of Glomerella but was not classified [23].Ascospores are produced in abundance when paired with other isolates of C. orbiculare, but few ascospores develop when isolates are selfed [23].
There is a close relation between C. orbiculare spread and wet weather conditions like rain, morning dew, and overhead irrigation.Conidia are mainly dispersed by rain splashing, but also by wind, instruments, and workers [4].Spore heaps in acervuli are surrounded by a sticky water-soluble matrix [8,18], explaining the need for moisture for spread.The importance of moisture for C. orbiculare was observed and established in the early 20th century when anthracnose epidemics were new.A wetness period of 16 h or more shows maximum disease development [24].Further, temperatures from 18 • C to 27 • C (65 • to 80 • F) are ideal for the establishment and growth of C. orbiculare on watermelon [25].C. orbiculare overwinters by surviving on the debris of infected plants.
Cucurbit anthracnose was more severe on fields that had melons as previous crops [26].Sheldon documented the spread of C. orbiculare by the transportation of diseased fruit and contaminated seeds [26].Overall, C. orbiculare spreads by rain, irrigation, seeds, fruit, and overwintering and survives between seasons on infected plant debris, on volunteer plants, and in and on seeds from infected fruits [4].
Infection Process
Colletotrichum orbiculare is a hemibiotrophic fungus; during the initial stage of infection, it behaves as a biotrophic pathogen, keeping the host cells alive, and later it takes nutrients from dead host cells, switching to the necrotrophic stage [27].C. orbiculare penetrates host leaves using two entry modes: turgor-mediated invasion (TMI) via melanized appressoria and hyphal tip-based entry (HTE).During TMI, C. orbiculare penetrates the adaxial epidermis [8].After landing, the spores adhere to the plant surface and then germinate to produce germ tubes and further form melanized appressoria.The appressoria penetrate plant epidermal cells directly through the cuticle and cell wall but not from the stomata [28].The epidermal cell wall below the appressorium swells, mostly due to cell-wall-degrading enzymes secreted by C. orbiculare [27,28].After penetration, biotrophic intracellular hyphae develop inside the host cells, infecting via intracellular colonization at the cellular level.The intracellular hypha is surrounded by an intact host cell plasma membrane, growing within the plant cell lumen, i.e., between the plant plasma membrane and plant cell walls [29].The infection then proceeds to the necrotrophic phase where the secondary necrotrophic hyphae arise from the intracellular hyphae, obtaining nutrients from dead host cells [27,28].HTE works independently of the melanized appressoria and is a morphogenic response at wound sites [30].The existence of these two invasive strategies implies a sensing system that induces the respective morphogenesis response on wound sites and intact leaf tissue for pathogenesis.
In watermelon fruits, the hyphae grow throughout the rind, and acervuli are formed after 4 to 5 days of infection.Conidiophores form conidia masses rupturing the rind epidermis.In resistant watermelon plants, the appressorium entry during foliar disease is the same as in a susceptible plant but the hyphae are only able to infect a few cells around the penetration site [28].Plant cells around infected leaf sites elongate, divide, and form a raised compact mass to resist fungal growth [28], most likely through lignification.Fruits from resistant plants develop raised areas that are greener as compared to the surrounding rind and remain darker even when the remaining rind starts to bleach [28].Like what is seen in leaves of resistant plants, C. orbiculare only infects one to two epidermal cells in the fruit rind after penetration [28].
Disease Symptoms
Colletotrichum orbiculare causes anthracnose in all cucurbits, and the symptoms on each of the species vary.All the above-ground parts of plants are susceptible to anthracnose.Photosynthetic cells are more susceptible than non-photosynthetic tissue [28].Lesions gradually increase in size with abundant acervuli formation (acervuli are conidiophores producing mycelium aggregates).The descriptions of symptom were added later [4].On watermelon leaves, anthracnose produces blackish-brown lesions (Figure 1A).Centers of older lesions on leaves fall out, giving it a 'shot hole' appearance.Petioles and stems show sunken and dark-color spindle-shaped lesions, which penetrate deeply and finally grid the stem (Figure 1D).Infected young fruits show aborted growth or are abnormal.Lesions on young fruit are small, black depressed spots.On mature fruits, lesions start as yellow translucent centered elevated pimples, which later turn into flat-topped, circular, water-soaked elevations (Figure 1B,C).Lesions on mature fruit further sink and show pink spore masses on a black or cream-colored background.The black lesions are the result of the black stroma left behind after the washing of spores, whereas the pink masses are like the spore masses found in culture media.
finally grid the stem (Figure 1D).Infected young fruits show aborted growth or are abnormal.Lesions on young fruit are small, black depressed spots.On mature fruits, lesions start as yellow translucent centered elevated pimples, which later turn into flat-topped, circular, water-soaked elevations (Figure 1B,C).Lesions on mature fruit further sink and show pink spore masses on a black or cream-colored background.The black lesions are the result of the black stroma left behind after the washing of spores, whereas the pink masses are like the spore masses found in culture media.
Pathogenesis Genes and Effectors
The average genome size of Colletotrichum species is 40 Mb, but C. orbiculare has a surprisingly large genome of 90 Mb [27].C. orbiculare expresses a large arsenal of genes during the infection process, including 287 protease-encoding genes, 327 plant cell-walldegrading enzymes, 700 small secreted proteins (SSPs), and many secondary metabolite backbone-forming proteins [27].All of these are expressed at a higher level than in other species such as C. graminicola and C. higginsianum.SSPs and secondary metabolite synthesis genes are upregulated during the initial biotrophic stage of infection, whereas degrading enzymes are upregulated during the later necrotrophic stage of infection.The upregulation of SSP genes during early infection in C. orbiculare suggests their importance in maintaining biotrophy during infection.As C. orbiculare is a hemibiotroph, there is an orchestrated expression of the effector genes during the shift from biotrophy to necrotrophy [31].
Although through genomic and transcriptomic studies it is known that C. orbiculare expresses an arsenal of genes involved in pathogenesis, only a few effectors and pathogenic pathways are known yet.C. orbiculare produces several effectors, which include necrosis-and ethylene-inducing peptide1-like protein1 (NLP1) [32,33], DN3, necrosis-inducing secreted protein1 (NIS1) [34,35], MC69 [36], and suppression of immunity in Nicotiana benthamiana (SIB1, SIB2) [37].NLP1 is expressed specifically in necrotrophic invasive hyphae [33], has cytotoxic activity, induces cell death, and triggers immune response [38].The effector DN3 of C. orbiculare suppresses NLP1-triggered plant cell death [39], which was also previously observed in the C. higginsianum DN3 homolog [40].NIS1 induces cell death and is expressed in bulbous biotrophic primary hyphae, but its activity reduces in necrotrophic hyphae.Homologs of NIS1 are present in C. higginsianum, C. graminicola, and Magnaporthe oryzae, suggesting it is a conserved sequence in Colletotrichum species.Although NIS1-knockout mutants are virulent on tobacco [35], the transgenic expression of NIS1 in Arabidopsis made the plant susceptible to C. orbiculare [41].Intuitively, the expression of cell-death-inducing NIS1 during the biotrophic phase suggests it is a
Pathogenesis Genes and Effectors
The average genome size of Colletotrichum species is 40 Mb, but C. orbiculare has a surprisingly large genome of 90 Mb [27].C. orbiculare expresses a large arsenal of genes during the infection process, including 287 protease-encoding genes, 327 plant cell-walldegrading enzymes, 700 small secreted proteins (SSPs), and many secondary metabolite backbone-forming proteins [27].All of these are expressed at a higher level than in other species such as C. graminicola and C. higginsianum.SSPs and secondary metabolite synthesis genes are upregulated during the initial biotrophic stage of infection, whereas degrading enzymes are upregulated during the later necrotrophic stage of infection.The upregulation of SSP genes during early infection in C. orbiculare suggests their importance in maintaining biotrophy during infection.As C. orbiculare is a hemibiotroph, there is an orchestrated expression of the effector genes during the shift from biotrophy to necrotrophy [31].
Although through genomic and transcriptomic studies it is known that C. orbiculare expresses an arsenal of genes involved in pathogenesis, only a few effectors and pathogenic pathways are known yet.C. orbiculare produces several effectors, which include necrosisand ethylene-inducing peptide1-like protein1 (NLP1) [32,33], DN3, necrosis-inducing secreted protein1 (NIS1) [34,35], MC69 [36], and suppression of immunity in Nicotiana benthamiana (SIB1, SIB2) [37].NLP1 is expressed specifically in necrotrophic invasive hyphae [33], has cytotoxic activity, induces cell death, and triggers immune response [38].The effector DN3 of C. orbiculare suppresses NLP1-triggered plant cell death [39], which was also previously observed in the C. higginsianum DN3 homolog [40].NIS1 induces cell death and is expressed in bulbous biotrophic primary hyphae, but its activity reduces in necrotrophic hyphae.Homologs of NIS1 are present in C. higginsianum, C. graminicola, and Magnaporthe oryzae, suggesting it is a conserved sequence in Colletotrichum species.Although NIS1-knockout mutants are virulent on tobacco [35], the transgenic expression of NIS1 in Arabidopsis made the plant susceptible to C. orbiculare [41].Intuitively, the expression of cell-death-inducing NIS1 during the biotrophic phase suggests it is a recognized avirulence (AVR) protein.Recently, it has been shown that NIS1 associates with the receptor-like kinases (RLKs) such as brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1) and receptor-like cytoplasmic kinases (RLCKs) such as botrytis-induced kinase1 (BIK1) in the host, inhibits pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation, and ultimately impairs pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) [42].DN3 suppresses NIS1-triggered HR-like cell death [35].MC69 is one of the characterized effectors in C. orbiculare.Although the MC69 mutants of C. orbiculare had normal colony morphology and conidiogenesis, they had reduced lesion development on cucumber and tobacco [36].C. orbiculare expresses MC69 predominantly during the biotrophic phase of infection.A recent study has proposed two novel effectors of C. orbiculare (SIB1 and SIB2), where SIB1 was found to be expressed at both the primary and later stages of host invasion in tobacco and cucumber, respectively [37].
Few signaling infection-related morphogenesis and pathogenesis pathways have been identified in C. orbiculare.C. orbiculare has three cascades important for virulence: CMK1, MAF1, and RPK1.The CMK1 cascade is involved in conidial germination, infection, growth, and appressorium maturation.The MAF1 cascade is required for appressorium differentiation, whereas the RPK1 pathway is essential for vegetative growth and conidiation.The melanization of appressoria is important for the normal function of C. orbiculare [21], and three melanin biosynthesis enzyme genes, PKS1, SCD1, and THR1, and one regulatory gene, CMR1, have been characterized [43][44][45][46][47].Mutants of melanin-related genes showed defects in the melanization of appressoria and penetration ability.Further, fatty acid oxidation of peroxisomes is also required for melanization and metabolic processes involved in turgor generation for penetration [41].
Plants have receptors that recognize PAMPs.An example of a PAMP is chitin, a major cell wall component in filamentous fungi.C. orbiculare has the SSD1 gene involved in cell wall integrity.Mutants of ssd1 were not virulent and induced host defense response along with papillae formation.The ssd1 mutants had increased induction of salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) activity as compared to the wild type.The effector secretion mechanism has also been studied.C. orbiculare shows a strong ring-like signal of the effectors around the primary biotrophic hyphae.The ring signal is present in the interfacial region between the host and C. orbiculare, and not inside C. orbiculare [31,33].C. orbiculare continuously secretes effectors towards the interfacial region.Effectors are also secreted in a single-dot fashion at the bottom of the appressoria and at putative interfaces on the hyphal surface.
Pathogen Races
The first commercial anthracnose-resistant watermelon cultivars were released in 1949 and were widely used to manage the rampant anthracnose outbreaks at that time.Since then, C. orbiculare has emerged and overcome host resistance.In 1958, severe anthracnose symptoms on resistant watermelon cultivars were observed in North Carolina [48].Pathogen isolates from the new symptoms were observed to be undistinguishable from earlier C. orbiculare isolates.Isolates were defined into races based on the differential host reactions on different cucurbit cultivars.Most isolates before 1954 were defined as race 1. Isolates found in 1954 and 1955 were defined as race 2 and were highly pathogenic on all cucurbit cultivars of that time.Some isolates were defined as race 3. The difference between race 1 and race 3 was pathogenicity on squash cultivars.The new isolates had no morphological and cultural differences and were considered as different races [48].Race 4 [49] and races 5, 6, and 7 [50] were also identified later.
Overall, from 1954 to 1964, seven pathological races of C. orbiculare were identified based on differential host reactions.The seven races were based on virulence differences on different cucurbit species and cultivars.Race diversity was reevaluated, and the seven races of C. orbiculare were combined into three vegetative compatibility groups (VCGs) by using 92 isolates from the U.S. [15].Based on vegetative compatibility (a phenomenon where fungi with certain genetic similarity can fuse together to form a single heterokaryon), 11 VCGs were formed among the 92 C. orbiculare isolates.Out of the 11 VCGs, only 3 were pathogenic on cucurbits, namely VCGs 1, 2, and 3. VCGs 1 and 3 showed virulence on similar hosts.The watermelon cultivar 'Charleston Gray' was resistant to both VCGs 1 and 3, but susceptible to VCG 2. Similar resistance was shown by cucumber cultivars 'Poinsett 76' and 'Gy 14'.VCGs 1 and 3 were classified as race 1 virulence phenotype, and VCG 2 as race 2 virulence phenotype.A race 2B has also been somewhat characterized through vegetative compatibility and virulence [51].Race 2B has been found on watermelon, bottle gourd, and muskmelon and belongs to VCG 2 [4].
Disease Management
Field management practices for anthracnose include planting disease-free seed material, deep plowing of crop residue immediately after harvest, crop rotation with noncucurbit crops for a minimum of 1 year (2 to 3 years is optimum), avoiding usage of farm machinery among fields when the foliage is wet, fungicide applications with effective active ingredients such as quinone outside inhibitors (QoIs), and resistant plant cultivars [4].To reduce fruit damage by anthracnose, growers are recommended to avoid mechanical injury to fruits, inspect for infected fruits during harvest and discard them, disinfect the fruit surface with chlorinated water, and refrigerate the fruit after harvest to prevent or delay anthracnose development postharvest [4].
Host Resistance
Host resistance screening was conducted either on cotyledons [15] or the two-to four-true-leaf stage [52][53][54][55][56][57].The disease inoculum used for the seedling screening varied from 2.5 × 10 3 [57] to 5 × 10 5 [54] conidial spores/mL.The incubation time also varied from 3 to 14 days post-inoculation (DPI).A recent study reported the optimum inoculum concentration and DPI for screening two-to four-leaf-stage seedlings for race 2 anthracnose resistance [53].The authors of this study found that 1 × 10 5 spores/mL and a single disease rating of percent leaf lesion at 9 DPI were optimal.
In 1937, Layton started breeding for anthracnose resistance and identified sources of resistance to develop commercial cultivars.Five cultivars from Africa with high resistance to anthracnose were identified, out of which three had edible fruit and desired horticultural traits [5].The three cultivars were named Africa 8, 9, and 13, and were further used as parents.Homozygous anthracnose-resistant selections from Africa 8, 9, and 13 were crossed with commercial cultivars Iowa Belle and Iowa King and a few other cultivars [5].The commercial Iowa cultivars were wilt-resistant, large-fruited, and crisp-fleshed.The first widely accepted anthracnose-resistant watermelon cultivars were 'Congo' (1949), 'Fairfax' (1953), and 'Charleston Gray' (1955), released by Andrus [57,58].Charleston Gray, Congo, and Fairfax are resistant to races 1 and 3 but susceptible to race 2 [48].Cultivars resistant to race 1 were also resistant to race 3 [57,59].
Resistance to race 2 was first found in a citron, W695, which was also resistant to races 1 and 3 [57].PI 326515 was the first PI reported to have resistance only to race 2 [56].More resistance sources to race 2 including PI 189225, 271775, 271778, and 512385 were identified [52,55].Resistance to anthracnose race 2 was also identified in Citrullus colocynthis, designated as R309 [60].Interestingly, two studies found that resistance in Citrullus colocynthis, R309, did not follow the single gene inheritance and was suggested to be multigenic [60].These studies suggested that a dominant single gene confers major resistance, but there are other genes contributing to the phenotype.R309 has been the only source of multigenic resistance; no other multigenic resistance sources have been reported.
The first inheritance work on anthracnose resistance was performed in 1937 [5].Resistance to race 1 is dominant over susceptibility and segregates as a single gene.Resistance to races 1 and 3 is controlled by the same gene, Ar-1 [57].Inheritance of race 2 resistance is like race 1 resistance, dominant and segregating as a single gene [56].In Korea, Jang et al. used a biparental population, 'DrHS7250' (female parent, resistant breeding line) and 'Oto9491' (male parent, susceptible breeding line), to identify a C. orbiculare race 1-resistance quantitative trait locus (QTL) on chromosome 8 and further conducted transcriptomics via RNAseq on the parents to identify a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene in the QTL region that conferred resistance to the disease [61].They hypothesized that residue 18 of a conserved motif, IxxLPxSxxxLYNLQTLxL, could govern resistance in 'DrHs7250'.An independent study conducted in the U.S. using a biparental mapping population, 'Charleston Gray' (female parent, resistant) and 'New Hampshire Midget' (male parent, susceptible), found a major C. orbiculare race 1-resistance QTL in the same region on chromosome 8 [62].A PACE SNP marker designed from the SNP marker CL 14-27-9, identified earlier [61], was also the diagnostic marker for the QTL (LOD = 14.06) in the study.Even though the resistance source for the breeding line 'DrHS7250' was not reported, it seemed that both 'DrHS7250' and 'Charleston Gray' may have the same resistance gene for C. orbiculare race 1.Both studies in different watermelon resistance sources validated that the race 1 anthracnose resistance is governed by a single dominant gene.Even today, anthracnose is a problem and a major research priority in watermelon [17].Most of the current commercial cultivars with anthracnose resistance were developed by private industry (Table 1).These commercial cultivars have intermediate to high levels of resistance to anthracnose race 1, and some descriptions do not specify the race.Many hybrid watermelon cultivars are resistant to races 1 and 2B and susceptible to race 2 [4].The SNP marker CL 14-27-9 could be utilized as a diagnostic marker to develop race 1-resistant cultivars via marker-assisted selection in watermelon breeding programs.
Prospects and Challenges
Although anthracnose has been an important watermelon disease for around a century, there are still many unanswered questions regarding pathogen biology and disease management.Anthracnose races have been identified for over 60 years now, but the genetic bases of those races remain unknown, forcing researchers to rely on differential phenotypic responses for race identification.In genomic studies like that of [27] the genome of C. orbiculare was sequenced, comparative studies were conducted, and transcriptomic analysis unraveling candidate effectors involved during different stages of infection was performed.Recently, the core effector NIS1 was shown to attack the conserved immune kinases in the host system and disrupt the first layer of plant immunity (PTI) [42].We are yet to understand how plant resistance genes interact with the C. orbiculare effectors through the second layer of defense, i.e., effector-triggered immunity (ETI).
Many genomic resources have been developed for cucurbit crops and watermelon in particular [68][69][70].Nonetheless, genetic determinants of anthracnose resistance have not been clearly identified.Cucurbit breeders still rely on genetic maps, loci, and QTLs to describe genetic resistance for conventional breeding, but specific resistance genes and the mechanism of anthracnose resistance have not been characterized.The whole genome sequence available for watermelon and advancements in genomics, transcriptomics, proteomics, and metabolomics offer the opportunity to identify genes responsible for the anthracnose-resistant phenotype; however, these approaches are sensitive to noise from phenotyping and genotyping and do not always result in a clear candidate gene.The occurrence of races in C. orbiculare is an indication that pyramiding resistance will be required to ensure the durability of the trait and minimize the risk of new isolates overcoming individual resistance genes as has occurred with other cucurbit diseases [48,71].Establishing the resistance gene repertoire in watermelon and characterizing the interactions of such proteins with pathogen proteins that result in a resistant phenotype will be needed to achieve durable anthracnose resistance in watermelon.Likewise, continued identification of new and improved resistance sources will remain a priority for breeding anthracnose resistance.
†Resistance to anthracnose race was not specified in the varietal description by the company. | 2023-10-15T15:10:34.936Z | 2023-10-13T00:00:00.000 | {
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252213539 | pes2o/s2orc | v3-fos-license | Highly pathogenic coronavirus N protein aggravates inflammation by MASP-2-mediated lectin complement pathway overactivation
Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.
The nucleocapsid (N) protein of SARS-CoV, a 46-kDa viral RNAbinding protein, 5 shares 91, 51, and~30% homology with those of SARS-CoV-2, MERS-CoV and other β-coronaviruses, respectively. 3,6,7 As one of the most abundant viral structural proteins, SARS-CoV N protein can also be detected in serum as early as 1 day after the onset of symptoms and has been used as an early diagnostic marker. 8 The complement system functions as an immune surveillance system that rapidly responds to infection and can be activated via the classical pathway (CP), the lectin pathway (LP), or the alternative pathway (AP). 9 In the LP, mannan-binding lectin (MBL) (or ficolins) binds carbohydrate arrays of mannan and N-acetylglucosamine residues on the surfaces of pathogens or virus-infected cells, resulting in the activation of MBL-associated serine protease-2 (MASP-2). Activated MASP-2 then cleaves C4 and C2, leading to the formation of the C3 convertase (C4bC2b), and C3, the central and most abundant component of the complement system, is cleaved by C4bC2b into C3a and C3b. C3b converts C4bC2b to C5 convertase (C4bC2bC3b), which cleaves C5 to yield C5a and C5b fragments and initiates the terminal pathway. 10,11 The N-linked glycosylation site N330 on the SARS-CoV spike (S) protein is critical for specific interactions with MBL. 12 Dysregulated complement activation has been implicated in the development of acute lung diseases induced by highly pathogenic viruses. 13,14 Higher levels of activated complement C3 and C4 fragments were found in SARS patients, 15,16 and increased C5a and terminal complement complex C5b-9 were detected in MERS-CoV-infected hDDP4-Tg mice. 17 In COVID-19 patients, extensive deposition of C5b-9, C4d and MASP-2 was observed in the lungs, microvascular endothelium, and kidneys. [18][19][20] Ali et al. reported that the N protein of SARS-CoV-2 directly binds MASP-2. 21 In addition, narsoplimab (OMS721), a fully humanized immunoglobulin gamma 4 (IgG4) monoclonal antibody against MASP-2 that inhibits LP functional activity, has been used successfully in the treatment of critically ill, mechanical ventilation-dependent COVID-19 patients in a phase II clinical trial. 22 These reports indicate that the complement pathways are activated in highly pathogenic human coronavirus infection, but the detailed mechanism underlying these excessive complement activations is not understood.
In this study, we found that the N proteins of highly pathogenic coronaviruses, including SARS-CoV, MERS-CoV and SARS-CoV-2, bind MASP-2 and significantly potentiate complement activation through MASP-2 activation. The crucial roles of MASP-2-involved complement activation in viral pathogenesis may direct the development of therapies for COVID -19.
RESULTS
N proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV interact with MASP-2 To substantiate the physical interaction between the SARS-CoV N protein and MASP-2, Flag-tagged SARS-CoV N protein (Flag-SARS N)-conjugated agarose beads were incubated with human or mouse serum, and MASP-2 in the sera was immunoprecipitated with the N protein (Fig. 1a). Similar to SARS-CoV N, exogenously expressed MERS-CoV N and SARS-CoV-2 N were both associated with MASP-2 (Fig. 1b). The association between MASP-2 and the N protein of SARS-CoV or SARS-CoV-2 was observed only in the presence of CaCl 2 but not EDTA (Fig. 1c, d), which is consistent with the requirement for Ca 2+ in MASP-2-MBL binding and MASP-2 autoactivation. 23 Furthermore, the binding affinity of recombinant MASP-2 and the N protein of SARS-CoV or SARS-CoV-2 was determined by surface plasmon resonance (SPR) analysis, with K D values of 2.678 × 10−7 M and 5.184 × 10 −7 M, respectively (Fig. 1e). The SARS-CoV-2 N:MASP-2 association was also confirmed in lung tissue sections from COVID-19 victims but not cancer patients by an in situ proximity ligation assay (PLA), and the N protein:MASP-2 complexes (red fluorescent spots) were mainly distributed in pulmonary epithelial cells ( Fig. 1f and Supplementary Fig. 1a). These results collectively demonstrate that the interaction between the N proteins and MASP-2 occurred in vitro and in SARS-CoV-2infected lung tissues.
Subsequently, anti-Flag immunoprecipitates of full-length or truncated Flag-MASP-2 ( Supplementary Fig. 1b) were incubated with lysates of 293T cells expressing GFP-tagged SARS-CoV N protein (GFP-SARS N) or its mutants, and the adsorbates were analyzed with anti-Flag or anti-GFP antibodies by immunoblotting. Compared with other truncated regions, the CCP1-CCP2-SP region of MASP-2 ( Fig. 1c) and the N-terminal domain (residues 1-175) of the N protein (Fig. 2a) were indispensable for the association.
More detailed analysis showed that amino acid residues 116-124, but not residues 321-323, which are critical for SARS-CoV N protein dimerization, were essential for the interaction with MASP-2 ( Fig. 2b and Supplementary Fig. 1c). The 116-124 motif of the SARS-CoV N protein shares a high identity with the corresponding sequence in SARS-CoV-2 N (residues 115-123) and MERS-CoV N (residues 104-112) ( Supplementary Fig. 1b). As expected, both the SARS-CoV-2 NΔ115-123 and MERS-CoV NΔ104-112 mutants exhibited a seriously impaired or abolished MASP-2 association (Fig. 2c, d). These results collectively demonstrate that a consensus motif (TGPEAXLPY) across the N proteins of highly pathogenic human coronavirus is conserved for MASP-2 binding. N proteins of highly pathogenic human coronavirus potentiate MASP-2-dependent complement activation The CCP1-CCP2-SP domains of MASP-2 are responsible for selfactivation and substrate binding, which, in turn, mediate complement LP activation. [24][25][26] To investigate MASP-2 dimerization, which charges LP activation, 27 MASP-2-Flag-conjugated beads were incubated with lysates of cells expressing MASP-2-Myc in the presence or absence of N protein and MBL. The homodimerization of MASP-2 was potentiated by the existence of both SARS-CoV N protein and MBL ( Fig. 3a and Supplementary Fig. 2a). Next, purified MASP-2-Flag was incubated with mannan and MBL with or without SARS-CoV N protein. Higher levels of the cleaved MASP-2 fragments released by MASP-2 autoactivation were detected in the presence of SARS-CoV N protein as well as MBL ( Fig. 3b and Supplementary Fig. 2b).
Furthermore, the dynamics of MASP-2:MBL binding were assessed with purified MBL and MASP-2 in mannan-coated wells. Compared with human serum albumin (HSA), the binding of MASP-2 to MBL was significantly enhanced by the SARS-CoV N protein, and the potentiation was greatly abrogated by the anti-N protein antibody (Fig. 3c). Moreover, the SARS-CoV N protein bearing the Δ116-124 or Δ321-323 deletion or N protein from the less pathogenic human coronavirus 229E (HCoV-229E) showed little if any effect on MASP-2:MBL binding (Fig. 3c). These results indicate that SARS-CoV N protein-potentiated MASP-2 activation depends not only on their association but also on the dimerization of the N protein.
N-regulated complement activation was further investigated by complement deposition assays in cell-ELISA and mannan-coated microplates. Significant MASP-2 deposition was observed on the surface of the cells infected with SARS-CoV-2 or SARS-CoV-2 S protein-pseudotyped human immunodeficiency virus (HIV/SARS-CoV-2 S) with additional N protein added compared with the noninfected cells (mock) (Fig. 4a, b), and the N protein potentiated MASP-2 deposition was significantly suppressed by anti-MASP-2 or anti-SARS-CoV-2 N antibody (nCoV396 29 ) (Fig. 4a, b). Accordingly, Fig. 1 The N proteins of SARS-CoV, MERS-CoV, and SARS-CoV-2 bind MASP-2. a Lysates of 293T cells expressing Flag-SARS N or Flag were mixed with human serum (HS) or mouse serum (MS) and subjected to immunoprecipitation with anti-Flag beads. The absorbates were probed with the indicated antibodies. Purified recombinant MASP-2 was loaded as a marker. b Lysates of 293T cells expressing GFP-SARS-CoV-2 N, GFP-SARS-CoV N, or GFP-MERS-CoV N were subjected to immunoprecipitation with MASP-2-Flag-conjugated beads in the presence of CaCl 2 and analyzed with the indicated antibodies. c Lysates of 293 T cells expressing GFP-SARS N and GFP were subjected to immunoprecipitation with Flag-tagged full-length (FL) MASP-2 or truncated MASP-2 (CUB1-EGF-CUB2 and CCP1-CCP2-SP)-conjugated beads in the presence of CaCl 2 (2 mM) or EDTA (1 mM). Immunoblotting was performed with anti-GFP and anti-Flag antibodies. IgG beads and Flag beads incubated with the indicated lysates were used as negative controls. d Lysates of 293T cells expressing Myc-SARS-2 N were subjected to immunoprecipitation with Flag-tagged MASP-2 conjugated beads in the presence of CaCl 2 (2 mM) or EDTA (1 mM). Immunoblotting was performed with anti-Myc and anti-Flag antibodies. Flag beads incubated with the indicated lysates were used as negative controls. Data in a-d are representative of two independent experiments. e SPR assays were used to detect the direct association between MASP-2 and SARS-CoV N/SARS-CoV-2 N with the indicated purified proteins. The K D values were calculated using software. f Paraformaldehyde-fixed lung tissues from COVID-19 patients from postmortem autopsies were used for paraffin tissue sections. SARS-CoV-2 N:MASP-2 complex formation in the lung was determined by in situ PLA as indicated by the red signals. Scale bar, 10 μm the N protein of SARS-CoV, MERS-CoV, and SARS-CoV-2, but not the HCoV-229E N protein, potentiated C4b deposition in a dosedependent manner in C1q-depleted serum (to eliminate the classical pathway 30 ) (Fig. 4c-e) but not in heat-inactivated or MBLdepleted serum ( Supplementary Fig. 3a-c). Furthermore, SARS-CoV-2 N protein potentiated C4b deposition could be blocked by anti-MBL, anti-MASP-2 or anti-N protein (Fig. 4f). Much less but detectable C4b deposition was observed in the wells without mannan coating (Fig. 4f). Taken together, these results suggest that the S protein or mannan and MBL are very important for Highly pathogenic coronavirus N protein aggravates inflammation by. . . Gao et al. MASP-2 activation, and the activation is significantly potentiated by the N protein.
The deposition of activated C3 was also evidently increased along with the increase in SARS-CoV N protein levels up to~40 nM (Fig. 4g), suggesting an enhanced activity of C3 convertase. In addition, the SARS-CoV N protein had little or no effect on activated C3 deposition in calcium-free buffer containing EGTA, suggesting that SARS-CoV N protein-potentiated C3 activation occurs through the LP but not the AP, in which C3 activation is Ca 2+ -independent (Fig. 4h). C3b deposition was decreased in the presence of a high concentration of N protein (Fig. 4g, h), possibly due to the further cleavage of C3b by soluble inhibitors in serum (such as factor H and factor I) when the surface was coated with high densities of C3b. 31,32 Subsequently, as a result of amplified complement cascades, a significantly increased deposition of the C5b-9 complex was induced by SARS-CoV N protein at a much lower concentration ( Fig. 4i), similar to that observed in patient sera. 8 Activated complement plays a crucial role in the efficient phagocytosis of pathogens and cellular debris by C3b-or C5bmediated opsonization. 33 Complement-dependent phagocytosis of E. coli by mouse peritoneal macrophages was remarkably enhanced by SARS-CoV N protein but not HSA ( Supplementary Fig. 3d), demonstrating that SARS-CoV N protein effectively potentiated complement-mediated opsonization.
Complement cascade was activated by the N protein of highly pathogenic coronavirus in mice and COVID-19 patients To investigate the effect of the N protein on complementmediated inflammatory responses in vivo, mice were pre-infected with adenovirus expressing SARS-CoV N (Ad-SARS N), and the N protein was detectable in mouse serum within one week ( Supplementary Fig. 4a). Then, we injected SARS-CoV spike protein-pseudotyped HIV (HIV/SARS-CoV S) to induce complement activation in vivo ( Supplementary Fig. 4b, upper panel). The production of leukotriene B4 (LTB4), an arachidonic acid metabolite and a potent inflammatory factor agent induced by membrane damage, including membrane attack caused by complement activation, 34,35 was significantly increased in the Ad-SARS N pre-infected mice compared with that in the Ad preinfected mice, reflecting the higher inflammation level induced by N protein (Fig. 5a, lanes 1 and 2). Importantly, N proteinpotentiated LTB4 production was significantly suppressed by the anti-MASP-2 antibody and C1INH (Fig. 5a, lanes 3 and 4). As a control, little, if any, induction of LTB4 production was observed following vesicular stomatitis virus G protein-pseudotyped virus (HIV/VSV-G) challenge (Fig. 5a, lanes 5 and 6), which is consistent with the findings that VSV-G did not bind MBL. 36 N proteinmediated complement overactivation was further evaluated in an LPS-induced inflammatory mouse model in which the MASP-2involved LP can be activated by LPS. 37 Mice pre-infected with Ad-SARS N were challenged with LPS and sacrificed 6 h after the challenge ( Supplementary Fig. 4b, lower panel). Similar to COVID-19 patient lung tissues (Fig. 1f), the in vivo association between MASP-2 and the SARS-CoV N protein was demonstrated by in situ PLA in the lungs of the Ad-SARS N pre-infected, LPS-challenged mice ( Fig. 5b and Supplementary Fig. 4c), and a higher level of the cleaved MASP-2 fragment was also observed in these mice ( Supplementary Fig. 4d). Immunohistochemical (IHC) analysis also demonstrated that the deposition of C4b and activated C3 staining in the lung were evidently increased in the mice expressing the SARS-CoV N protein (Fig. 5c, upper panel and Fig. 5b) compared with the weak staining observed in the control (Fig. 5c, lower panel and Supplementary Fig. 4c). These results show that the SARS-CoV N protein could promote MASP-2 and, thus, LP activation in an LPS-induced inflammatory mouse model.
Next, the paraformaldehyde-fixed lung tissue of COVID-19 victims was collected and subjected to IHC staining. Strong positive signals of the SARS-CoV-2 N protein and complement cascade components involved in LP 38 were detected in lung tissue from COVID-19 patients (Fig. 5d). Meanwhile, significantly higher serum C3a (Fig. 5e) and C5a ( Fig. 5f) levels were observed in critical, but not mild, COVID-19 patients. 39,40 These data collectively indicate that the complement pathways could be aggressively activated in mice infected with Ad-SARS N and critical COVID-19 patients.
N proteins aggravate LPS-and coronavirus-induced pneumonia by MASP-2-involved complement activation Furthermore, to assess whether the N protein potentiated complement activation in pneumonia, mice pre-infected with Ad-SARS N or Ad-vector were challenged with LPS to induce pneumonia. 41 All mice pre-infected with Ad-SARS N died within 12 h after the LPS challenge, while 8 of 10 mice pre-infected with Ad survived ( Fig. 6a and Supplementary Fig. 4b, lower panel). Severe lung damage and massive inflammatory cell infiltration were observed in the dying mice (Fig. 6b). In accordance, the pre-infection with the Ad-229E N or Ad-SARS N mutants (NΔ116-124 or NΔ321-323) had far fewer effects on mortality (Fig. 6a). Accordingly, when an anti-N antibody, an anti-MASP-2 antibody or C1INH was administered simultaneously with LPS to the Ad-SARS N pre-infected mice, both the mortality and lung tissue inflammation induced by LPS were significantly improved (Fig. 6a, b). Similarly, the mice preinfected with adenovirus expressing MERS-CoV N (Ad-MERS N), but not its Δ104-112 mutant, 100% died within 24 h after the LPS challenge, which could also be partially rescued by C1INH and the anti-MASP-2 antibody (Fig. 6c). Then, Masp2 −/− mice ( Supplementary Fig. 5a, b) were subjected to the same treatment as shown in the lower panel of Supplementary Fig. 4b. The LPS challenge induced high mortality in wild-type mice pre-infected with Ad-SARS N or Ad-MERS N, and a significantly lower mortality was observed in the Masp2 −/− littermates ( Fig. 6d and Supplementary Fig. 5c).
Furthermore, compared to the uninfected mice, the mice preinfected with mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA) showed a significantly decreased percentage of lymphocytes (Lym%) and an increased percentage of neutrophils (Neu%) (Fig. 6e), which are frequently observed in SARS-CoV-2-infected patients. 42 The administration of a recombinant anti-MASP-2 antibody (OMS721) or anti-SARS-CoV-2 N antibody (nCoV396) resulted in a compromised Lym% decrease and Neu% increase Fig. 2 A consensus motif across the N proteins of the highly pathogenic human coronavirus TGPEAXLPY is indispensable for MASP-2 binding. a MASP-2-Flag-conjugated agarose beads were incubated with lysates of 293T cells expressing full-length or truncated N protein in the presence of 2 mM CaCl 2 , and the absorbates were analyzed by immunoblotting with the indicated antibodies. b Lysates of 293T cells expressing full-length GFP-SARS N or its mutants Δ321-323 and Δ116-124 were subjected to immunoprecipitation with MASP-2-Flagconjugated beads in the presence of CaCl 2 and analyzed with the indicated antibodies. c Lysates of 293T cells expressing HA-SARS-CoV-2 N or its truncated mutant Δ115-123 were subjected to immunoprecipitation with MASP-2-Flag-conjugated beads in the presence of CaCl 2 and analyzed with the indicated antibodies. d Lysates of 293T cells expressing full-length GFP-MERS N or its truncated mutant Δ104-112 were subjected to immunoprecipitation with MASP-2-Flag-conjugated beads in the presence of CaCl 2 and analyzed with the indicated antibodies. Data in a-d are representative of two independent experiments compared with saline treatment (Fig. 6e). Accordingly, a significantly lower Lym% decrease and Neu% increase were observed in the Masp2 −/− mice compared to those observed in the wild-type littermates upon SARS-CoV-2 MA infection (Fig. 6f). Taken together, these results suggest that suppression of MASP-2-mediated complement overactivation may provide an approach for the treatment of pneumonia in pathogenic coronavirus-infected patients.
DISCUSSION
In this study, the N proteins of highly pathogenic coronaviruses, including SARS-CoV N, MERS-CoV N, and SARS-CoV-2 N, were demonstrated to bind MASP-2 and potentiate the MBL-dependent activation of MASP-2, leading to hyperactivation of the LP complement cascade in vitro, in mice, and in COVID-19 patients (a schematic summary is shown in Fig. 7). Coronavirus N proteinmediated MASP-2 and thereby complement cascade overactivation can aggravate pneumonia in mice (Fig. 6).
The findings were supported by a panel of clinical and basic investigations showing that complement activation plays important roles in the pathogenesis of COVID-19, and thus, complement may serve as a driver of and therapeutic target for SARS-CoV-2. [43][44][45] Both the S and N proteins of SARS-CoV-2 were observed to be directly recognized by components of the LP, leading to complement activation. 21,46 Accordingly, the complement components involved in the LP (MBL, MASP-2, C3, and C5b-9) were increased in lung tissue from COVID-19 patients (Fig. 5d). Significantly higher levels of serum C3a and C5a were present in critical COVID-19 patients (Fig. 5e, f), which was supported by the findings that high levels of C5a and C5b-9 were present in severe COVID-19 patients, and the levels were correlated with clinical severity. 39,40 Moreover, pre-infection with Ad-SARS N or Ad-MERS N evidently increased the fatality of LPS-induced pneumonia (Fig. 6a-c), underlining the potential role of the N protein in the inflammatory injury to tissue caused by the massive LPS released from secondary bacterial infections frequently occurring in SARS, COVID-19 and MERS patients. 1,47,48 Our findings showed that the SARS-CoV N protein potentiated the binding of MASP-2 to MBL in mannan-coated microplate wells (Fig. 3c), and the CCP1-CCP2-SP region of MASP-2 was responsible for the interaction with SARS-CoV N (Fig. 1c). These results suggest that the N protein potentiated the MASP-2:MBL interaction by inducing conformational changes or MASP-2 activation, since the CUB1-EGF domains of MASP-2 are responsible for the MBL association. 49 Ali et al. reported that SARS-CoV-2 N directly interacts with MASP-2 and promotes MASP-2-mediated C4 cleavage. 21 Our results also show that the N protein induced the autoactivation of MASP-2 in vitro (Fig. 3b). These data raise the question of whether MASP-2 serves as a pattern recognition molecule (PRM). As shown in Fig. 4a, b, the N protein of SARS-CoV-2 potentiated MASP-2 deposition in SARS-CoV-2-infected cells or HIV/SARS-CoV-2 S-infected cells, but little if any effects were observed in the cells that did not express the S protein. The N protein failed to potentiate complement activation in MBLdepleted serum (Supplementary Fig. 3b, c). In addition, the SARS-CoV-2 N protein potentiated C4b deposition was also dependent on the presence of mannan, which could be blocked by anti-MBL, anti-MASP2 or anti-SARS-CoV-2 N antibodies (Fig. 4f). These findings indicate that the S protein on the cell surface (or mannan in microplate wells) and MBL are largely indispensable for MASP-2 activation and that activation is significantly potentiated by the N protein. These results suggest that the MBL-bound S protein of SARS-CoV-2 served as a PRM and that the N protein potentiated LP activation. Stravalaci et al. also suggested that SARS-CoV-2 S protein-bound MBL served as a PRM. 46 However, the N protein of SARS-CoV-2 can still partially activate the complement pathway in the absence of mannan (Fig. 4f), suggesting that N protein-potentiated MBL-independent MASP-2 autoactivation may play a minor role in complement activation.
The lethality of LPS-exposed mice by SARS-CoV N and MERS-CoV N could be blocked by the corresponding anti-N antibody treatments but not completely by C1INH or anti-MASP-2 antibody (Fig. 6a, c), suggesting that in addition to promoting complement activation, SARS-CoV and MERS-CoV N protein may affect other pathways. Virus-specific CD8 + T cells can greatly ameliorate recovery from respiratory diseases such as avian influenza, SARS, and MERS and persist long-term as cross-reactive memory pools. [50][51][52][53] In addition, B cells elicit an early response against the N protein and N-specific neutralizing antibody production at the early stage of highly pathogenic coronavirus infection. 52,54 These studies suggest that the highly pathogenic coronavirus N protein may not only activate complement but also affect CD8 + T cells and antibody responses to promote inflammatory responses. However, the specific mechanism remains to be further studied.
The hyperactivation of complement was found to contribute to endothelial cell injury, thrombosis and intravascular coagulation, ultimately leading to multisystemic organ failure and excessive coagulation in COVID-19 patients. 18,55,56 The potent anaphylatoxin C5a attracts neutrophils and monocytes to the infection site, causes tissue damage by oxidative radical formation and enzyme release, and leads to the activation of the coagulation system. 43 These studies suggest that the coagulation-related symptoms in COVID-19 may also be associated with the abnormal activation of complement. Thus, complement cascade-targeted immunomodulation may provide new approaches against highly pathogenic coronavirus-induced inflammation, complement-related coagulopathy, and secondary bacterial infection-related diseases. Our findings provide a rationale for the clinical use of anti-MASP-2 antibodies and anti-C5a antibodies for COVID-19 treatment. Based on our primary data, 57 narsoplimab, an anti-MASP-2 antibody also known as OMS721 developed by Omeros Corporation, was first compassionately administered to critical COVID-19 patients. 22 The study found that all six patients requiring mechanical ventilation prior to treatment survived and were discharged from the hospital after the treatment, while substantial mortality rates of 33% and 53% were observed in the two control groups with similar entry criteria and baseline characteristics used for retrospective comparison. 22 Rapid and sustained improvement was observed across all assessed markers of cell damage and inflammation following narsoplimab treatment, including circulating endothelial cell (CEC) counts, interleukin-6 (IL-6), IL-8, C-reactive protein (CRP) and lactate dehydrogenase (LDH). 22 In another randomized, open label phase II clinical trial completed in the Netherlands by InflaRx (NCT04333420), an anti-C5a antibody (IFX-1, also known as vilobelimab 58 ) showed potential efficacy in critical COVID-19 patients, improved lymphocytopenia, and reduced lactate dehydrogenase concentrations. 58,59 Recently, a phase III clinical trial of vilobelimab showed a 43% reduction in 28-day all-cause mortality compared with placebo treatment in a prespecified subgroup Purified MASP-2-Flag was incubated with/without SARS-CoV N, MBL, and mannan at 37°C. Cleaved MASP-2 was probed with an anti-Flag antibody. c Recombinant MASP-2 and N protein were diluted in buffer and incubated with preconjugated MBL in mannan-coated plates. The binding of MASP-2 to MBL in plates was detected with an anti-MASP-2 antibody. The data are presented as the mean ± SD of three tests. Statistical analysis vs. HSA was performed using unpaired two-tailed Student's t-test. d Complement C4 was incubated with MASP-2, MBL, mannan, N proteins, C1INH, or an anti-MASP-2 antibody at 37°C for 1 h. C4 and the cleaved truncated C4 fragment were detected with an anti-C4alpha antibody. e Densitometric analysis of C4 cleavage (d) is shown. f Complement C4 was incubated with/without MASP-2, MBL, mannan, MERS-CoV N protein, or mutant N protein at 37°C for 1 h or 2 h. C4 cleavage was detected with an anti-C4alpha antibody. Data in a, b, d, and f are representative of two independent experiments. g The C4 cleavage rate in (f) was calculated by densitometric analysis with ImageJ software (n = 3) Highly pathogenic coronavirus N protein aggravates inflammation by. . . Gao et al. analysis of patients in Western Europe with more severe disease (https://www.inflarx.de/Home/Investors/Press-Releases/03-2022-InflaRx-Announces-Encouraging-Phase-III-Topline-Results-from-PANAMO-Trial-of-Vilobelimab-in-Severe-COVID-19-Patients.html). These data further support that N protein-potentiated complement overactivation plays important roles in the pathogenesis of SARS-CoV-2.
Our findings have uncovered the mechanism underlying the excessive complement activation induced by highly pathogenic coronavirus infection, which has largely contributed to aggravated Highly pathogenic coronavirus N protein aggravates inflammation by. . . Gao et al. inflammatory lung injury in severe COVID-19 patients. Either blocking the N protein:MASP-2 interaction or suppressing complement activation can significantly alleviate N proteininduced complement hyperactivation and lung injury in vitro and in vivo. Complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.
Purification of SARS-CoV and MERS-CoV N protein As previously described, 60 pET22b-SARS/MERS-CoV N was transformed into the E. coli expression strain BL21 (DE3). After induction with 1 mM IPTG for 8 h, the bacteria were harvested by centrifugation and resuspended in buffer A (25 mM Na 2 HPO 4 / NaH 2 PO 4 (pH 8.0), 1 mM EDTA, and 1 mM DTT) before sonication. Soluble N protein in the lysate was purified with ion-exchange chromatography with SP-Sepharose Fast Flow (25 mM Na 2 HPO 4 / NaH 2 PO 4 (pH 8.0), 1 mM EDTA, 1 mM DTT, and 0.35-0.5 M NaCl), followed by Superdex 200 gel filtration (GE Healthcare) and elution with buffer A. E. coli transformed with the vector pET22b was lysed as described above, and the eluate was used as a negative control for the purified N protein. Purified SARS-CoV-2 N-His was obtained from General Biosystems (Anhui) Co. Ltd.
Purification and renaturation of MASP-2 Recombinant protein expression and renaturation were performed as previously described. 61,62 In brief, pET22b-MASP-2 was transformed into the expression strain BL21 (DE3). After induction with 1 mM IPTG, the cells were harvested and sonicated. The inclusion bodies were solubilized in 6 M GuHCl, 0.1 M Tris-HCl (pH 8.3), and 100 mM DTT at room temperature; then, the solubilized proteins were diluted into refolding buffer containing 50 mM Tris-HCl, 3 mM reduced glutathione (Sigma), 1 mM oxidized glutathione (Sigma), 5 mM EDTA, and 0.5 M arginine at 4°C and incubated for 24 h. The renatured protein was dialyzed against 20 mM Tris-HCl pH 7.4 and 140 mM NaCl at 4°C, concentrated with PEG8000, aliquoted, and stored at −70°C.
To obtain high-activity MASP-2, Flag-tagged MASP-2 was expressed in 293T cells, precipitated with anti-FLAG magnetic beads in lysis buffer with 1 mM EDTA at 4°C for 2 h, eluted with Flag peptide (Sigma), and concentrated with a 10,000 MWCO ultrafiltration tube (Millipore). The concentration of MASP-2 was assessed using a BCA kit (Thermo), SDS-PAGE analysis and Fig. 4 N proteins activate the lectin complement pathway via MASP-2. a, b Vero E6 cells infected with or without SARS-CoV-2 (MOI = 1) or pseudotyped virus expressing SARS-CoV-2 S (HIV/SARS-CoV-2 S, MOI = 0.2) were treated with 20% diluent serum, OMS721, nCoV396, IgG4 or SARS-CoV-2 N protein. Then, the MASP-2 deposition was analyzed. The OD450 value of non-infection was set to 1 to calibrate the relative MASP-2 deposition level. The data are presented as the mean ± SEM. c-e C4b deposition correlates with the N protein concentration of SARS-CoV, HCoV-229E, SARS-CoV-2, and MERS-CoV. The OD450 value of the control at 0 nM was set to 1 to calibrate the relative C4b deposition level. The data are presented as the mean ± SD of three tests. The statistical analysis was performed using an unpaired two-tailed Student's ttest. f The effect of the SARS-CoV-2 N protein on C4b deposition in the presence or absence of mannan is regulated by anti-MBL antibodies, OMS721, and nCoV396. The OD450 value of SARS-2 N at 0 nM was set to 1 to calibrate the relative C4b deposition level. The data are presented as the mean ± SEM. g Activated C3 deposition was assessed in the presence of C1q-depleted serum (diluted in Ca 2+ -Mg 2+ buffer) with/without N protein, and activated C3 was detected by an activated C3 antibody. h C1q-depleted serum was diluted in Ca 2+ -Mg 2+ (LP+AP) or Mg 2+ -EGTA buffer (AP only) and incubated with the indicated concentration of N protein. Activated C3 was detected using an activated C3 antibody. i C5b-9 deposition in relation to the N protein concentration. The C5b-9 complex was detected with a C5b-9 antibody. Data in a-f are representative of three independent experiments Highly pathogenic coronavirus N protein aggravates inflammation by. . . Gao et al. immunoblot analysis with purified prokaryotically expressed MASP-2 as a standard control. a BALB/c mice were pre-infected with Ad-SARS N or Ad (1 × 10 9 PFU) on days 1, 2, and 3, and the SARS-CoV S protein or VSV-G pseudotyped HIV virus was injected on the 4th day via the tail vein. Sera were collected at 0 and 6 h post-pseudovirus injection, serum LTB4 was detected, and an increase in the serum LTB4 levels was observed. b Mice were infected with 1 × 10 8 PFU Ad-SARS N, and LPS was injected on the 7th day post-infection. The mice were sacrificed 6 h after the LPS challenge. SARS-CoV N and MASP-2 complex formation in frozen lung sections was measured by in situ PLA (red signals). Deposited C3 fragments were stained with a FITC-labeled anti-C3c antibody (green). Scale bar, 50 μm. c Frozen lung sections from the indicated mice in (b) were stained with an anti-C4alpha antibody. Data in a-c are representative of two independent experiments. d Paraformaldehyde-fixed lung tissues from COVID-19 postmortem autopsies were used for paraffin tissue sections and IHC staining with the indicated antibodies. Microphotography was carried out by an Olympus BX52 microscope under a 10× objective. Scale bar, 200 μm. Serum C3a (e) and C5a (f) levels in critical COVID-19 patients were analyzed by ELISA. The data are presented as the mean ± SEM. The statistical analysis was performed using an unpaired two-tailed Student's t-test
MASP-2 autoactivation and C4 cleavage assay
C4alpha' fragments, which are left by C4a release after C4 cleavage and can indicate C4 activation, and MASP-2 were detected by an immunoblot analysis with an anti-C4alpha antibody (Santa Cruz) or anti-Flag antibody (Sigma). Purified C4b (Calbiochem) containing C4alpha', but not C4a, was used to indicate the C4alpha' band position.
Complement deposition assay The C4b deposition assay was performed using a human MBL/ MASP-2 assay kit (Hycult Biotech). 63 In brief, C1q-depleted serum (Calbiochem, 1:100 dilution) or MBL-depleted serum (obtained by immunoprecipitation with an anti-MBL antibody from human serum (HS, Gemini)) was incubated in mannan-coated plates with high salt binding buffer (10 mM Tris-HCl, 10 mM CalCl 2 , 1 M NaCl, and 0.05% Triton X-100, pH 7.4) overnight at 4°C and removed by washing, and the MBL:MASP-2 complex was captured. Purified C4 in the kit and purified N protein were added and incubated at 37°C for 1.5 h, and the deposited C4b was detected following standard protocols. The functional activity of LP and AP was analyzed by ELISA as previously described. 32,63 Nunc Maxisorb plates were coated with 10 μg mannan per well in 100 mM Na 2 CO 3 /NaHCO 3 (pH 9.6) at room temperature overnight. After each step, the plates were washed three times with TBST-Ca 2+ (10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 0.05% Tween-20, and 5 mM CaCl 2 ). The residual binding sites were blocked by incubation with 10 mM Tris-HCl (pH 7.4), 140 mM NaCl, and 1 mg/ml HSA for 2-3 h at room temperature. Serum samples were diluted in 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 0.5 mM MgCl 2 , 0.05% Tween-20, and 0.1% gelatin with or without 2 mM CaCl 2 and N protein. All samples and buffers were prepared on ice. Then, the plates were sequentially incubated for 1 h at 4°C and 1.5 h at 37°C, followed by washing. All incubation volumes were 100 μl. Complement binding was detected using antibodies followed by washing. The detection of C4, activated C3, and C5b-9 was performed using an anti-C4α chain antibody (Santa Cruz), antiactivated C3 antibody (Santa Cruz), and anti-C5b-9 antibody (Calbiochem), respectively. Antibody binding was detected using an HRP-conjugated sheep anti-mouse antibody or donkey antirabbit antibody (R&D). The enzyme activity of HRP was detected Fig. 6 The administration of anti-MASP-2 antibodies recovered the abnormal inflammation in SARS-CoV-2-infected mice. a BALB/c mice (10/ group) were infected three times (days 1, 2, and 3) with 1 × 10 9 PFU Ad-SARS N, Ad or saline, and LPS was administered on the 7th day. Antibodies or C1INH were injected 30 min before LPS injection. The survival rate of the mice is presented, and the statistical analysis was performed using a log-rank test. b Lung paraffin sections from the indicated mice in (a) were analyzed by HE staining. Scale bar, 100 μm. c BALB/c mice were pre-infected with 1 × 10 9 PFU Ad-MERS N or Ad and treated with LPS, an anti-MASP-2 antibody or C1INH as mentioned above. The mouse survival curves were plotted, and the statistical analysis was performed using a log-rank test. d Masp2 −/− (KO) and Masp2 +/ + (WT) C57BL/6N mice were infected with N-expressing adenovirus or Ad and injected with LPS (10 mg/kg) as mentioned above. The mouse survival curves were plotted, and statistical analysis was performed using the log-rank test. e BALB/c mice pre-infected with 10 5 PFU mouseadapted SARS-CoV-2 (SARS-CoV-2 MA) were treated with saline or an anti-MASP-2 antibody (OMS721) or anti-MASP-2 antibody (nCOV396) as mentioned. Six days after infection, the lymphocyte percentage (Lym%) and neutrophil percentage (Neu%) in the mice were measured. Error bars, mean ± SEM. The statistical analysis was performed using an unpaired two-tailed Student's t-test. f Masp2 +/+ (WT) and Masp2 −/− (KO) C57BL/6N mice were infected with 10 5 PFU SARS-CoV-2 MA. Six days after infection, the Lym% and Neu% in the mice were measured. Error bars, mean ± SEM. The statistical analysis was performed using an unpaired two-tailed Student's t-test using TMB incubation for 30-60 min at RT, and the reaction was stopped with 2 M H 2 SO 4 . The OD was measured at 450 nm using a microplate reader.
Cell-ELISA The quantification of complement system activation was performed using a previously described method based on cell-ELISA technology, with modifications. 64 Briefly, Vero E6 cells were infected with or without SARS-CoV-2 (MOI = 1) or the SARS-CoV-2 S protein-pseudotyped virus (HIV/SARS-CoV-2 S, MOI = 0.2) for 48 h. Then, 20% diluent human serum (HS), anti-MASP-2 antibodies (OMS721 from Omeros prepared by Sino Biological), anti-N antibodies (nCoV396) or IgG4 antibodies (0.5 μg) and SARS-CoV-2 N protein (100 nM, only in HIV/SARS-CoV-2 S infection experiments) were added to each well, and the samples were incubated for 3 h at 37°C. Then, the cells were washed and fixed with 4% paraformaldehyde for 30 min. Subsequently, each well was washed and blocked with HSA (1 mg/ml) for 1 h, biotinconjugated anti-MASP-2 antibodies were added, and the deposition of MASP-2 was analyzed by ELISA.
MASP-2:MBL binding assay
The binding of MASP-2 to MBL was assessed by ELISA. As mentioned above, Nunc Maxisorb plates were coated with 10 μg mannan per well in 100 mM Na 2 CO 3 /NaHCO 3 (pH 9.6) at room temperature overnight and blocked with 1 mg/ml HSA. MBL protein (1 μg/ml) was incubated in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , 100 μg/ml HSA, and 0.5‰ Triton X-100 at 4°C for 2 h. Purified MASP-2 and N (or control) proteins were added to the wells at different times to obtain final concentrations of 0.2 mg/ml and 200 ng/ml, respectively. The plates were washed after 32 h of incubation at 4°C, and the binding of MASP-2 was detected with an anti-MASP-2 antibody followed by an HRPconjugated rabbit anti-goat antibody. The enzyme activity of HRP was detected using TMB incubation for 30-60 min at RT, and the reaction was stopped with 2 M H 2 SO 4 . The OD was measured at 450 nm using a microplate reader.
Opsonocytophagic assay The effect of the SARS-CoV N protein on opsonization by the products of complement C3, such as C3b and iC3b, was performed using a previously described method based on the opsonocytophagic assay, with modifications. 65,66 Briefly, mouse cells isolated from the peritoneal cavity were washed and inoculated with RPMI 1640 media (10% FBS) in 96-well plates for 2 h at 37°C. The serum was diluted by 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50, and 100% with 1×PBS, 1 mM CaCl 2 , and 2 mM MgCl 2 . The diluted serum, SARS-CoV N protein or HSA (100 ng/ml) and E. coli (the ratio to cells was 10:1) were added to each well, and the samples were incubated for 30 min at 37°C. The reaction was terminated by incubation with 4% paraformaldehyde for 30 min. Complement C3 deposition was detected with a FITC-C3c antibody (Abcam), and the stained cells were counted. The points represent the mean values of two repeated wells. Error bars, mean ± SD. Exact P-values are shown by an unpaired two-tailed Student's t-test between two groups.
Immunofluorescence and immunohistochemistry Postmortem autopsy of 4 patients who died in Huoshenshan Hospital was carried out by Dr. Xiuwu Bian under the approval of the hospital ethics committee and the family members of the patients. Paraformaldehyde-fixed lung tissues were used for paraffin tissue sections and immunohistochemical staining with SARS-CoV-2 N (Sino Biological Inc.), MBL (Santa Cruz), MASP-2 (Santa Cruz), C4α chain (Santa Cruz), C3 (Santa Cruz) or C5b-9 (Calbiochem) antibodies as described in the instruction manual. The in situ PLA of mouse and human lung tissue sections was performed using a Duolink™ In Situ PLA kit (Sigma).
Detection of C3a and C5a in COVID-19 patients Sera were collected from mild or critical COVID-19 patients under the approval of the hospital ethics committee. Critical patients who were in the ICU were defined as having fever or suspected respiratory infection plus one respiratory rate >30 breaths/min, severe respiratory distress, or SpO2 < 90% in room air. Patients with pneumonia and no signs of severe pneumonia were defined as mild cases. Sera collected from 10 mild patients, and 18 critical patients were assayed. Sera from 10 healthy people were collected from the clinical laboratory. The serum C3a and C5a levels were detected by double antibody sandwich ELISA (R&D Systems). The serum C3a in some samples was not tested because of insufficient sample volume.
Statistical analysis
The statistical analyses were performed using GraphPad Prism software version 8.4.0.
Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.
DATA AVAILABILITY
All the data shown in this paper are available from the corresponding authors upon reasonable request.
Highly pathogenic coronavirus N protein aggravates inflammation by. . . Gao et al. | 2022-09-14T13:40:33.185Z | 2022-09-14T00:00:00.000 | {
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245056431 | pes2o/s2orc | v3-fos-license | Transient ascending ST‐segment depression and widening of the S wave in 3‐channel Holter monitoring—A sign of dromotropic disturbance in the right ventricular outflow tract in the Brugada syndrome: A report of five cases
Abstract Background Brugada syndrome (BrS) is somewhat a challenging diagnosis, due to its dynamic pattern. One of the aspects of this disease is a significant conduction disorder located in the right ventricular outflow tract (RVOT), which can be explained as a consequence of low expression of Connexin‐43. This decreased conduction speed is responsible for the typical electrocardiographic pattern. Opposite leads located preferably in inferior leads of the electrocardiogram may show a deep and widened S wave associated with ascending ST segment depression. Holter monitoring electrocardiographic (ECG) aspects is still a new frontier of knowledge in BrS, especially in intermittent clinical presentations. Methods We describe, as an exploratory analysis, five case series of intermittent type 1 BrS to demonstrate the appearance of ascending ST segment depression and widening of the S wave, during 3‐channel 24h‐Holter monitoring (C1, C2 and C3) with bipolar leads. Results In the five cases described, the ST segment depression was observed mainly in C2, but in some cases also in C1 and C3. Only case 1 presented concomitant intermittent elevation of the ST segment in C1. All cases were intermittent. Conclusion The recognition of an ECG pattern with ascending ST‐segment depression and widening of the S wave in 3‐channel Holter described in this case series should raise a suspicion of the BrS and suggests the counterpart of a dromotropic disturbance registered in the RVOT and/or reciprocal changes.
| INTRODUC TI ON
is an inherited arrhythmia disorder characterized by an increased risk of unexplained syncope and/or sudden cardiac death (SCD), without macroscopic structural heart disease.
Currently, two electrocardiographic (ECG) patterns are recognized as follows: type 1 (coved) and type 2 (saddleback), although only the type 1 has a definitive diagnostic value, whether spontaneous or induced by sodium channel blockers. The type 1 is characterized by J-point ST-segment elevation ≥2 mm of superior convexity or rectilinear descent followed by a negative symmetrical T wave in at least one right precordial lead in conventional position or in the second or third intercostal space (ICS) (Priori et al., 2013).
Due to their dynamic nature, these ECG patterns can be transient or augmented by multiple factors such as precordial electrode positions, variation in autonomic tone or body temperature, or multiple drugs (Brugada et al., 2018).
ST-segment depression in the limb leads was already described as one of the ECG characteristics of BrS related to reciprocal changes or mirror image of the ST-segment elevation in the right precordial leads, and when transient in 3-channel Holter, these ECG changes may help to identify the intermittent type 1 Brugada pattern and a dromotropic disturbance in the right ventricular outflow tract (RVOT) (Crea et al., 2015).
| C A S E REP ORTS
In this series of cases, Holter with three channels and four electrodes placed on the chest was used, as shown in Figure 1a. According to the manufacturer, channel 1 (C1) corresponds to modified leads V1-2, channel 2 (C2) to modified V4-5 while channel 3 (C3) has no specific correspondence to ECG leads.
| Case 1
A 72-year-old man with brown skin color had no cardiovascular comorbidities or family history of sudden death. He underwent a colonoscopy procedure complicated by colon perforation. During surgical correction, he had an episode of atrial fibrillation detected in the rhythm monitor. At the end of the procedure, an ECG was performed and showed elevation of the ST segment in the right precordial leads (Figure 2c). He was transferred to our hospital, where he underwent coronary angiography that did not show a significant luminal reduction. An echocardiogram was performed, and no struc-
| Case 2
A 46-year-old male farmer with brown skin color was admitted to the hospital in September 2019, due to palpitation, acute precordial pain, and dizziness while he was working. He denied syncopal episodes, but his wife reported that 2 weeks before the admission, she found her husband unconscious on his bed with noisy breathing, drooling over, and with tonic movements. The initial ECG showed a could be induced. The event reported by the wife was interpreted as "nocturnal agonal breathing" secondary to a probable arrhythmic event, which was considered as an indication for an implantable cardioverter-defibrillator (ICD).
| Case 3
A 33-year-old man with brown skin color and no history of comorbidities was admitted to the emergency department in April 2016, with palpitations associated with dyspnea and precordial pain.
| Case 4
A 35-year-old man with brown skin color, referred to the hospital
| Case 5
A 64-year-old Caucasian woman was referred for exercise testing The patient has undergone follow-up without invasive procedures.
| DISCUSS ION
In the five cases described, the ST-segment depression was observed mainly in C2, but in some cases also in C1 and C3. Only case 1 presented concomitant intermittent elevation of the ST segment in C1. All cases were intermittent. They were preceded by a broadbased S wave with a slowly ascending morphology in a straight or slightly concave line, followed by a positive T wave, resulting in QRS broadening-it was difficult to determine the end of the QRS and the beginning of the T wave. In Figure 2b, e, h, k, and n we can see a ramp signal, represented by a straight line between the nadir of the S wave and the peak of the T wave representing the loss of transition between the QRS and the T wave. Bipolar leads used in the chest were directed downwards and/ or to the left (Figure 1a), resulting, particularly in C2, in a vector that opposes the vector of RVOT (Figure 4). The widening of the S wave, occurring mainly in periods of lower heart rate, can also be explained by a dromotropic disturbance registered in RVOT, due to interstitial fibrosis and reduced gap junction expression (connexin-43).
In line with our Holter findings, a wide S wave in lead I in the ECG has previously been described as a risk marker for ventricular fibrillation and/or sudden cardiac death in the BrS (Calò et al., 2016).
The foundation of this ECG finding is similar to that of the aVR sign (R (Crea et al., 2015).
An important characteristic of the BrS is the dynamic nature of the ECG manifestations. Strategies, such as placing electrodes superiorly in the second or third ICSs, seem to increase the sensitivity of the diagnosis (Nakazawa et al., 2004). The pattern is related to the autonomic tone and is more prevalent in periods of vagal predominance, such as during sleep or in the postprandial period. Fever can unmask the type 1 pattern and is associated with the risk of triggering an arrhythmic event (Brugada et al., 2018). The appearance or worsening of the pattern in the recovery phase of an exercise test can be a diagnostic tool and even correlate with a worse arrhythmic prognosis (Makimoto et al., 2010). Vector IV (final ventricular depolarization located in the RVOT area) pointing to the right shoulder near aVR (−150°) and concomitantly moving away from channel 2 (near +60°). Note a deep/broad S wave in the C2/II lead, and an opposite pattern in aVR they can present with an ascending ST-segment depression and wid- | 2021-12-11T06:16:18.881Z | 2021-12-09T00:00:00.000 | {
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53373496 | pes2o/s2orc | v3-fos-license | Determination of Diabetic Retinopathy Using Fractals
: The intention of this paper is to determine the retinal blood vessel system of diabetics. The retinal blood vessel system has been determined using Reynold’s number and Poiseuille’s law. A new algorithm has been established to traverse through the retinal blood vessel system in a robust manner.
Introduction
In the recent articles fractal model for the blood vessels in different parts of human body has been analysed where as this exertion deals with fractal retinal blood vessel trees. The retinal blood vessels have been modeled using fractals. The pressure drop has been found out according to Poiseuille"s law [13].
Fractals
A Fractal is an object which appears self-similar under varying degrees of magnification. Self-similarity is the major characteristic of the fractal objects [7]. Recent studies have attempted with some success to characterize certain parts of the body using fractal geometry where the retinal blood vessel system exhibits fractal characteristic. There are many definitions for fractal, among them a) "A fractal is a shape made of parts similar to the whole in some way". b) "A fractal is by definition for which the Hausdorff dimension strictly exceeds the Topological dimension" Mandelbrot (1975) introduced the term "FRACTAL" to characterize spatial or temporal phenomena that are continuous but not differentiable. Fractal objects and processes are therefore said to display "Self-invariant" (Selfsimilar or Self-affine) properties [5]. Fractal structures do not have a single length scale, while fractal processes (timeseries) cannot be characterized by a single-time scale [10].
Retina
The retina is a thin layer of tissue that lines the back of the eye on the inside. It is located near the optic nerve. The purpose of the retina is to receive light that the lens has focused, convert the light into neural signals, and send these signals on to the brain for visual recognition. The retina processes light through a layer of photoreceptor cells. These are essentially light-sensitive cells, responsible for detecting qualities such as color and light-intensity. The retina processes the information gathered by the photoreceptor cells and sends this information to the brain via the optic nerve. (Fig-1)
Diabetic Retinopathy
Diabetic retinopathy occurs as a result of high blood sugar, and can cause blindness if left untreated.Diabetic retinopathy is an eye condition that affects people with diabetes who have high blood glucose, or blood sugar, over a prolonged period of time. Too much blood sugar can damage the blood vessels in the back of the eye, preventing the retina from receiving the proper amount of nutrients it needs to maintain vision.
The retina is light-sensitive nerve tissue at the back of the eye. As light enters the front of the eye, the retina converts the light rays into electrical impulses that travel along the optic nerve to part of the brain called the visual cortex. The brain then combines images sent from both eyes to interpret them as a single, three-dimensional image. This allows us to perceive depth and distance. Without the retina, the eye cannot communicate with the brain, making vision impossible. Diabetic retinopathy occurs when diabetes damages the tiny blood vessels in the retina. In the early stages of diabetic retinopathy, called non-proliferative retinopathy, these blood vessels leak fluid and distort sight.
In the more advanced stage of diabetic retinopathy, called proliferative retinopathy, fragile new blood vessels grow around the retina and in the vitreous humour.
Fractal model for retinal blood vessels
Fractal model of blood vessel system is a certain geometrical simplification but it suffices for acceptable blood flow analysis [4]. This analysis permits understanding influence of hemodynamic forces. [8]. In real blood vessels system, vessel walls are elastic and change its diameters [6]. In this way resistance of blood vessel system is regulated. This process is known as autoregulation and corrects nutrition of all cells in human body [9][10][11].
Methods
Blood flow in the retinal blood vessel system has been mathematically calculated by the following methods. These results are very useful to investigate the defects in the above system.
Rate of flow (Q)
It is defined as the quantity of the fluid flowing per second through a section of a pipe or a channel. For an incompressible fluid the rate of flow is expressed as the volume of fluid flowing across the section per second. Mathematically the rate of flow is defined as where Q is the Rate of blood flow (ml/sec), A is the crosssecional area (cm 2 ), V is the velocity of the blood.
Poiseuille's law
The quantity of blood that flows through a vessel in a given period of time i.e. per unit of time, is equal to the velocity of flow times the cross sectional area according to the following equations: Viscosity of the Blood (Poises), L is the Length of the Vessel (Cms). The equation (2) is known as Hagen-Poiseuille"s equation. This equation shows that the rate of blood flow is directly proportional to the fourth power of the radius of the vessel and also illustrates that the diameter of a vessel plays a great role in determining the rate of blood flow.The most effective factor controlling blood flow is radius of the blood vessel. High blood pressure can be caused by narrowing blood vessel and is reduced by relaxing the smooth muscle tension that controls the blood vessel radius. This process is known as an auto regulation.
Pressure Drop
Pressure drop is a positive function depending on the blood flow, which passes through the different parts of the retinal blood vessel system. Pressure drop have been calculated according to Poiseuille"s law, the pressure drop P for rate of flow Q through a blood vessel with radius a over a length L is
Reynold's number
When normal laminar blood flow becomes turbulent flow The rate of blood flow (i.e) the velocity of flow, is high It passes by an obstruction in a vessel (as in case of compression by cuff of Sphygmomanometer). It makes a sharp turn. It passes over a rough surface Turbulence is also related to the diameter of the vessel and the viscosity of the blood which can be expressed by ratio of inertia to viscous forces.
where e R is the Reynold"s number, L is the length of the blood vessel, V is the Average velocity and is the Viscosity of the blood. This is the most important dimensionless number; it describes the fluid flow regime. If e R is higher, there is a greater probability of the turbulence (Table2). The above factors has been analysed for diabetic patient and is given in the Table 2.
Algorithm
The algorithm traverses through the blood vessels of the retina in a recursive manner Name: Flood Fill algorithm Step 1 − Initialize the value of seed point (seed x, seed y), fvessel and dvessel.
Step 2 − Define the boundary values of the retina.
Step 3 − Check if the current seed point is of dvessel, then repeat the steps 4 and 5 till the boundary is reached.
Step 4 − Change the default vessel with the fvessel at the seed point.
Numerical Simulation Results
The human retinal blood vessel system features a tremendous variety of components with different geometrical dimensions and mechanical properties. A synthetic view is given in the following Table.1.
Conclusion
The blood vessels have been numerically calculated for the parameters such as area of the cross section, rate of flow, Pressure drop and Reynold"s number for a diabetic patient, in our proposed system . Reynold"s number provides us the accurate result of the affected blood vessels of the retinal blood vessel system in an effectual conduct. This numerical simulation helps us in understanding the affected blood vessels of the retina. The recursive strategy is a useful tool to go through the system in an effective manner. | 2018-10-14T16:20:48.077Z | 2016-01-01T00:00:00.000 | {
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199006996 | pes2o/s2orc | v3-fos-license | Design and Implementation of Water Saving Irrigation System Based on Zigbee Sensor Network
This paper divides the water-saving irrigation system into five component modules: acquisition node module, control node module, aggregation node module, routing node module and host computer management software module. Design and analysis are carried out for each module. Finally, the design of the system is tested, mainly including sensor sensitivity test, reliable control of control equipment and the influence of weather conditions on system operation. This water-saving irrigation system is of great significance for saving water resources and improving the degree of automation control. In the longer term, it has improved the degree of mechanization of agriculture and realized the high efficiency of planting.
Introduction
In recent years, precision agriculture and the improvement of agricultural automation are the development trend of modern agriculture. Harvesters and rice transplanters in farmland have replaced the labor methods that people used to cultivate. Nowadays, it is not uncommon to use wireless sensors to measure crop ecological environment. To design a water-saving irrigation system, we must first start with its overall structure, analyze the overall design of the system from hardware and software, and select the appropriate chip and other hardware according to technical indicators, expected functions and design purposes.
Principle of water saving irrigation system
The system mainly uses the soil temperature and humidity sensor to detect the soil entropy information in the crop growth environment. By arranging a large number of micro-sensors in the field, according to the different demand for water in different growth stages of crops, watering is implemented by manual control or computer intelligent control of the switching state of the control device. The main control part is shown in Fig. 1.
Intelligent irrigation is reflected in the combination of software and hardware, as well as the temperature and humidity of the soil at that time, using reasonable watering methods, abandoning previous planting patterns relying on experience, furrow irrigation or flood irrigation, using wireless measurement and control methods to detect whether plants are in a state of water shortage, Rational use of natural precipitation or ditch water for irrigation. This will not only enable the plant to maintain a good growth state, increase the yield per mu, obtain the best economic benefits, but also reduce manpower waste and reduce production costs. Figure 1. System function chart The system is divided into three parts. The control node and the acquisition node are the terminals of the system. The control node is connected to the control device to control the working condition of the pump. Each collection node is connected with a sensor. The function is to collect the entropy information of the soil and transmit the information. To the data processing center, the PC is composed of a data storage module and a display processing module. The display module mainly displays the current temperature and humidity values of each terminal and the working state information of the control device.
Water saving irrigation system hardware solution
Considering the objective conditions of field planting, this paper designs a ZigBee-based intelligent irrigation system, which consists of two parts: the upper computer monitoring center and the monitored irrigation area. The coordinator is the aggregation node, and the router is responsible for controlling the node network. Joining, the collection node is responsible for the monitoring of soil temperature and humidity, and the control node is responsible for controlling the operation of the control equipment. The system mainly realizes the following functions: a large number of terminals are arranged in the irrigation area, and the sensors connected by the collection nodes detect the soil entropy information and the working state information of the control equipment at intervals. Because it is planted in the field, the plot is widely distributed and scattered, and the effective transmission distance of the ZigBee network is about 75 meters. Therefore, the routing node is added in the system. For the stable operation of the system and large-area data transmission, the mesh topology is adopted. The structure of the network is set up, the collection node can join the network in a multi-hop manner, and the information is processed and transmitted to the aggregation node. The RS-232 interface is used between the aggregation node and the upper computer for wired data transmission; the upper computer has three component modules. The temperature and humidity information display module, the soil humidity threshold setting module and the historical database module can see the real-time soil temperature and humidity value and the control device working state information in the display screen of the upper computer monitoring center, and compare with the preset threshold value, if Below the threshold, the host computer sends instructions to the control node through the sink node, so that the control device enters the working state. According to different plant demand for water in different periods, the threshold value can be flexibly set to keep the plant in good growth state; the machine will make the information of each transmission into a historical number. Library for users to understand the last few days of soil information and whether the system is working properly. The overall structure design of the system is shown in Figure 2: The hardware design of this paper is mainly the aggregation node, the collection node and the control node. The aggregation node is responsible for the "bridge" of information exchange between the upper computer and the collection node, and maintains the ZigBee network; the collection node is responsible for collecting soil information and transmitting it to the upper computer for control. The node is responsible for controlling the operation of the device and executing the instructions sent by the host computer. a). Aggregation node hardware The aggregation node mainly refers to the ZigBee coordinator, which is responsible for maintaining the ZigBee network, performing bidirectional transmission with the host computer and the acquisition control node, and displaying the soil entropy information of the acquisition control node, since it is constantly receiving the Information exchange, so a stable power supply is required, and its structural block diagram is shown in Figure 3: . Acquisition node hardware The collection node is responsible for the collection of soil temperature and humidity information in the system. When no information is collected, it enters the sleep mode, so its power consumption is small, and the dry battery is used as the power supply. The block diagram of the composition is shown in Figure 4: The control node is composed of relay and irrigation equipment. The switch state of the relay determines the working condition of the irrigation equipment. Because the working energy of the relay is large, the normal power supply is adopted. At the same time, the relay can also automatically change the switch state according to the threshold change of the upper computer. The composition block diagram is shown in Figure 5:
Water saving irrigation system software program
The water-saving irrigation system designed in this paper is based on the information of soil temperature and humidity detected by the terminal, and makes corresponding judgments to realize intelligent control. The whole system is divided into communication protocol module, hardware control module and upper computer management software module. The overall design of system software is shown in Figure 6: The design of the protocol stack is the most basic design of the system. The establishment of ZigBee network, data transmission and reception, and the joining of terminal nodes are all based on this. On top of this, it is a hardware control module, which is mainly used to control the soil of the acquisition control node. Information detection, relay status detection, etc., the host computer provides a flexible user visual control interface to achieve remote supervision, the role of the communication protocol is to ensure the safe and reliable transmission of information.
Temperature and humidity sensor sensitivity test
The system uses the lawn of the school as a test site. The lawn is planted with various trees and flowers, and the water content of the soil is different. The sensors carried by the terminal should be 5 arranged next to different plants. It is vast, and because the soil quality of each piece is different, it is just that the accuracy of the test system is affected by the change of soil quality.
The function of the sensor is to detect the real-time temperature and humidity information of the soil. If the sensor is not sensitive enough, it may not perceive subtle changes in the surrounding area. Therefore, the sensitivity of the sensor needs to be tested. Figure 7 shows the temperature and humidity information under normal conditions: In this paper, the sensor of terminal 3 is breathed and tested whether it will change. As a result, as shown in Fig. 8, it can be clearly seen that the humidity value of terminal three has changed due to the water molecules in the exhaled gas. In order to make the final measured humidity value larger, the DHT11 soil temperature and humidity detector has a very sensitive response.
System accessibility test
The terminal nodes of this system act on the field. Due to objective reasons, it is inevitable to withstand wind and rain, even lightning strikes, sun exposure, etc. are normal weather changes. After experimental observation, you can know when the weather is compared. In the worst case, the performance of the system will be affected, and the stability is not as stable as during normal operation. Since the wireless communication network is also interfered by electromagnetic waves, this is also inevitable. This is a shortage of this intelligent irrigation system. In the future work, I will continue to study to enhance its anti-jamming performance.
The barrier-free operation test is to verify the stability of the entire system. The whole system is turned on in the test field for one month, and the test system components can operate normally according to requirements, such as the battery life time, whether the relay can start and stop normally, whether the sensor can collect data normally, whether the host computer can send the corresponding As can be seen from the above table, the system is stable in operation, and the monitoring of soil moisture is instantaneous and accurate, and the soil water content can be stabilized within a range.
By testing the integrity of the system, from the sensitivity of the sensor, the impact of the weather on the operation of the system and the system's accessibility time, the test results show that the sensor can also make sensitive response according to changes in the external environment; From the longterm system operation results, the system can reliably monitor the soil in the monitoring area, and the running time is long, so that timely and accurate detection can be achieved to achieve the expected goal.
Summary
This paper mainly completes the ZigBee-based intelligent water-saving irrigation system, and transmits the soil entropy information to the user through the sensor, and the user sends corresponding instructions to the terminal for execution. The system is divided into five parts: the collection node, the control node, the aggregation node, the routing node, and the upper computer monitoring center. | 2019-08-02T11:23:40.739Z | 2019-07-09T00:00:00.000 | {
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17419083 | pes2o/s2orc | v3-fos-license | PET Imaging with [18F]FSPG Evidences the Role of System xc- on Brain Inflammation Following Cerebral Ischemia in Rats
In vivo Positron Emission Tomography (PET) imaging of the cystine-glutamate antiporter (system xc-) activity with [18F]FSPG is meant to be an attractive tool for the diagnosis and therapy evaluation of brain diseases. However, the role of system xc- in cerebral ischemia and its involvement in inflammatory reaction has been scarcely explored. In this work, we report the longitudinal investigation of the neuroinflammatory process following transient middle cerebral artery occlusion (MCAO) in rats using PET with [18F]FSPG and the translocator protein (TSPO) ligand [18F]DPA-714. In the ischemic territory, [18F]FSPG showed a progressive binding increase that peaked at days 3 to 7 and was followed by a progressive decrease from days 14 to 28 after reperfusion. In contrast, [18F]DPA-714 evidenced maximum binding uptake values over day 7 after reperfusion. Ex vivo immnunohistochemistry confirmed the up-regulation of system xc- in microglial cells and marginally in astrocytes. Inhibition of system xc- with sulfasalazine and S-4-CPG resulted in increased arginase (anti-inflammatory M2 marker) expression at day 7 after ischemia, together with a decrease in TSPO and microglial M1 proinflammatory markers (CCL2, TNF and iNOS) expression. Taken together, these results suggest that system xc- plays a key role in the inflammatory reaction underlying experimental stroke.
Introduction
Cystine-glutamate antiporter (system xc -) is a heterodimer composed by 4F2hc and xCT that mediates the cellular import of cystine used in glutathione production and oxidative protection in exchange for export of glutamate [1][2][3]. Indeed, the need for oxidative protection in the pathologic brain induces extrasynaptic glutamate release through system xccontributing to neuronal damage [4].
System xcis expressed in a wide variety of cells both outside and inside the central nervous system (CNS) including immature cortical neurons [5], astrocytes [6] and microglia [7][8][9]. The activation of microglia from a resting state and migration into injured brain areas has not only beneficial neuroprotective and neurothrophic effects, but also a detrimental action through excitotoxic glutamate release [7,[10][11][12][13][14].
Ivyspring International Publisher
Likewise, the involvement of microglia on excitotoxic stress has been linked to a wide variety of neurodegerative diseases such as Alzheimer's disease [14], amyotrophic lateral sclerosis [8], glioma-derived epileptic seizures [15] and multiple sclerosis [9,16,17]. Therefore, in vivo imaging of system xcwith positron emission tomography (PET) technique might be crucial to further understanding the role of these receptors on inflammation underlying brain diseases. BAY 94-9392 ((4S)-4-(3-[ 18 F]fluoropropyl)-L-glutamate (herein referred to as [ 18 F]FSPG), a fluorine-18-labeled L-glutamate derivative, is taken up by system xcdue to the lack of discrimination between its natural substrate cystine and glutamate for the inward transport [18,19]. [ 18 F]FSPG has shown to be a promising radiotracer to image the amino acid antiporter system xcin patients with cancer [20,21] and infectious lesions [22]. In this sense, the PET imaging of amino acid transporters with [ 18 F]BAAs (boramino acids) have also showed high tumor-specific accumulation, suggesting that PET imaging of amino acid transporters are promising targeted radiotracers for molecular imaging in oncology and mental disorders [23]. Within the CNS, [ 18 F]FSPG has been used to explore cystine-glutamate antiporter activity after early acute cerebral ischemia [4] and multiple sclerosis [24] in rats. Recently, we have demonstrated with PET imaging that the system xcwas overexpressed in microglial cells in those areas more severely affected in experimental autoimmune encephalomyelitis (EAE) in rats [24], suggesting the potential of this radioligand to monitor neuroinflammatory damage. Despite this, the use of [ 18 F]FSPG to evaluate the contribution of system xcon the neuroinflammatory response following cerebral ischemia has not been explored before. The purpose of the present study was to investigate the levels of system xcin the rat brain after cerebral ischemia using [ 18 F]FSPG-PET and immunohistochemistry. In particular, we were interested in clarifying the relationship of the system xcactivity with the activation of glial cells after cerebral ischemia in rats. For this reason, ischemic rats were subjected to PET studies with [ 18 F]DPA-714, a specific radioligand for the translocator protein (18kDa) (TSPO), to image brain inflammation [25,26]. Likewise, [ 18 F]DPA-714 binding and RT-PCR were used to evaluate the effect of pharmacological inhibition of system xc -(SAS and S-4-CPG) on the neuroinflammatory reaction after ischemic stroke in rats. The results reported here provide novel information about the activity of cystine/glutamate antiporter in microglial cells after cerebral ischemia and might ultimately contribute to a better design of anti-inflammatory strategies for the treatment of ischemic stroke.
Cerebral ischemia and treatment
Adult male Sprague-Dawley rats (300 g body weight; Janvier, France) (n=52) were used. Animal studies were approved by the animal ethics committee of CIC biomaGUNE and local authorities and were conducted in accordance with the Directives of the European Union on animal ethics and welfare. Transient focal ischemia was produced by a 90 minutes intraluminal occlusion of the middle cerebral artery (MCA) followed by reperfusion as described elsewhere [27]. Briefly, rats were anaesthetized with 4% isofluorane in 100 % O2 and a 2.6-cm length of 4-0 monofilament nylon suture was introduced into the right external carotid artery up to the level where the MCA branches out. Animals were then sutured and placed in their cages with free access to water and food. After 90 minutes, the animals were re-anesthetized and the filament was removed to allow reperfusion. Twelve rats were repeatedly examined before (day 0) and at 1, 3, 7, 14, 21 and 28 days after ischemia to evaluate the temporal PET binding of both xcsystem and TSPO. The animals studied at day 0 have been considered as the baseline control group.
Two groups of seven and eight rats, respectively, were inoculated daily for a total of seven days from one hour following MCAO with 0.1 mL Sulfasalazine (SAS) (8 mg/Kg, i.p.) or 0.1 mL (S)-4-Carboxyphenylglycine (S-4-CPG) (1 mg/Kg, i.p.). A control ischemic group of five rats received daily the same volume of vehicle (normal saline). At day seven, treated and control rats were imaged with PET to determine the effect of both SAS and S-4-CPG on the TSPO expression. Sixteen rats were used to perform ex vivo studies (immunohistochemistry) for xcsystem at 0, 3, 7 and 28 days after cerebral ischemia. Rats used in imaging studies for the assessment of the effect of the treatments were used to perform immunohistochemistry for TSPO (n=20). Therefore, MCAO was induced to forty-four rats.
Magnetic resonance imaging
T2-weighted (T 2 W) MRI scans were performed in ischemic animals at 24 hours after reperfusion to select the rats (n=12) presenting cortico-striatal lesions to be included in the PET studies. Before the scans, anesthesia was induced with 4% isofluorane and maintained by 2-2.5% of isofluorane in 100% O2 during the scan. Animals were placed into a rat holder compatible with the MRI acquisition systems and maintained normothermic using a water-based heating blanket at 37ºC. MRI experiments were performed on a 7 Tesla Bruker Biospec 70/30 MRI system (Bruker Biospin GmbH, Ettlingen, Germany), interfaced to an AVANCE III console. The BGA12-S imaging gradient (maximum gradient strength 400 mT/m switchable within 80 µs), an 82 mm inner diameter quadrature volume resonator for transition and surface rat brain coil for reception were used. T2W images were acquired with a RARE sequence with the following parameters: RARE factor 2, TR/TE = 4400/40 ms, FOV = 25 mm x 25 mm, ACQ Matrix =256 x 256, Slice Thickness = 1 mm, 2 averages and 24 contiguous slices. Contiguous slices covering all the infarcted volume were acquired and fat suppression was used.
Magnetic resonance imaging image analysis
MRI (T 2 W) images were used to calculate the lesion volume. Regions of Interest (ROIs) were manually defined using the Open Source software 3D Slicer image analysis software (Version 3.6.3 www.slicer.org) for each rat on the region of increased signal in the ipsilateral hemisphere. The total lesion volume was calculated by summing the area of the infarcted regions of all slices affected by the lesion.
Radiochemistry
Fluorine-18 production: Fluorine-18 (T 1/2 = 109.8 min) as [ 18 F]fluoride was cyclotron-produced (Cyclone-18/9, IBA, Louvain-la-Neuve, Belgium) by proton irradiation of an 18 was performed using a TRACERlab FX FN synthesis module (GE Healthcare), based on a two-step procedure, as previously described [18] with minor modification: i) 18 F-fluorination of the appropriately protected precursor for labeling (di-tert-butyl (2S,4S)-2-tert-butoxycarbonylamino-4-(3-nitrophenyls ulfonyloxy-propyl)-pentanedioate), followed by ii) removal of the protective groups. Briefly, once transferred into a dedicated (ventilated and lead-shielded) hot-cell, [ 18 F]fluoride was first trapped on a pre-conditioned Sep-Pak® Accell Plus QMA Light cartridge (Waters, Milford, MA, USA), then eluted from the cartridge with a solution of Kryptofix K2.2.2/K 2 CO 3 in a mixture of water and acetonitrile. A solution of the precursor for labeling (5 mg) in acetonitrile (1 mL) was then added and the mixture heated at 80 º C for 10 minutes. The crude reaction mixture was then diluted with a solution of acetonitrile and water (1/1, 2 mL) and purified by high performance liquid chromatography (HPLC) using a Nucleosil 100-7 C18 column (Macherey-Nagel, Düren, Germany) as stationary phase and water/acetonitrile (30/70) as the mobile phase at a flow rate of 8 mL/min. The desired fraction (11-13 min) was collected and diluted with water (50 mL). The labeled specie was retained on a C-18 cartridge (Sep-Pak® Light, Waters, Milford, MA, USA) and further eluted with ethanol (1 mL). This solution was treated with 0.5 ml of 4M HCl (500µL); after hydrolysis, the solution was neutralized with 2N NaOH (650 µL) and phosphate buffer solution (PBS, pH=7.4, 3 mL). After filtration through a 0.22 μm filter, the radiotracer was ready for injection. Radiochemical yields (decay-corrected) were in the range 30-35% and radiochemical purity was always >93% at the time of injection.
Positron emission tomography scans and data acquisition
PET scans were repeatedly performed before (day 0) and at 1, 3, 7, 14, 21 and 28 days after reperfusion using a General Electric eXplore Vista CT camera (GE Healthcare). Scans were performed in rats anaesthetized with 4% isofluorane and maintained by 2-2.5% of isofluorane in 100% O 2 . The tail vein was catheterized with a 24-gauge catheter for intravenous administration of the radiotracer. Animals were placed into a rat holder compatible with the PET acquisition system and maintained normothermic using a water-based heating blanket. Animals were subjected to PET scans to assess system xc -([ 18 F]FSPG) and TSPO binding ([ 18 F]DPA-714) at each time point before and after ischemia onset. The appropriate radiotracer ([ 18 F]FSPG or [ 18 F]DPA-714, ~70 MBq) was injected concomitantly with the start of the PET acquisition and dynamic brain images were acquired (24 frames: 1x5, 1x15, 3x30, 5x60, 5x120, 3x180, 6x300 seconds) in the 400-700 keV energetic window, with a total acquisition time of 57.5 minutes. For evaluation of SAS and S-4-CPG treatment efficacy after ischemia, ~70 MBq of [ 18 F]DPA-714 were injected concomitantly with the start of the PET acquisition and brain dynamic images were acquired for a total of 30 minutes (23 frames 3x5, 3x15, 4x30, 4x60, 4x120, 5x180 seconds). After each PET scan, CT acquisitions were also performed (140 µA intensity, 40 kV voltage), providing anatomical information of each animal as well as the attenuation map for the later image reconstruction.
Dynamic acquisitions were reconstructed (decay and CT-based attenuation corrected) with filtered back projection (FBP) using a Ramp filter with a cutoff frequency of 0.5 mm -1 .
Positron emission tomography image analysis
PET images were analyzed using PMOD image analysis software (PMOD Technologies Ltd, Zürich, Switzerland). To verify the anatomical location of the signal, PET images were co-registered to the anatomical data of a MRI rat brain template. Two type of Volumes of Interest (VOIs) were established as follows: (i) A first set of VOIs was defined to study the whole brain [ 18 F]FSPG and [ 18 F]DPA-714 PET signal over time. Whole brain VOIs were manually drawn in both the entire ipsilateral and contralateral hemispheres containing the territory irrigated by the middle cerebral artery on slices of a MRI (T 2 W) rat brain template from the PMOD software. (ii) A second set of VOIs was automatically generated in the cortex and the striatum by using the regions proposed by the PMOD rat brain template, to study the evolution of PET signal in these specific regions in both the ipsilateral and contralateral cerebral hemispheres. The last four frames in steady state were used to calculate the summed PET binding uptake during the last 15 minutes of acquisition for both radiotracers. PET signal uptake was averaged in each ROI and expressed as percentage of injected dose per cubic centimetre (%ID/cc).
Immunohistochemistry and cell counts
Immunohistochemistry staining was performed at (i) control (day 0), day 3, day 7 and day 28 after reperfusion and (ii) at day 7 after ischemia in animals treated with SAS and S-4-CPG and non-treated rats. Animals were terminally anesthetized and killed by decapitation. The brain was removed, frozen and cut in 5-μm-thick sections in a cryostat. Sections were fixed in acetone (−20°C) during 2 minutes, washed with phosphate-buffered saline (PBS) and saturated with a solution of bovine serum albumine (BSA) 5%/Tween 0.5% in PBS during 15 minutes at room temperature, and incubated during 1 hour at room temperature with primary antibodies BSA (5%)/Tween (0.5%) in PBS. The first set of sections were stained for xCT with rabbit anti-rat xCT (1:100, AbCam, Cambridge, UK), for CD11b with mouse anti-rat CD11b (1:300; Serotec, Raleigh, NC, USA) and for the glial fibrillary acidic protein (GFAP) with chicken anti-rat GFAP (1:500; AbCam, Cambridge, UK). The second set of sections was stained for TSPO with a rabbit anti-rat TSPO (NP155, 1:1000), for CD11b and for GFAP. Sections were washed (3 × 10 minutes) in PBS and incubated for 1 hour at room temperature with secondary antibodies Alexa Fluor 350 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-chicken IgG (Molecular Probes, Life Technologies, Madrid, Spain, 1:1,000) in BSA 5%/Tween 0.5% in PBS, washed again (3 × 10 minutes) in PBS, and mounted with a prolong antifade kit in slices (Molecular Probes Life Technologies, Madrid). Standardized images acquisition was performed with an Axio Observer Z1 (Zeiss, Le Pecq, France) equipped with a motorized stage. The number of microglial and astrocytic cells immunopositive for xCT and TSPO within the ischemic area was assessed at 0, 3, 7 and 28 days after ischemia (xCT + /CD11b + and xCT + /GFAP + ) and at day 7 to evaluate the inflammatory effect of xcsystem inhibition (TSPO + /CD11b + and TSPO + /GFAP + ). Cells were manually counted in ten representative and different fields at 100x magnification by using Image J (NIH) software.
Real-time quantitative PCR (qPCR)
Frozen tissue was cut into 40 µm slices on a cryostat and a pool of 3-4 different slices of ipsilateral or contralateral hemispheres was used per each condition. Total RNA was isolated from tissue using Trizol (Invitrogen) according to the manufacturer's instructions. Subsequently, cDNA synthesis was conducted using SuperScript III retrotranscriptase (200 U/µL; Invitrogen) and random hexamers as primers (Promega). Primers specific for the rat M1 and M2 markers were designed using Primer Express software (Applied Biosystems) at exon junctions to avoid genomic DNA amplification (see Table 1). Data was normalized to HPRT gene (best M coefficient using GeNorm Software v3.5; Vandesompele et al., 2002). Real-time quantitative PCR reactions were performed with SYBR-Green using a BioRad CFX96 Real-time PCR detection system as described previously [30].
Statistical analyses
For PET signal values, the statistical analysis was performed as follows: The percentage of injected dose per cubic centimeter (%ID/cc) for each animal, brain hemisphere (ipsilateral and contralateral) and region (cortex and striatum) were calculated at each time point after cerebral ischemia. Values of %ID/cc within each region and time point following cerebral ischemia and after treatment were averaged and compared with the averaged baseline control values (day 0) and non-treated ischemic rats respectively using one-way ANOVA followed by Dunnet's multiple-comparison tests for post-hoc analysis.
Likewise, cellular expression of both microglial/xCT and astrocytic/xCT receptors before (day 0) and at days 3, 7 and 28 after ischemia and microglial/TSPO and astrocytic/TSPO after treatment were compared using the same statistical analysis than that for PET imaging. Neurological outcome comparisons were performed as follows: animals were subjected to the 9-neuroscore test before MCAO and at 1, 3, 7, 14, 21 and 28 days after cerebral ischemia. The results within each time point and after treatment were averaged and compared with baseline average values using Mann-Whitney U-tests. The effect of treatments in the mRNA levels for M1/M2 microglial phenotypes in sham, treated and non-treated ischemic rats were compared using a one-way ANOVA followed by Bonferroni's multiple-comparison tests for post hoc analysis. Finally, the level of arginase mRNA in treated ischemic rats was compared with non-treated ischemic rats using an unpaired t-test. The level of significance was regularly set at P<0.05. Statistical analyses were performed with GraphPad Prism version 6 software.
Results
Hyperintensities of T 2 W images showed similar infarct extents as well as locations affected. All ischemic rats subjected to nuclear studies showed cortical and striatal MRI alterations (mean ± s.d. 344.49 ± 59.44 mm 3 , n=12) ( Figure 1).
[ 18 F]FSPG PET after cerebral ischemia
The coronal brain images with normalized color scale shown in Figure 1 illustrate the evolution of the [ 18 F]FSPG-PET signals in ischemic animals at day 0 (control) and at 3, 7, 14 and 28 days after reperfusion. Quantification of the images provided information related to the time-course activity of system xcin both the ipsilateral and contralateral cortex, in the striatum and the whole brain at days 0 (control) and 1, 3, 7, 14, 21 and 28 days after MCAO (Figure 2, n=6). All brain regions considered evidenced a similar [ 18 F]FSPG binding evolution after long-term focal cerebral ischemia. In the ipsilateral whole brain (cerebrum), the PET signal for [ 18 F]FSPG showed a non-significant increase at day 1 followed by a significant uptake increase from days 3 to 7 after ischemia in comparison to control (day 0) values (P<0.001, Figure 2A). In fact, the highest binding value was reached at day 3 after MCAO. Subsequently, the PET signal evidenced a progressive decrease from day 14 to day 28 compared to days 3 and 7. In the contralateral whole brain, [ 18 F]FSPG PET signal at day 1 showed similar values to those at day 0 (control) followed by a non-statistically mild increase from days 3 to 7 compared to control values. This was followed by a progressive decline from days 14 to 28 after cerebral ischemia ( Figure 2B). The cerebral cortex in the ipsilateral hemisphere evidenced a statistically [ 18 F]FSPG PET signal progressive increase from day 1 to day 3 and 7 followed by a progressive decrease at days 14 and onwards (P<0.05; P<0.01, with respect to control animals, Figure 2C). In contrast, non-statistically significant differences were observed in the contralateral hemisphere despite the weak increase of PET signal observed from day 3 to day 7 following reperfusion ( Figure 2D). Likewise, the non-ischemic cortex showed higher PET signal values than those observed in the non-infarcted striatum, however, this situation was reverted after cerebral ischemia where striatum displayed higher [ 18 F]FSPG binding levels at days 3 and 7 after reperfusion than those observed in the cerebral cortex (P<0.001 with respect to control animals, Figure 2E).
[ 18 F]DPA-714 PET after cerebral ischemia
The coronal brain images with normalized color scale shown in Figure 3A-E illustrate the evolution of the [ 18 F]DPA-714 PET signals in the ischemic lesion at 0, 3, 7, 14 and 28 days after reperfusion. Quantification of the images provided information related to the time-course activity of TSPO in both the ipsilateral and contralateral cortex, in the striatum and the whole brain at days 0 (control) and 1, 3, 7, 14, 21 and 28 days after MCAO (Figure 3, n=6). Quantification of the images at day 0 (control) and 1, 3, 7, 14, 21 and 28 days after MCAO show low TSPO expression in healthy cerebral tissue, followed by a dramatic up-regulation due to the inflammatory reaction, evidencing the usefulness of this radiotracer to evaluate neuroinflammation by PET imaging [31]. All brain regions considered evidenced a similar [ 18 F]DPA-714 binding profile during the following month to cerebral ischemia. In the ischemic territory, [ 18 F]DPA-714 uptake displayed a progressive increase during the first week after MCAO, from circa 0.20-0.30 %ID/cc at days 0 and 1, to 0.50 at day 3, peaking to ca. 1 at day 7. Uptake of [ 18 F]DPA-714 then decreased from 0.70 at day 14 to 0.30 at days 21-28 (P<0.001, with respect to control animals, Figure 3F). In the contralateral hemisphere, [ 18 F]DPA-714 PET uptake showed a significant increase at day 7 after reperfusion in relation to day 0 (P<0.05, Figure 3G). The cerebral cortex in the ipsilateral hemisphere evidenced a statistically [ 18 F]FSPG PET signal progressive increase from day 1 to day 3 and 7 followed by a progressive decrease at days 14 and onwards (P<0.001, with respect to control animals, Figure 3H). Likewise, the contralateral cortex showed a significant binding increase at day 7 after reperfusion (P<0.05, Figure 3I). The ipsilateral and contralateral striatums evidenced a similar TSPO expression than that observed in the cerebral cortex after cerebral ischemia; (i) a progressive increase from day 3 to day 7 and followed by a progressive decrease from day 14 to 21 (P<0.05; P<0.001, with respect to control animals, Figure 3J) in the ischemic striatum and (ii) a significant TSPO over-expression at day 7 after ischemia in the contralateral striatum (P<0.05, with respect to control animals, Figure 3K).
Time course of neurologic score after cerebral ischemia
Ischemic animals subjected to [ 18 F]DPA-714 PET studies (n=6) presented a major impairment from days 1 to day 7 after MCAO in relation to control (day 0). The neurologic impairment showed a significant increase with respect to control at days 1, 3, 7, 14, 21 and 28 (P<0.01). After day 3, rats exhibited a trend to progressive functional recovery over time at days 7, 14, 21 and 28 in relation to day 1 after ischemia (P<0.05; P<0.01, Figure 3L). selected brain ROIs for the quantification defined on a slice of a MRI (T2W) template. Rats (n=6) were repeatedly examined by PET before (day 0) and at 1, 3, 7, 14, 21 and 28 after ischemia. The neurologic score shows a neurofunctional improvement over time (L). *p<0.05 and ***p<0.001 compared with control; # p<0.05 and ## p<0.01 compared with day 1.
Expression of system xcin microglia and astrocytes after MCAO
Immunofluorescence staining exhibited xCT expression in both microglia/macrophages and astrocytes after ischemia (Figure 4). At days 3 and 7, cells with the morphology of amoeboid reactive microglia/macrophages showed intense CD11b immunoreactivity in the lesion (in red; Figure 4B) followed by a decrease at day 28 (in red; Figure 4B). The over-reactivity of microglia co-localized with the cellular expression of xCT (in blue and red; Figure 4D) at days 3, 7 and 28 after ischemia. The number of xCT + /CD11b + cells displayed a significant increase at days 3 and 7 in relation to day 0 followed by a sharp decrease at day 28 after reperfusion (P<0.001, Figure 4E). Likewise, astrocytes displayed an increase of the GFAP immunoreactivity from days 3-7 to 28 (in white; Figure 4A) after ischemia, forming a thin astrocytic rim in the vicinity of the lesion. In addition, reactive astrocytes showed co-localization with xCT expression (in blue and white; Figure 4D) after cerebral ischemia (3, 7 and 28 days). The number of xCT + /GFAP + cells showed a significant progressive increase from days 3-7 to day 28 after MCAO (P<0.01; P<0.001, Figure 3F).
Effect of system xcinhibition on neuroinflammation following cerebral ischemia
The effect of system xcinhibition with SAS and S-4-CPG on neuroinflammation was explored by PET imaging of TSPO with [ 18 F]DPA-714 at day 7 following MCAO ( Figure 5). All the images were quantified in standard units, i.e., %ID/cc. Coronal and axial images with normalized color scale illustrate the [ 18 F]DPA-714 PET uptake in vehicle (MCAO) ( Figure 5A and B), SAS (MCAO+SAS) (Figure 5C and D) and S-4-CPG (MCAO+S-4-CPG) ( Figure 5E and F) ischemic rats. Both SAS and S-4-CPG treatments evidenced a significant decrease of [ 18 F]DPA-714 binding in the ischemic cerebral hemisphere in relation to non-treated ischemic rats (P<0.05, Figure 5G). Neurological score at day 1 after ischemia showed similar values reflecting that the groups presented similar neurological impairment before the start of the treatments (Figure 5H). At day 7, treated ischemic rats with SAS and S-4-CPG displayed a superior neurofunctional improvement in relation to that showed by non-treated ischemic rats (P<0.05; P<0.01, Figure 5H). CD11b-reactive microglia/ macrophages increase at days 3 to 7 (B) corresponding to the temporal xCT immunoreactivity (C). (D) Merged images of three immunofluorescent antibodies at different time points. The number of CD11b-reactive microglia/ macrophages expressing xCT increase at days 3 and 7 after cerebral ischemia (E). The number of GFAP-reactive astrocytes expressing xCT increase in the ischemic area following reperfusion (F). **p<0.01 and ***p<0.001 compared with control. Scale bars, 5 μm. vehicle (A, B), SAS (C, D) and S-4-CPG-treated (E, F) rats at day 7 after cerebral ischemia. PET images are co-registered with a MRI (T2W) rat template to localize anatomically the PET signal. [ 18 F]DPA-714 uptake was quantified as %ID/cc (mean ± SD) in the entire ipsilateral cerebral hemisphere. Vehicle (n=5), SAS (n=7) and S-4-CPG-treated rats (n=8) were examined by PET 7 days after ischemia (G). The neurologic score show similar neurologic outcome at day 1 after ischemia (before the start of treatments) followed by a higher neurological outcome improvement at day 7 after SAS and S-4-CPG treatments than vehicle treated rats (H). *p<0.05 compared with vehicle; # p<0.05 and ## p<0.01 compared with day 1.
Expression of TSPO receptors in microglia and astrocytes after treatments in ischemic rats
Immunofluorescence staining exhibited TSPO expression in both microglia/macrophages and astrocytes after ischemia in vehicle and treated-rats ( Figure 6). At day 7, cells with the morphology of amoeboid reactive microglia/macrophages showed intense CD11b immunoreactivity (in red; Figure 6B) in the lesion that co-localized with TSPO receptor expression (in green and red; Figure 6D). The number of TSPO + /CD11b + cells displayed a significant decrease in treated ischemic rats with both SAS and S-4-CPG at day 7 after ischemia in comparison with control ischemic rats (P<0.05; P<0.01, Figure 6E). Likewise, rats treated with S-4-CPG showed a non-significant decrease of cerebral TSPO + /CD11b + cells in comparison to SAS treated ischemic rats. In contrast, equivalent number of TSPO + /GFAP + cells was observed for non-treated and treated ischemic rats at 7 days after ischemia ( Figure 6F).
Effect of system xcinhibition on the expression of M1 and M2 microglia/ macrophages
We next investigated the impact of system xcinhibition on microglia phenotype after stroke. Polarized microglia/macrophages after cerebral ischemia are commonly distinguished by their expression of signature genes for surface markers and cytokines/chemokines [32]. Reverse-transcription polymerase chain reaction (RT-PCR) was used to measure the effect of system xcinhibition in the expression of mRNA for M1 (CCL2, TNF and iNOS) and M2 (arginase, mannose receptor and TGFβ) markers in the region of the infarction and the in contralateral brain hemisphere at sham control and at day 7 after MCAO in vehicle-, SAS-and S-4-CPG-treated rats (Figure 7). M1 gene markers (CCL2, TNF and iNOS) evidenced a significant overexpression following cerebral ischemia in vehicle-treated rats (MCAO) (P<0.01, Figure 7A to C). M1 markers expression displayed a significant decrease in ischemic rats after system xcinhibition with SAS and S-4-CPG (P<0.05; P<0.01, Figure 7A to C) with respect to non-treated rats. In contrast, M2 markers showed more complex patterns of expression after stroke. Firstly, arginase expression displayed no expression changes following cerebral ischemia and was significantly increased in ischemic rats treated with SAS but not-significantly with S-4-CPG ( Figure 7D). In contrast mannose receptor and TGFβ mRNA expression evidenced a significant increase after MCAO that was slightly decreased in rats treated with SAS and S-4-CPG (P<0.01, Figure 7E and F).
Altogether, data showed here demonstrated that system xc-modulates microglial inflammatory reaction.
Discussion
Following brain injury, system xchas not only a detrimental function via glutamate release but also a beneficial neuroprotective and neurothrophic effect through the import of L-cysteine for glutathione production and oxidative protection [33]. Indeed, Piani and Fontana showed that microglial cells overexpressed system xcas a response to the oxidative stress underlying CNS disease [7] and the conversion to excitotoxic stress was related to the exacerbation of neurodegeneration [14,16]. Therefore, the identification of new up-regulated transporters in reactive glial cells, such as system xcis of great interest to the in vivo imaging research of cerebral ischemia. Because of this, we have assessed in parallel the in vivo expression of the system xcand TSPO in reactive glial cells using PET imaging procedures, in combination with immunohistochemistry, RT-PCR and neurofunctional evaluation after experimental stroke in rats.
PET imaging of system xcwith [ 18 F]FSPG
Recent preclinical and clinical PET studies performed with [ 18 F]FSPG have shown a low background uptake in the normal brain [34] that improves the visualization of the system xcfunction increase following pathological situations [4,35]. These findings are consistent with the low [ 18 F]FSPG binding uptake in the rat brain before the induction of the cerebral ischemia. In the present study, the PET [ 18 F]FSPG binding uptake in the healthy rat brain was mainly concentrated in the leptomeninges and ventricles (Figure 1) which are in agreement with the well-known distribution of system xcin the rat healthy brain [36,37].
In vivo and ex vivo evidence of system xcoverexpression after cerebral ischemia
Previous in vivo PET imaging of system xcwith [ 18 F]FSPG evidenced a significant progressive increased uptake at early phase following MCAO in rats. Indeed, these results evidenced the role of system xcon excitotoxic damage after reperfusion [4]. In the present study, [ 18 F]FSPG binding was evaluated from day 1 to day 28 after MCAO in rats, to show its relationship to the neuroinflammatory reaction after cerebral ischemia. In the infarcted brain hemisphere, [ 18 F]FSPG binding experienced a non-significant increase at day 1 followed by a maximum binding at days 3 to 7 that was followed by a progressive decline to pseudo-control values at days 21 to 28 after reperfusion (Figure 1 and 2). Nevertheless, these results stand in contrast with the in vivo PET imaging distribution of TSPO, a well-known marker for neuroinflammation, following cerebral ischemia [26,38] (Figure 3). In our study, the ischemic brain hemisphere experienced a gradual [ 18 F]DPA-714 binding increase from days 1 to 3 which peaked at day 7, followed by a slight decline afterwards. These results are in agreement with Martin and collaborators who, after monitoring the in vivo post-ischemic TSPO receptor expression with [ 18 F]DPA-714 PET, observed a progressive binding increase that peaked at day 11 followed by a progressive decline later on [26]. Likewise, the peak of the inflammatory reaction observed by PET imaging during the first week after ischemia runs in parallel with the gradual recovery of neurological handicap ( Figure 3L). Thus, animals experienced a quasi-recovery of functional outcome one month after stroke. Likewise, such capacity to undergo motor and cognitive recovery has been monitored in adult human brain following stroke [39].
Altogether, these results evidence an earlier overexpression of system xccompared to TSPO that could suggest the existence of a different involvement of both receptors on the neuroinflammatory response after stroke. The first step to confirm this hypothesis was the characterization of system xcby ex vivo immunohistochemistry at days 3, 7 and 28 after reperfusion (Figure 4). These results verified the expression of system xcin both microglia/macrophages and astrocytes following cerebral ischemia. Moreover, immunohistochemistry showed an over-increase of microglial cells expressing system xc -(xCT + /CD11b + ) at days 3 and 7 in relation to day 0 followed by a dramatic decrease at day 28 after cerebral ischemia. Likewise, astrocytes expressing system xc -(xCT + /GFAP + ) evidenced a mild progressive increase from day 3 to day 28 after cerebral ischemia ( Figure 4). Hence, the present results supported the previous PET imaging results obtained with [ 18 F]FSPG evidencing that the system xcover-expression at days 3 to 7 was mainly promoted by the system xcexpression increase by microglial cells. Indeed, these findings are in concordance with the role of system xcon microglial reactivity following amyotrophic lateral sclerosis [8] and multiple sclerosis [24] in rodents. Despite this, little is known concerning the role of system xcon inflammation underlying experimental ischemic stroke.
System xcinhibition attenuates neuroinflammatory after cerebral ischemia
Pharmacological inhibition of system xcwith sulfasalazine depletes the inflammatory response following EAE [17] and amyothrophic lateral sclerosis [8]. Likewise, since sulfasalazine is an inhibitor of the antiinflammatory NF-Κ B factor [40], other blockers of system xcthat do not have any effect on NF-Κ B such as S-4-CPG have also been tested to reduce the inflammatory reaction after EAE in mice [17]. Additionally, the depletion of microglial population using clodronate induced a reduction on system xcexpression following experimental multiple sclerosis in rats [24]. Altogether these studies confirmed the role of system xcon the inflammatory reaction underlying neurodegenerative diseases. In our study, we explored the effect of SAS and S-4-CPG on neuroinflammation by using in vivo [ 18 F]DPA-714 PET binding following cerebral ischemia ( Figure 5). The inhibition of system xcwith both SAS and S-4-CPG induced a significant decrease of the TSPO expression following seven days after reperfusion ( Figure 5G). Besides, treatment with system xcinhibitors evidenced a major neurological handicap improvement at day 7 after reperfusion in relation to the non-treated rats. These findings support those showed by Evonuk and collaborators who observed a clinical improvement after system xcinhibition in EAE mice [17]. Besides, rats treated with SAS and S-4-CPG displayed a significant decrease of microglial population expressing TSPO (Figure 6), confirming the results obtained with [ 18 F]DPA-714 PET and the role of the system xcon microglial reactivity after cerebral ischemia in rats.
Effect of system xcinhibition on M1/M2 microglial state
Microglial activation can be influenced by both extracellular glutamate and intracellular glutathione, by acting through microglial expressed glutamate receptors [41,42]. Thus, system xcinhibition might have a direct influence on the microglial function through the control of microglial M1/M2 polarization state following stroke. For this reason, we evaluated the mRNA expression of typical M1 and M2 markers after inhibition of system xcwith both sulfasalazine and S-4-CPG in ischemic rats. The expression of known M1 proinflammatory/neurotoxic markers; CCL2, TNF and iNOS at day 7 after cerebral ischemia showed a significant increase in relation to sham-operated rats (Figure 7). These findings stand in agreement with the expression profile experienced by proinflammatory markers after MCAO in mice [32]. The inhibition of system xcshowed a significant decrease of CCL2, TNF and iNOS (Figure 7). On the contrary, the antiinflammatory M2 mRNA markers arginase, mannose receptor and TGFβ showed an asymmetrical temporal expression after cerebral ischemia in mice. While mannose receptor and TGFβ mRNA showed high expression at day 7 after reperfusion, arginase evidenced a peak at day 3 followed by a sharp decline from day 5 to day 7 afterwards [32]. Our results support these previous findings since arginase showed similar mRNA values at day 7 after ischemia to those obtained for sham-operated rats, and both mannose receptor and TGFβ mRNA levels displayed a significant increase in ischemic animals in relation to non-ischemic rats ( Figure 7). Therefore, this may be a possible explanation why only arginase experienced an increase in mRNA expression following treatment with both sulfasalazine and S-4-CPG after cerebral ischemia in rats. Hence, these results suggest that system xcinhibition in stroke leads to a change in microglial phenotype markers. Interestingly, similar results have also been described in ALS in mice, indicating the regulation of microglial functions by system xc - [8].
Summary and conclusions
We report here [ 18 F]FSPG PET imaging to assess system xcactivity and its relationship with neuroinflammatory response after cerebral ischemia in rats. Our results confirmed the [ 18 F]FSPG binding increase in the ipsilateral hemisphere during the first week after cerebral ischemia followed by a progressive decrease later on. In addition, our findings displayed an earlier expression of system xccompared to TSPO with [ 18 F]DPA-714 imaging, evidencing its novel role on inflammation after cerebral ischemia. Besides, the overexpression of system xcwas identified in microglial cells by ex vivo immunohistochemistry and the inhibition of system xcwas able to promote a decrease of TSPO expression and a shift of pro-inflammatory microglia state towards an anti-inflammatory phenotype after MCAO in rats. These findings provide novel insights on the inflammatory reaction underlying cerebral ischemia that might contribute to the identification of a novel target at improving both diagnosis and therapy evaluation of stroke. | 2018-04-03T04:57:02.291Z | 2016-07-09T00:00:00.000 | {
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244057491 | pes2o/s2orc | v3-fos-license | Topological edge solitons and their stability in a nonlinear Su-Schrieffer-Heeger model
We study continuations of topological edge states in the Su-Schrieffer-Heeger model with on-site cubic (Kerr) nonlinearity, which is a 1D nonlinear photonic topological insulator (TI). Based on the topology of the underlying spatial dynamical system, we establish the existence of nonlinear edge states (edge solitons) for all positive energies in the topological band gap. We discover that these edge solitons are stable at any energy when the ratio between the weak and strong couplings is below a critical value. Above the critical coupling ratio, there are energy intervals where the edge solitons experience an oscillatory instability. Though our paper focuses on a photonic system, we also discuss the broader relevance of our methods and results to 1D nonlinear mechanical TIs.
We study continuations of topological edge states in the Su-Schrieffer-Heeger model with on-site cubic (Kerr) nonlinearity, which is a 1D nonlinear photonic topological insulator (TI). Based on the topology of the underlying spatial dynamical system, we establish the existence of nonlinear edge states (edge solitons) for all positive energies in the topological band gap. We discover that these edge solitons are stable at any energy when the ratio between the weak and strong couplings is below a critical value. Above the critical coupling ratio, there are energy intervals where the edge solitons experience an oscillatory instability. Though our work focuses on a photonic system, we also discuss the broader relevance of our methods and results to 1D nonlinear mechanical TIs. Topological insulators (TIs) are exotic materials that behave like an insulator in the bulk and a conductor on the edge [1][2][3]. Prototypical TIs exhibit nontrivial topology in their bulk wave functions, which guarantees the existence of robust edge states in the bulk band gap(s) [4]. Though TIs were originally proposed in 2D and subsequently generalized to 3D, the simplest TI already exists in 1D as the Su-Schrieffer-Heeger (SSH) model [5]. Here we consider an orbital version of the SSH model on a 1D lattice with two sites per unit cell and different intra-(t) and inter-cell (t ′ ) hopping amplitudes [6]; see Fig. 1. This model is described by the following Hamiltonian with chiral symmetrŷ
I. INTRODUCTION
whereâ † j (b † j ) is the creation operator on the site a j (b j ). A standard calculation shows that the bulk wave functions exhibit a phase Φ that is a function of the momentum k, and the bulk spectrum consists of two bands ±(|t − t ′ |, t + t ′ ) separated by a gap. The winding number * Electronic address: yiping.m@gmail.com † Electronic address: hadi.susanto@ku.ac.ae W of the phase Φ (k) over the Brillouin zone can be shown to be a topological invariant that is trivial (W = 0) for t > t ′ and non-trivial (W = 1) for t < t ′ [7]. The principle of bulk-edge correspondence then guarantees that in the latter case only, there exists a topologically protected edge state in the semi-infinite chain in Fig. 1 that terminates at a 1 . This edge state exists in the bulk band gap with zero energy, and has an exponentially decaying envelope |a j | ∼ Λ j and |b j | = 0, where Λ = t/t ′ . Note that the chiral symmetry of the Hamiltonian in Eq. (1) is essential for the existence of the winding number W as a topological invariant [4].
The principles underlying quantum TIs have been fruitfully adapted to photonic (electromagnetic) [8,9] and phononic (mechanical) [10,11] systems. The governing differential equations of these systems often exhibit nonlinearity, which can interact with topology to create new dynamical effects. A prominent feature of such nonlinear TIs is localized states that bifurcate from the topologically protected edge state with zero-energy in the linear limit. Hereafter these are referred to as topological edge solitons and will be the focus of this paper. Though photonic TIs only gained popularity in recent years, there are earlier studies on 1D waveguide arrays with alternating spacings [12,13]. This setup is essentially identical to the SSH model, but the topological properties of such systems remain largely unexplored in these earlier studies. In [12], bulk gap solitons are found in the SSH model with on-site cubic (Kerr) nonlinearity, and their stability is analyzed systematically. In [13], both bulk and surface (edge) gap solitons are found in the SSH model with on-site saturable nonlinearity, but there are no explicit discussions of topological edge solitons.
More recently, there have been several proposals to include inter-site or on-site nonlinearity in the SSH model, with the latter considered particularly relevant to photonics [9]. In [14,15], self-induced topological transitions are achieved in SSH models whose inter-cell coupling depends on the local dimer energy. In [16], topological gap solitons with nontrivial chirality are found in the SSH model with on-site cubic nonlinearity. This model reduces to the nonlinear Dirac model in the continuum limit, where topological edge states and gap solitons are found to share a common origin [17]. In the presence of chiral symmetry breaking, this model is found to exhibit rich topological physics that entail new edge and bulk solitons [18]. Topological gap solitons are also found in a mechanical SSH model with on-site cubic nonlinearity [19] and a photonic SSH model with on-site saturable nonlinearity [20]. Other nonlinearities also find prominent applications including topological lasers [21,22] and topological circuits [23,24] among others.
The SSH model with on-site Kerr nonlinearity can be regarded as the prototypical model for realizing nonlinear topological localized states. Here we explain the origin of topological gap solitons in this model, including both bulk and edge solitons, based on the phase space geometry of the underlying discrete spatial dynamical system (DS). We use numerical continuation to obtain the complete set of topological edge solitons, and compute their stability to reveal the inherent limit of energy storage on the edge. We also present analytical approximations in the small coupling ratio limit and the continuum limit. Our aim is to characterize edge solitons systematically, since only particular instances of this solution set have been reported to our knowledge [17,18]. Our method can be generalized to many other nonlinear TIs to expand their tunability through the discovery of new solutions.
II. SPATIAL DYNAMICS
The governing equation of the SSH model (1) with Kerr nonlinearity is j ∈ Z + , b 0 = 0, and˙ denotes derivative in the propagation distance (time) z. By scaling, we have taken the nonlinear coefficient to be 1 and will take t ′ = 1 in our numerical computations below. Eq. (2) conserves the power P = j |a j | 2 + |b j | 2 and the Hamiltonian Hereafter we assume t < t ′ such that the linear limit of Eq. (2) is topologically nontrivial. We look for standing waves of Eq. (2) in the form of , where E is the propagation constant (energy). Assuming that A j and B j are both real without loss of generality [25], Eq. (2) reduces to the following 2D invertible map in j: [26,27]. In the band gap |E| < t ′ − t, the only equilibrium on the fix(R) section is the origin O : (0, 0) with eigenvalues and the corresponding eigenvectors Since θ ∈ (0, π/2), the equilibrium O is a saddle whose stable and unstable eigenspaces are respectively defined by ( The origins of both bulk and edge solitons can be explained in the (A, B) phase plane. In Fig. 2, we show the stable manifold W s and the unstable manifold W u of the saddle O at E < 0 (top panel) and E > 0 (bottom panel). A bulk soliton is a reversible homoclinic orbit consisting of the backward and forward iterates of a transverse intersection between W s (O) and fix(R) [28], while an edge soliton consists of the forward iterates of a transverse intersection between W s (O) and the section B = 0 that represents the boundary condition. We note that for E > 0, although the bulk soliton B and the edge soliton E belong to the same invariant manifold W s (O), their spatial profiles generally do not coincide except in the continuum limit [17]. We also note that for E < 0, W s (O) wiggles after crossing fix(R) at B and intersects fix(R) and B = 0 to form additional bulk and edge solitons, some of which have been discovered computationally in [18]. Finally, we note that Eq. (2) with t ′ = t is simply the focusing discrete nonlinear Schrödinger (NLS) equation [29]. Therefore, our analysis shows that "topologisation" of classical models in nonlinear waves can greatly enrich their solution sets.
III. NUMERICAL CONTINUATION
Hereafter we focus on the primary family of edge solitons E that bifurcate towards E > 0. We can use numerical continuation to obtain these solutions, which essentially tracks E as E varies without explicitly computing W s (O). Once a solution is found, its linear stability is computed through solving the corresponding eigenvalue problem obtained from substituting a j = (A j + ǫâ j exp(λz)) exp(−iEz), b j = (B j + ǫb j exp(λz)) exp(−iEz) and linearizing about ǫ = 0. In Fig. 3, we present the power P for varying E with t = 0.1. An example profile and its spectrum in the complex plane are also shown. As E increases from 0 to t ′ − t, the stable spatial eigenvalue λ 1 in Eq. (4) decreases from −t/t ′ to −1. This implies that the edge soliton decays more slowly in space as E increases and becomes a bulk state as E tends to t ′ − t, where the solution branch terminates as (7), respectively. The insets show the solution profile and its spectrum for E = 0.5 (lines are from numerics, and circles are from approximation (6)).
shown in Fig. 3. Here we note the different conventions that we have used for defining the spectrum: the spectrum of an edge soliton consists of growth rates λ, while the spectrum of the HamiltonianĤ in Eq. (1) consists of angular frequencies; these are consistent with the standard conventions in the nonlinear waves and quantum physics literature. Thus, in the limit E → 0, the spectrum of the edge soliton reduces to i times the spectrum of the HamiltonianĤ in Eq. (1). We also study the effect of varying the coupling t and the energy E in general. For every t ∈ (0, t ′ ) and E ∈ (0, t ′ − t), there exists a unique edge soliton. We show in Fig. 4 the critical spectrum of these edge solitons in the (t, E) plane, defined as the maximum of the real part of the spectrum. The edge soliton is stable for all E when t < t c ≈ 0.35, but becomes unstable over certain interval(s) of E when t > t c . The nature of the instability is oscillatory as seen in the inset of Fig. 4. The critical eigenvalue appears following the collision of two eigenvalues that bifurcate from the lower edge of the uppermost band and the upper edge of the next-uppermost one. This is typical of the oscillatory instability appearing in gap solitons [30,31]. As t increases, we obtain more and more unstable eigenvalues appearing. We simulate the typical dynamics of the oscillatory instability. Using an exact edge soliton as the initial condition with random perturbation that is proportional to the field amplitude, we show the time evolution of Eq. (2) in Fig. 5. At t = 0.43, the initial edge soliton with E = 0.5, which is inside the unstable region in Fig. 4, gradually loses energy by releasing a localized wave packet into the bulk, and eventually evolves into a stable edge soliton with smaller power, which is just outside the unstable region in Fig. 4. This release process approximately in 100 < z < 150 features temporal oscillations with an angular frequency of about 1.08, consistent with the imaginary part of the unstable eigenvalues in the inset of Fig. 4. This process is almost the exact opposite of the mechanism discussed in [17,32], where a bulk soliton is launched towards the edge to increase the energy of an edge soliton. Our result implies that such proposals to couple the bulk and edge modes are robust only when the edge soliton is stable. We also note that a similar instability is observed using a different realization of the Kerr nonlinearity in coupled fiber loops that implement a discrete 1D quantum walk [33].
IV. ANALYTICAL APPROXIMATIONS
For the coupling ratio |Λ| ≪ 1, we can approximate the edge state by with, using the assumption that only the equation for a 1 is nonlinear and keeping the remaining sites linear, In the limit E → 0, we can calculate that L → Λ = t/t ′ . Using (6), the power is given by This compares well with the numerics as seen in Fig. 3. The stability in the limit Λ → 0 can be established using perturbation theory, exploiting dimers of the system; see a similar problem considered in, e.g., [34]. The method, that has been standard by now, can be applied here to show that in that limit the edge soliton is stable.
On the other hand, near the Dirac point (t, E) = (t ′ , 0), the edge soliton becomes slowly varying, i.e., we have the continuum limit [35]. In that case, denoting = a, b, we introduce a small parameter ǫ and take: t = t ′ − E − ǫδ, that j±1 = (x ± ǫ) = (x) ± ǫ x + O(ǫ 2 ), E = ǫ E, and a slow time scale z = ǫz. After dropping the hats, Eq. (2) becomes the nonlinear Dirac (NLD) equation [36] iȧ As shown in [17], Eq. (8) admits an exact analytical solution on the infinite domain that describes traveling bulk solitons. At zero velocity, the stationary bulk soliton has a spatial orbit that passes through b = 0, so part of this orbit describes a stationary edge soliton. An example stationary profile is plotted in Fig. 6(a); note that the bulk soliton can be identified as a reversible homoclinic orbit invariant under (x, a, b) → (−x, b, a) [28].
The stability of solitons in the NLD equation (8) is harder to analyze than in the NLS equation due to the absence of the Vakhitov-Kolokolov criterion [37]. This task can be challenging even numerically because simple discretization (finite difference) of the eigenvalue problem can generate a large number of spurious unstable eigenvalues; see, e.g., [30,38] for the stability of gap solitons in a similar system. Delicate methods, such as Chebyshev interpolation method [39] and Evans function techniques [31], can be used to capture the actual instability. Here, we have used the Chebyshev method to solve the eigenvalue problem corresponding to Eq. (8). We show the spectrum in Fig. 6(b), where we obtain that the edge soliton in Fig. 6(a) experiences an oscillatory instability, which is consistent with the top left corner of Fig. 4.
V. COMPARISON WITH MECHANICAL SYSTEMS
In view of recent work on edge/interface solitons in 1D nonlinear mechanical TIs [19,40], we compare our 1D nonlinear photonic TI with its mechanical counterpart, namely the mechanical SSH model with on-site cubic nonlinearity [19]. The key difference is that a photonic system can be reduced to a spatial DS using a simple ansatz with a single harmonic in time (with angular frequency E in our notation) even for strong nonlinearity, but this is generally impossible in a mechanical system except for weak nonlinearity. In particular, edge solitons in [19] (referred to as nonlinear normal modes in [40]) generally consist of infinitely many harmonics in time.
Still, the model in [19] can be viewed as a spatial DS for the Fourier components in time on each lattice site. After a Galerkin truncation that keeps only the lowest harmonic, the resulting spatial DS is similar to Eq. (3) in our model. Without this truncation, the spatial DS in [19] is higher-dimensional and therefore exhibits bifurcation diagrams different from Eq. (3) in our model. Most notably, the branches of edge solitons in Figs. 2 & 3 of [19] penetrate into the bulk bands, while edge solitons in our model exist strictly within the band gap.
Though the concept of spectral stability of nonlinear waves is similar in photonic and mechanical systems, the details of the analyses are different. The stability of an edge soliton in our model depends on the eigenvalues of a time-independent linear operator, while the stability of an edge soliton in [19] depends on the Floquet multipliers of a time-periodic linear operator. Interestingly, the stability trend is exactly opposite between our model and the model in [19] with stiffening nonlinearity: edge solitons in our model are generally stable below a critical energy (frequency), while edge solitons in [19] are generally stable above a critical energy (frequency) as shown in Fig. 3 of [19]. Thus, a natural question is whether edge solitons can be stabilized for all energies (frequencies) using a hybrid between these two models.
VI. CONCLUSION AND OUTLOOK
We have studied the existence and stability of topological edge solitons in the SSH model with on-site Kerr nonlinearity. The phase space topology implies that edge solitons exist for all positive energies in the topological band gap. These edge solitons are completely stable below a critical coupling ratio, but can experience an oscillatory instability above it. Our results are pertinent to photonic experiments using, for example, 1D topological arrays of coupled nonlinear resonators [41]. Our results can also be realized in the 1D Gross-Pitaevskii equation with a periodic dimer potential, which reduces to the nonlinear SSH model in the tight-binding limit [16].
The topological edge solitons in the nonlinear SSH model correspond directly to extended edge states in 2D nonlinear TIs such as nonlinear photonic graphene [42]. These 2D extended edge states inherit the instability of their 1D counterparts longitudinally, but are subject to an additional transverse instability [43]. In addition to these essentially 1D localized states, we expect that new intrinsically 2D localized states in 2D nonlinear TIs can also be discovered using numerical continuation in the spatial dynamics framework. | 2020-10-26T01:00:15.184Z | 2020-10-23T00:00:00.000 | {
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118504808 | pes2o/s2orc | v3-fos-license | Certifying entangled measurements in known Hilbert spaces
We study under which conditions it is possible to assert that a joint demolition measurement cannot be simulated by Local Operations and Classical Communication. More concretely, we consider a scenario where two parties, Alice and Bob, send each an unknown state to a third party, Charlie, who in turn interacts with the states in some undisclosed way and then announces an outcome. We show that, under the assumption that Alice and Bob know the dimensionality of their systems, there exist situations where the statistics of the outcomes reveals the nature of Charlie's measurement.
We study under which conditions it is possible to assert that a joint demolition measurement cannot be simulated by Local Operations and Classical Communication. More concretely, we consider a scenario where two parties, Alice and Bob, send each an unknown state to a third party, Charlie, who in turn interacts with the states in some undisclosed way and then announces an outcome. We show that, under the assumption that Alice and Bob know the dimensionality of their systems, there exist situations where the statistics of the outcomes reveals the nature of Charlie's measurement.
I. INTRODUCTION
Quantum nonlocality, the fact that entangled quantum systems separated in space can violate Bell inequalities [1], is one of the most profound discoveries in science. This nonlocal nature of entanglement has also been identified as an essential resource for various quantum information processing tasks. It allows to reduce communication complexity [2] or makes it possible to devise deviceindependent protocols for quantum key distribution [3], genuine random number generations [4], and state tomography [5]. Indeed, these tasks can be performed without resorting to the actual inner working of the devices, which is a feat without counterpart in the classical world.
In all of the above instances, quantum nonlocality arises when space-like separated measurements are performed over jointly prepared entangled states. However, the existence of nonlocality can also be manifested in a dual setup [6], [7]: quantum states which are prepared separately (hence unentangled) exhibit anomalous correlations when measured jointly. In this case, more information can be revealed by joint measurements than can be gained by using any sequence of Local Operations assisted by Classical Communication (LOCC). In particular, the superiority of globally entangled measurements over LOCC ones has been proved conclusively [8]. The improved performance is due to entanglement occurring in the process of measurement and again it is not associated with the states.
For quantifying the effectiveness of entangled measurements, different figures of merit have been proposed, like the Shannon mutual information [6] or the quantum fidelity [8]. A common feature of those is, though, that one must rely both on the precise form of the states prepared and on the a priori probabilities of the preparations; otherwise, the estimations on the figure of merit would be not reliable. Hence, in order to ascertain that a measurement is truly globally entangled and not only an LOCC one, one must resort to a detailed knowledge of the preparation procedure. Clearly, this issue sheds doubt on the reliability of the obtained results.
In the present paper we take another approach, more in the spirit of a black box scenario, and tackle the problem of certifying entangled measurements by introducing a kind of dual Bell inequalities. These inequalities involve correlation terms which can be gathered from experimental data, and do not depend on the specific form of the quantum states to be prepared. Actually, the only necessary condition entering in the derivation is the Hilbert space dimension of the prepared particles. Taking into account that entangled measurements enable increased classical capacity of quantum channels [9] and efficient quantum state estimation [10], in the future it would also be interesting to find applications related to these tasks within a black box approach.
The notation we use along the article is introduced in the next section. Section III provides an instance of certifiable entangled measurements based on a nonlinear inequality. Section IV is devoted to a numerical study where linear inequalities are applied to detect entangled measurements in the simplest possible scenarios. Section V summarizes our conclusions.
II. PRELIMINARIES AND NOTATION
Let us imagine the following communication scenario, close to the spirit of the simultaneous message passing model [11]. Two separated parties, Alice and Bob, have each some preparation device with N possible inputs. Alice (Bob) is thus able to prepare any of the unknown qudit states {ρ x ∈ C D } N x=1 ({σ y ∈ C D } N y=1 ). Alice and Bob then send their states to a third party, Charlie, who performs a measurement over those two states, announcing a dit a of dimension K at the end of the process. Denote by {M a : a = 0, ..., K − 1} the elements of Charlie's Positive Operator Valued Measure (POVM). Then, for each pair of inputs x and y, several repetitions of this primitive would allow Alice and Bob to estimate the frequency of occurrence of each outcome a = 1, 2, . . . , K. Depending on the form of the POVM elements {M a : a = 0, ..., K − 1}, we will distinguish four different types of measurements: Classical measurements. Suppose that Charlie's measurement is fixed in advance to a given complete projective measurement on each system (say, in the computational basis), followed by some random processing of the raw output. Any set of distributions P (a|x, y) so generated can be reproduced in a classical setting where Alice and Bob send each one dit of information to Charlie, who, in turn, applies a random function f : {0, ..., D −1}×{0, ..., D −1} → K and then announces the result. For each N DK scenario, we will denote bỹ P C the set of all possible correlations P (a|x, y) that can be attained in such a way.
It is easy to see that any point ofP C is a convex combination of deterministic classical strategies. As we will see, though, the converse is not true. On the other hand, if Alice, Bob and Charlie share random variables, then any convex sum of deterministic strategies can be attained. Since for any N DK scenario the number of deterministic classical strategies is finite, the set of correlations attainable through classical maps and shared randomness is a polytope, that we will denote by P C . This classical set is symbolized in Figure 1 by a square.
Unentangled and LOCC measurements. If each of the POVM elements M a is a separable operator, we will say that the measurement M is unentangled. We will say that Charlie's measurement can be attained via Local Operations and Classical Communications (LOCC) if M corresponds to a sequence of local measurements on Alice's and Bob's individual qubits, with each measurement possibly depending on the outcomes of earlier measurements. It is known that the class of unentangled measurements is strictly greater than the class of LOCC measurements [7]. This implies thatP unent ⊇P LOCC , whereP unent ,P LOCC resp. denote the sets of distributions P (a|x, y) attainable through unentangled and LOCC measurements in a given N DK scenario. It is an open question whether such an inclusion is strict.
As before, if we allow Alice, Bob and Charlie share some prior random variables, the resulting sets of correlations P unent ⊇ P LOCC are convexifications of the former. A two-dimensional slice of the unentangled set P unent is symbolized in Figure 1 by a circle.
General measurements. Here the measurement operators are only limited by positivity and normalization constraints, i.e., M a 0, where the operators M a ∈ B(C D ⊗ C D ) may very well be entangled. These are the most general measurements allowed by quantum mechanics. In analogy with the former sets, we will denote byP ent and P ent the set of distributions accessible through general joint measurements and its convex hull. The latter is depicted as an ellipse in Figure 1.
The numerical study of the setsP C ,P unent andP ent is much more convoluted than that of their convexifications P C , P unent and P ent . Consequently, most of this article is devoted to the numerical analysis of the latter.
If we reflex a bit about the previous definitions, it will soon be clear that the above sets of correlations can only be distinguished experimentally in a black box way if the N DK scenario is such that N > D. Otherwise, by forcing Alice and Bob to send orthogonal states, Charlie could always infer the values of x, y through an appropriate projective measurement, and so any conceivable distribution P (a|x, y) could be classically realized.
The simplest scenario where we may expect to find an interesting structure is thus the 322 scenario. And, indeed, the next example shows that there the setsP C and P unent differ fromP ent . It also proves that neither of the former two sets is convex, and so the convexifications P C , P LOCC , P unent are non-trivial.
III. AN INSTANCE OF ENTANGLEMENT DETECTION
Consider the 322 scenario, and denote by P the matrix of probabilities P xy ≡ P (0|x, y). Suppose now that This set of probabilities can be attained if the states {ρ x , σ y } and the POVM element M ≡ M a=0 correspond to where |Ψ + = 1 √ 2 (|00 + |11 ). It hence follows that P ∈P ent .
We will prove that M must be an entangling measure (i.e., P ∈P unent ) by reductio ad absurdum.
Then, the condition P 11 = 0 implies that: 1) ρ 1 or σ 1 (or both) is a pure quantum state; and 2) there exists a subset of indices I ⊂ {1, 2, ..., K} such that Here ω ⊥ denotes I 2 − ω ⊥ , for any qubit state ω. From tr(M ρ 1 ⊗ σ 1 ) = P 11 = P 12 = tr(M ρ 1 ⊗ σ 2 ) we have that σ 1 = σ 2 , and thus tr( for some δ > 0. The condition P 22 = 0 therefore requires that tr(|v i v i |σ 2 ) = 0, i ∈ I, and tr(|u i u i |ρ 2 ) = 0, i ∈ I, so Using this last decomposition, we get with δ ′ > 0. But P 33 = 0, from which it follows that M is indeed entangling. Actually, it is possible to obtain a non-linear witness to detect M 's entanglement if we just follow the previous steps carefully while keeping track of the approximation errors (see the Appendix).
We have thus proven that P ∈P unent , i.e., the matrix of probabilities P cannot be observed if only unentangled measurements are allowed. However, the matrix P xy can be expressed as a convex combination of four deterministic classical strategies. Indeed, (10) The setsP C ,P LOCC ,P unent are hence not convex, since otherwise P would belong to them. Allowing the three parties to have shared randomness thus leads to a radically different scenario.
Since convex sets can be completely characterized by systems of linear inequalities, it is legitimate to investigate how different linear constraints on P C of the form x,y W xy P xy ≤ w c may be violated by elements of P unent and P ent .
This leads us to the problem of maximizing quantities like over all possible POVM elements M in the unentangled or the general class, and over all qubit states ρ x and σ y so as to get the values w unent and w ent . Clearly, if w ent > w unent for some matrix W , then there exist experimental situations where one can prove that the statistics observed in the lab cannot be simulated with unentangled measurements and shared randomness. Numerical optimization to obtain w unent is carried out very similarly to the iterative algorithm used in [14]. The steps are the following: 1. Generate some random pure qubit states ρ x , σ y , x, y = 1, 2, . . . , N .
2. In dimension D = 2, for fixed states ρ x , σ y , maximizing Eq. (11) reduces to a semidefinite problem: define thus F ≡ 4. For fixed M and ρ x , the optimal σ y can be found such as ρ x in step 3.
5. If convergence has not been achieved, go back to step 2.
This optimization algorithm may encounter several local optima, therefore we must iterate it many times in order to ascertain with reasonable confidence that the largest maximum has been found. In Sections IV B and IV C the semidefinite programs described in step 2 were solved using the SeDuMi package [15].
A very similar procedure can be applied if we wish to inquire about the maximal value w ent which can be achieved within the class of general quantum measurements. The only difference is in step 2, where we have to maximize tr(M F ) subject to the only constraint 0 M 1 1. However, in this case the maximum for a fixed F can be easily found by diagonal- Thereby the optimization algorithm for the case of entangled measurements is much faster.
B. The 322 scenario
We next describe the way linear witnesses were produced for entangled measurements. Our starting point is the classical polytope P C for the 322 scenario, consisting of 104 extremal points in dimension 9. All its facets can be enumerated by using the double description method implemented in Fukuda's cdd package [16]. The polytope P C has 1230 facets; among them, 9 describe positivity facets P x,y ≥ 0, x, y = 1, 2, 3. The rest of them define non-trivial inequalities W . However, most of them are equivalent under the following operations, • permuting Alice's inputs x • permuting Bob's inputs y
• exchanging parties Alice and Bob
• multiplying by −1 all coefficients W x,y of the matrix W .
It turns out that there are 13 inequivalent facets of the polytope P C . Those are listed in Table I, where we also give the maximum classical value w c , the value w unent achievable with unentangled measurements, the value w ent attainable with general measurements, and the number of vertices lying on the particular facet. We must stress that the values for w unent and w ent come from numerical optimization, and so only local optimality is guaranteed. Nevertheless, due to the small dimensionality of the problem and the number of iterations of the main algorithm, we are quite confident about their overall optimality as well. Interestingly, we found that in each of the 13 cases w unent could be achieved with rank-2 projective measurement operators.
We now focus on inequality #4 from Table I, which is the only inequality for which w ent > w unent , and hence it enables to witness entangled measurements. The inequality looks (in an equivalent form) as − P 11 − P 12 + P 13 + P 21 + P 23 + P 31 − P 32 − P 33 ≃ 2.3371, (12) and can be violated by entangled measurements up to the value of 2.5. Next, we present the actual measurements and states achieving w ent and w unent . Entangled case. The POVM element M is a rank-2 projector with |m = (0, cos θ, sin θ, 0) having θ = 2 arctan( ) and φ = π/3. The partial transpose of M has the following eigenvalues: with the first entry being negative. Both Alice's and Bob's states are pure and real valued, ρ x = |ψ x ψ x | and σ y = |φ y φ y |, respectively. Their explicit forms are, respectively, And, indeed, evaluating the left-hand side of inequality (12), we get w ent =2.5. Unentangled case. In appropriate local bases, a separable rank-2 projective operator can be written in the form where |m = (cos θ, sin θ), and in our particular case, θ = where P 12 = 5 4(4+ √ 6) ≃ 0.193814 and P 13 = P12 2 (5 + 2 √ 6) ≃ 0.959279. Plugging these numbers into the lefthand side of inequality (12), we get w unent = 2+3 √ 6 4 ≃ 2.3371. In some sense, the probability vector (19) approximates the best way among unentangled measurements the probability vector (16) corresponding to an entangled measurement.
C. The 422 scenario
The classical polytope P C for the 422 scenario is spanned by 520 non-redundant vertices in the 16 dimensional probability space. However, it turned out that the full characterization of this polytope in terms of facets is computationally an elusive task. Instead, the problem was approached in a different way. We scanned through all the inequalities with small integer coefficients (−1, 0, +1), and sorted out all of them which are facet defining. Several inequivalent facets have been found in this way (beyond the ones, which are just straightforward extensions of the 322-type inequalities in Table I).
In Table II, we list all those 10 inequivalent facets for which w ent > w unent , hence witnessing entangled measurements. Among them, #9 is equivalent to the single 322-type witness in (12). Table II contains results on w c , w unent , w ent , and also gives the sole negative eigenvalue λ 1 of P T (M ) achieving w ent . Since all the other eigenvalues are positive, the absolute value |λ 1 | defines negativity, a valid entanglement measure [17].
Inequality #2 is interesting on its own. In this case, w ent = 3.3195 is attained with a negativity of 0.1744, although a larger value of negativity of 0.1777 is found at another locally optimal point with w ′ ent = 3.145. This feature resembles the dual case obtained in a Bell scenario, where for several tight 2-party Bell inequalities the maximum quantum value was achieved with nonmaximally entangled 2-qubit states [18]. It has also been observed that for all the 422 witnesses for which unentangled measurements gave numerically maximal violation, the measurements could always be brought to a form of rank-2 projective matrices.
To conclude this section, the 422 scenario gave us a few new entangled measurement witnesses over the single witness of (12) of the 322 scenario. However, no significant improvement could be found in the efficiency of detecting entangled measurements with respect to unentangled measurements. Also, no simple generalization of inequality (12) for larger input alphabets was discovered among the 422 inequalities.
V. CONCLUSION
In this paper, we have studied how to translate the concept of witnessing entangled measurements to the deviceindependent arena. We found that even in a black-box scenario it is possible for a theorist to assert that a joint demolition measurement cannot be simulated with any sequence of local measurements assisted by classical communication: the only assumption one has to rely on is the dimensionality of the probe states. Following this line of thought, we derived correlation inequalities which allow to test the superiority of entangled measurements over unentangled ones in two-qubit experiments where 9 (Secs. III,IV B) and 16 correlation terms (Sec. IV C) are estimated.
Previous routes to certify the 'non-locality' of unknown measuring devices involved carrying out complete tomography of their associated POVM elements [19], or envisaging a state discrimination problem where entangled measurements have an advantage with respect to unentangled ones [6,8]. Either scheme has been conducted experimentally [19,20] with success. Both approaches, though, relied crucially on a detailed knowledge of the probe states to be measured, and so their conclusions cannot be considered definite.
In contrast, in this article we have proven that the experimental inaccuracies arising from an imperfect preparation of the probe states can be completely eliminated by certifying entangled measurements within a black box approach. Given the simplicity of our inequalities, we thus propose their experimental implementation as an interesting challenge.
We are now ready to derive the non-linear inequality. Suppose that M is of the form with λ i > 0. It is straightforward that the relation u i |ρ k |u i v i |σ k |v i (A14) holds for j = 1, 2, 3. Also, notice that i c i = P 11 + P 22 + P 33 . (A15) Identifying C = R 2 , with R ≡ max j =k min l f (P jl , P kl ), min l f (P lj , P lk ) , (A16) in Proposition 2, we thus arrive at the bound λ i ≤ 4ci (1−R) 2 . Putting all together, we have that where in the last step we made use of Eq. (A15). On the other hand, i λ i ≥ i λ i tr(|u u|ρ k ) · tr(|v v|σ l ) = P kl , (A18) for all k, l = 1, 2, 3, and so the inequality 4(P 11 + P 22 + P 33 ) with R given by Eq. (A16), must hold for all k, l.
It is immediate to see that the example P in Section III violates it by an amount of −1/4. | 2011-01-30T18:16:51.000Z | 2011-01-27T00:00:00.000 | {
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9016034 | pes2o/s2orc | v3-fos-license | 2D Lattice of coupled Sinai billiards: metal or insulator at g<<1
We investigate the transport in a two-dimensional (2D) lattice of coupled Sinai billiards fabricated on the basis of a high-mobility 2D electron gas in GaAs/AlGaAs heterojunction. For the states with low reduced conductivity g<<1 an anomalously weak temperature dependence of g was found. The large negative magnetoresistance described by the Lorentz line-shape of the width corresponding to the half magnetic flux quantum through the area of the billiard is observed. In going from g>1 to g<<1 it strongly increases. The Shubnikov-de Haas oscillations and commensurability magnetoresistance peak are preserved at g<<1. The data suggest that the system studied behaves more like a metal than an insulator at g<<1 and is not described by the generally accepted picture of Anderson localization.
Quantum and classical transport in systems with dynamic chaos has been intensively studied during last few years, since the successes of modern semiconductor technology has made it possible to obtain various experimental realizations of such systems with electron billiards as an example. At the present time two varieties of these systems are studied. The first one is unit billiards (regular or chaotic Bunimovich or Sinai billiards) [1][2][3][4][5], while the second one is macroscopic two-dimensional (antidot lattice) [6][7][8][9][10] or one-dimensional [11] Sinai billiards. A number of interesting phenomena resulting from the classical and quantum chaotic electron dynamics was found in these billiards (mesoscopic conductance fluctuations, commensurability magnetoresistance oscillations, weak localization effects, statistical and parametrical correlations of Coulomb blockade peaks).
However up to now, there have been practically no investigations of systems, in which separate electron billiards with reduced conductivity g = σ xx /(e 2 /h) ≫ 1 and the size L < l (l is the mean free path) compose a lattice with the coupling between them being weak in the sense that the conductivity of the lattice itself is low g ≪ 1. The study of such lattice is of interest mainly due to the fact that, on the one hand, this system should have some properties of a unit billiard (because the coupling is weak), and on the other hand, since the billiards form a regular structure, the systems may exhibit properties caused by that structure.
We report for the first time the results of experimental investigation of this new system with dynamical chaos. The system was built on the basis of a two-dimensional (2D) lattice of closely situated antidots fabricated from a high mobility 2D electron gas in GaAs/AlGaAs heterojunction with a metallic gate evaporated on the top, that permitted us to control the conductivity of the structure in a sufficiently wide range from g = 0.01 to g = 2.
The square lattice of antidots was fabricated on the basis of a two-dimensional electron gas with electron density N S = (2 − 3) × 10 11 cm −2 and mobility µ = (3−8)×10 5 cm 2 /V s corresponding to the mean free path l = (3−6) µm by means of electron lithography and subsequent plasma etching. Then the NiAu or TiAu gate was evaporated on the top of the device. We investigated three samples with the lattice period d = 0.6 µm and three ones with d = 0.7 µm. One of the samples had no metallic gate, and its conductivity was controlled using illumination by LED. The lithographic size of antidots a = 0.2 µm was the same for all samples. However, due to the depletion layers the actual size was larger, being approximately equal to the lattice period even before the gate evaporation. The experimental sample was made up of two Hall bars of the length of 100 µm and the width of 50 µm. The lattice of antidots was introduced into one of the bars. Measurement were carried out in the temperature range 50 mK-1 K in the magnetic field up to 2 T using ordinary four-terminal scheme at the frequency 6 Hz and with the current 0.01-0.1 nA in order to exclude heating effects. Fig. 1(a) shows the set of temperature dependences of the conductivity g(T ) for the sample AG219 with the period d = 0.6 µm (µ = 7 × 10 5 cm 2 /V s at N S = 2×10 11 cm −2 in the unpatterned part of the sample) for different values of the gate voltage. It is seen that for g > 1 conductivity is practically temperature independent. More exactly, weak logarithmic decrease of g is observed typical for the weak localization effects. It becomes more noticeable for lower values of conductivity, but it still remains weak even for g ≪ 1, and is well described by the power law dependence g(T ) ∝ T a with a < 1 for all of the tested samples. Specifically, for the dependences of Fig. 1(a) a = 0.1 − 0.27 at g ≪ 1. It should be mentioned that the value of a is larger for the samples with lower mobility of the initial 2D electron gas. As an example, the same set of dependences g(T ) for sample AG35 with the period d = 0.7 µm (µ = 3 × 10 5 cm 2 /V s at N S = 2 × 10 11 cm−2) is presented in the Fig. 1(b). It is seen that they are characterized by two times higher magnitude of a for the same values of g. Hence it may be inferred that the enhancement of fluctuation potential leads to stronger temperature dependence. The behavior of g(T ) described above is significantly different from that for the unpatterned 2D electron gas both in silicon MOS-transistors [12] and in AlGaAs/GaAs heterojunction [13], as well as for antidot lattices with short period [14,15]. In all of these cases at g ∼ 1 the transition from the weak logarithmic dependence (weak localization regime) to the strong exponential one (strong localization regime) is observed. In our case the weak logarithmic decrease of g (for g > 1) is followed by a weak power law, that has not been observed in other 2D systems before.
We now turn to description of the influence of magnetic field. It is well known that in AlGaAs/GaAs heterojunctions at g < 1 under the influence of magnetic field a transition from an insulator to the quantum-Hall-liquid is observed. This transition is characterized by the critical point B c and g c ≈ 0.5 − 1 [13]. A similar transition was recently observed in the triangular antidot lattice with a small period d = 0.2 µm [15]. Our samples exhibit a radically different picture. It is seen from Fig. 2 that for all values of g(B = 0) in the magnetic fields about B ≈ 1 T the transition takes place from weak power-law dependence g(T ) to no temperature dependence at all. Moreover, this transition is of different kind, for there is no critical point, and the metallic behavior extends for g ≪ 1. Fig. 2(a) also shows an interesting picture in weak magnetic field. For all values of g, the negative magnetoresistance (NMR) is observed for B < 0.05 T, followed by a peak at B ≈ 0.2 T corresponding to the condition 2R c = d. This peak is well-known for the antidot lattices at g > 1 and originates from the so-called pin-ball trajectory that surrounds an antidot not colliding with it. It is seen from Fig. 2(b) that the second commensurate peak is observed at g ≈ 1. The second peak corresponds to the condition 2R c = ( √ 2 − 1)d. We have recently established that this peak is associated with the non-colliding trajectory inside the billiard [16]. The positions of the commensurability peaks shows that we really deal with the lattice of closely situated antidots with d ≈ a and d ≫ d− a. The Sinai billiards between the antidots have the area S = d 2 (1 − π/4) and contain a large number of electrons N ≫ 1. In our case we have correspondingly S = 0.5 µm 2 , N ≈ 70 for d = 0.7 µm, and S = 0.36 µm 2 , N ≈ 50 for d = 0.6 µm. It is also important that both the main commensurability peak and NMR are conserved at the transition from g > 1 to g ≪ 1. The peak position is slightly shifted towards lower magnetic fields with decreasing g. This indicates a small change of electron density (from 1.4 × 10 11 to 0.9 × 10 11 cm −2 ) in the billiard within the whole range of g.
The behavior of NMR is shown in Fig. 3 in more detail. It is characterized by two distinguishing features: (i) for all states with 0.05 < g < 2 NMR is cut off at the same magnitude of magnetic field B ≈ 0.05 T; (ii) NMR noticeably increases with decreasing g (Fig. 3(a) shows that at g = 0.05 it reaches a considerable value about 40%), and its temperature dependence becomes stronger. For the states with the highest resistivity it was more stronger than g(T )). For g > 1 NMR can be attributed to the effects of weak localization in open chaotic billiards [2], because it has relatively small amplitude and Lorentzian line-shape. The behavior of NMR for g ≪ 1 is surprising. It increases by an order of magnitude reaching a considerable value comparable to the total resistance of the sample, while the line-shape of NMR is described by a Lorentz curve of the same width. The width is equal to ∆B 1 = 27 ± 2 mT for the sample AG219. It corresponds to a half magnetic flux quantum through the area of the billiard that is equal to d 2 (1 − π/4). The fact that the width is determined by the magnetic flux quantum is well seen from the comparison of NMR for the samples with two different periods 0.6 µm and 0.7 µm. As it is clearly seen from the Fig. 3(c) the width of NMR curve for the period 0.7 µm equals ∆B 2 = 20 ± 2 mT, that is ∆B 1 /∆B 2 = (0.7) 2 /(0.6) 2 . Thus at g ≪ 1 we observe NMR wich is very similar to weak localization NMR in chaotic open billiards [2]. But the value of NMR is much larger. It is necessary to add that the following two conditions should be satisfied in order to observe the effects described above: (i) the influence of the fluctuation potential should be as low as possible, and (ii) the area of billiard should be relatively large. That is because one or both these conditions were not met that these effects were not observed in [13,14,17] (except for the commensurability peak observed in [14] at g ≪ 1, that might still have been of different nature, for the temperature dependence was exponentially strong).
Let us discuss the results obtained. First turn to the temperature dependence at the transition from g > 1 to g ≪ 1. It differs from the accepted picture of the metal-insulator transition in 2D and 3D electron systems. This picture is based on the concept of Anderson localization of electrons, be it the model of minimal metallic conductivity (MMC) or the scaling theory (ST) [18]. According to it at k F l ∼ 1 or g ∼ 1 in a macroscopic 2D or 3D system the transition should occur from the metallic behavior (as in MMC model) or from the weak localization (as in ST) to the strong localization characterized by the exponential temperature dependence of the activation type (hopping conductivity) or of the Mott type (variable range hopping conductivity). In real systems the transition can be quite complicated, but for g ≪ 1 the state with hopping conductivity is always realized [12,13]. We know only one work [19] in which the linear temperature dependence for g ≪ 1 was observed in thin In 2 O 3 films. Recently a similar system was considered theoretically in [20] within the model of metal grains with g ≫ 1 coupled by tunneling in a way that the conductivity of the macroscopic sample was low g ≪ 1. At g ≪ 1, due to inelastic electron-electron scattering the linear dependence g(T ) was obtained in [20] in a wide temperature range. The dependence changes to the exponential one at T ≪ e 2 /C, where C is the capacitance of a metal grain. Our results are not in agreement with the predictions of this theory. Firstly, in our case g(T ) is weaker than linear. Secondly, it is observed at T ≪ e 2 /C, because the Coulomb energy for our samples e 2 /2C ≈15-25 K. This means that the low conductivity of the lattice of the Sinai billiards can not be explained by the model of metal lakes coupled by weak tunnelling junctions. The effects of Coulomb blockade are not manifested in the experiment, because one observe no features in g(V g ) dependence. So we have to assume that at g ≪ 1 the coupling between the billiards is stronger than that provided by tunneling. The behavior of the lattice in the magnetic field supports this assumption. In Fig. 2(a) one can see the Shubnikov-de Haas oscillations, which give the electron density in the lattice saddle points connecting the billiards. It coincides with the density determined from the Hall effect that should give the concentration in these points [21]. The electron density in these points weakly changes with the strong change of g. It decreases only by about 30% while g drops by a factor of 30, and its magnitude, equaled to 4.1 × 10 10 cm −2 , is only three times less than N S inside the billiard even for the state with the lowest value of g. This means that the Fermi level in the saddle point lies several meV above the barrier at g ≪ 1, and an electron should ballistically move through the "bottle neck" to go from one billiard to another. The behavior of commensurability peak and of NMR support this picture. Hence, we should come to a paradoxical conclusion that 2D lattice of Sinai billiards coupled via conducting "bottle-neck" can have very low conductivity g ≪ 1 but simultaneously exhibits the properties typical for metallic ballistic systems rather then for the insulators. This conclusion is in a drastic contradiction with the standard picture of Anderson localization. Let us discuss possible reasons for such a situation and consider it first from the weak localization side. The transition from the weak to strong localization, caused, for example, by the decrease of temperature, should be accompanied by the increase of the phase coherence length. Due to this increase, the number of localized trajectories should increase until at T = 0 all the trajectories become localized. In the system investigated this increase can be at least hampered, because an electron can loose the phase coherence inside a billiard before it leaves for the other billiard through the "bottle-neck". A simple estimation gives for the dwell-time of the electron inside a billiard τ = 10 −8 s. The collimation effects can only increase this time. The estimation shows that τ can be larger than the time of phase coherence, which is of the order of τ ϕ = 10 −9 s at 40 mK. This means that even at the lowest available temperature an electron can loose the phase coherence on the length scale L = d. Then the resistance is the classical sum of the resistances of individual billiards, which can yield any value of g. The situation discussed is to some extent similar to that considered in [20]. In contrast to [20] in our system the quantum dots are separated by the conducting "bottle-neck" instead of the tunnel barrier as in [20]. Obviously, this should lead to the weaker temperature dependence of the conductivity, that is just observed in our experiments. Nevertheless, it is not clear what happens at T → 0, because the Coulomb blockade effects are not observed in our case. The description of the system from the strong localization side is complicated, because one can not start from the ground state of electron in the well, for it represents a multilevel system with the large (∼ 100) number of electrons. In any case the description of 2D lattice of coupled Sinai billiards and the phenomena described in the present work presents a challenge to the modern theory of quantum transport in the condensed matter.
In conclusion, we investigate a new type of dynamic chaos system -2D lattice of coupled Sinai billiards. We find that its transport properties are very unusual. They characterize the system as a metal rather than an insulator at g ≪ 1. The whole set of experimental data (temperature dependence of conductivity, NMR, and commensurability peak of magnetoresistance) is in contradiction with the standard picture of Anderson localization, and testifies that the system possesses quite unusual transport properties the description of which requires new theoretical approaches.
Note added. Since the completion of this paper, D.P.Druist et al [22] have reported the observation of metallic behavior at g ≪ 1 in a completely different system -a layered three-dimensional semiconductor structure -under the conditions of IQHE.
We would like to thank M.Entin, V.Falko, A.Charplik, and E.Baskin for useful discussion and V.Alperovich for reading the manuscript. M.V.Budantsev acknowledges "Mission scientifique" from French Embassy in Moscow for the support. This work was supported by RFFI through Grant No 96-02-287 and by NATO Linkage through grant No HTECH.LG.971304. | 2014-10-01T00:00:00.000Z | 1998-02-18T00:00:00.000 | {
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248725459 | pes2o/s2orc | v3-fos-license | Diagnostic yield of inpatient capsule endoscopy
Background Capsule endoscopy (CE) provides a novel approach to evaluate obscure gastrointestinal bleeding. Yet CE is not routinely utilized in the inpatient setting for a variety of reasons. We sought to identify factors that predict complete CE and diagnostically meaningful CE, as well as assess the impact of inpatient CE on further hospital management.1 na d2 Methods We conducted a retrospective review of patients undergoing inpatient CE at a tertiary referral, academic center over a 3 year period. We analyzed data on patient demographics, medical history, endoscopic procedures, hospital course, and results of CE. The primary outcome was complete CE and the secondary outcome was positive findings of pathology on CE. Results 131 patients were included (56.5% were men 43.5% women, median age of 71.0 years). Overall, CE was complete in 77.1% of patients. Complete CE was not related to motility risk factors, gender, or administration modality. Patients with incomplete CE tended to be older, have lower BMI, and Caucasian, however results did not reach statistical significance (p = 0.06; p = 0.06; p = 0.08 respectively). Positive CE was noted in 73.3% of patients, with 35.1% of all patients having active bleeding. Positive CE was not associated with AVM risk factors or medication use. 28.0% of patients underwent subsequent hospital procedures, among which 67.6% identified the same pathology seen on CE. Conclusions Contrary to previous studies, we found the majority of inpatient CEs were complete and positive for pathology. We found high rates of correlation between CE and subsequent procedures. The use of CE in the inpatient setting helps to guide the diagnosis and treatment of hospitalized patients with obscure gastrointestinal bleeding.
Introduction
Video capsule endoscopy (CE) provides a novel noninvasive method to evaluate patients with obscure gastrointestinal bleeding. After initial endoscopy and colonoscopy, clinicians often struggle to determine the next appropriate diagnostic modality, including repeat endoscopy, radiographic modalities, or small bowel evaluation with push enteroscopy or balloon-assisted deep enteroscopy. In contrast to the above methods, CE provides a non-invasive modality to directly visualize the small bowel in its entirety. CE aids in the diagnosis and management of several small bowel diseases, including but not limited to inflammatory bowel disease, celiac disease, polyposis, and gastrointestinal bleeding [1][2][3].
In patients with suspected small intestinal bleeding, CE has demonstrated superiority in identifying sources of bleeding in comparison to both push enteroscopy and small bowel barium radiography [4,5]. In addition to the high rates of meaningful diagnostic findings, there are few contraindications to CE, unlike the other Open Access *Correspondence: ikeslevine@gmail.com modalities [6]. As such, current European Society of Gastrointestinal Endoscopy (ESGE) and American College of Gastroenterology (ACG) guidelines recommend CE as first-line investigation for patients with obscure gastrointestinal bleeding [7,8].
Despite these advantages, CE is not routinely used in the inpatient setting for several reasons. First, variable reimbursement and cost of equipment often precludes inpatient CE. Additionally, prior studies have demonstrated decreased completion rates among inpatient CE [9,10]. Hospitalized patients tend to be more sedentary and are prone to delayed transit time through the gastrointestinal tract [9]. However, while previous studies have noted the lower completion rates of CE among inpatients, they have failed to characterize patient and clinical factors that predict these incomplete CE.
Moreover, the diagnostic potential of CE carries tremendous importance. Patients with obscure gastrointestinal bleeding tend to have prolonged length of stay, frequent hospital admissions, and invasive procedures [11]. Determining an etiology of obscure gastrointestinal bleeding has the potential to decrease length of stay, prevent readmissions, and prevent repeat unnecessary invasive procedures.
Lastly, to our knowledge, previous studies have failed to correlate the results of inpatient CE and further inpatient management. CE has the potential to greatly impact the selection of further therapeutic interventions which is a decision that often challenges clinicians. CE can help differentiate the patients who would most benefit from further endoscopic procedures (or push enteroscopy or balloon-assisted deep enteroscopy), surgical intervention, medical management, or even no further intervention at all.
In January 2018 NYU Langone Health obtained the necessary technology to perform small bowel video capsule endoscopy for hospitalized patients onsite. The hospital had also recently transitioned to a gastroenterology (GI) hospitalist led consult service which established the in-house availability of a gastroenterologist trained in video capsule endoscopy and balloon-assisted deep enteroscopy. Thereafter small bowel evaluation with video capsule endoscopy became a regularly utilized resource in the evaluation of obscure gastrointestinal bleeding for hospitalized patients in accordance with recent guidelines [7,8].
In this study, we sought to (1) identify patient and clinical factors that predict complete inpatient CE; (2) identify predictors of yielding diagnostic results on inpatient CE; and (3) assess impact of inpatient CE on further hospital management and rates of 30-day readmission.
Study population and variables
We identified all patients age 18 and older undergoing inpatient CE at NYU Langone Health Tisch Hospital/ Kimmel Pavilion between January 1, 2018 and October 30, 2019. Charts were reviewed for patient demographic information, including age, gender, and medical history. Elderly status was defined as age over 65 years at the time of CE. Obscure gastrointestinal bleeding was defined as gastrointestinal bleeding without a known source despite esophagogastroduodenoscopy (EGD) and colonoscopy. Indication for CE was divided into overt obscure gastrointestinal bleeding (hematemesis, hematochezia, or melena) or occult obscure gastrointestinal bleeding (iron-deficiency anemia, chronic anemia). Motility risk factors were defined as comorbid diabetes mellitus or systemic disease affecting motility (i.e. scleroderma, hypothyroidism, amyloid). Arteriovenous malformation (AVM) risk factors were defined as having comorbid cardiac valvular disease or advanced kidney disease (CKD stage 3-5). Charts were reviewed for hospital course, including length of stay, laboratory findings, radiographic imaging, procedures, and rates of readmission. Thirty-day readmission was defined as non-elective hospital admission at NYU Langone Health within 30-days of discharge from index admission.
Capsule endoscopy protocol
CE with PillCam SB3 12 h capsule was used. All patients who underwent CE had either nothing by mouth (except for medication) during the 12 h before CE administration or had a bowel preparation (2 L polyethylene glycol or equivalent preparation, or 4 L if performed after colonoscopy preparation). CE was administered either orally or endoscopically, at the discretion of the administering gastroenterologist.
Capsule endoscopy results
All CE were reviewed by the same GI hospitalist to ensure consistency. CE were defined as complete (complete CE) if the capsule passed into the cecum within the recording period. The yield of diagnostically meaningful pathology on CE (positive CE) was defined as finding abnormal small bowel bleeding pathology. Abnormal small bowel bleeding pathology was characterized as arteriovenous malformation (AVM), small bowel ulceration or erosion, small or old blood in the lumen with no identifiable source, brisk active bleeding, gastritis/ duodenitis, or polyp/mass. Additionally, the presence or absence of active bleeding was identified.
Subsequent procedures
Charts were reviewed for subsequent procedures following inpatient CE during the index admission. Subsequent procedures included EGD, colonoscopy, surgery, interventional radiology procedure, double balloon enteroscopy (antegrade or retrograde), and push enteroscopy. All subsequent procedures operative notes and images were reviewed by a single gastroenterologist to determine if the findings correlated with CE findings.
The primary outcome was complete CE. Secondary outcome was positive CE.
Statistical analysis
Continuous variables were summarized using means and standard deviations and compared using the Student's t-test, whereas categorical variables were summarized using proportions and compared using Chi-square test. All tests were considered significant at a 2-sided p-value less than 0.05. STATA was used to perform all statistical analyses. The study was approved by the NYU Langone Health Institutional Review Board.
Study population
A total of 131 patients were identified. Overall, the median age was 71.0 years, 62.6% were elderly, 56.5% male and 43.5% female (Table 1). 87.0% of patients received bowel preparation (2-4 L of polyethylene glycol preparation solution or equivalent) prior to the CE. Motility risk factors were identified in 49.6% of patients.
AVM risk factors were identified in 37.4% of patients. The most common indication for CE was overt obscure bleeding (n = 81; 61.8%) followed by occult obscure bleeding (n = 48; 36.6%).
Positive CE
96 patients (73.3%) had a positive CE. (Table 2) Positive CE was not associated with AVM risk factors or NSAID use. Use of PPI, anticoagulation or antiplatelet were not associated with positive CE. BMI tended to be higher in patients with complete CE and those with positive CE, however this did not reach statistical significance. (Table 3).
A total of 46 patients (35.1%) were found to have active bleeding. The most common finding among positive CE was AVM (n = 46; 47.9% of positive CE, Table 4), followed by brisk active bleeding (n = 25; 26.0%).
Discussion
In this retrospective study of patients undergoing inpatient CE, we found high rates of complete and positive CE. The clinical significance of these findings suggests a prominent role of inpatient CE in identifying and diagnosing the etiology of obscure gastrointestinal bleeding. Furthermore, we found congruence between CE results and subsequent endoscopic procedures, suggesting CE can assist in the selection of appropriate subsequent procedures and therapeutic intervention. To our knowledge, our study is the first to correlate CE with subsequent inpatient procedures. Our finding of a 77.1% completion rate is higher than prior inpatient studies and comparable to outpatient studies [9,10,[12][13][14]. Ben-Soussan et al. [15] first identified inpatient status as a risk factor for gastric capsule retention among 29 inpatients. Subsequent studies have noted inpatient status as a risk factor for incomplete small bowel evaluation with CE, noting completion rates as low as 65-69% [9,10,16]. The proposed mechanism for decreased completion rates among hospitalized patients includes immobility, the supine position of most hospitalized patients, stress induced by acute illness, and gastroparesis, either attributed to an underlying medical condition (i.e. diabetes mellitus, hypothyroidism, or scleroderma) or medication use [9,15]. Yet all of these studies were limited by small numbers of inpatients, ranging from 29 to 70 patients. Our higher completion rate may relate to our standardized protocol for bowel preparation prior to CE, as 85.1% of patients had polyethylene glycol before the CE.
Our study found several patient demographics that trended towards incomplete CE, including older age, elderly status, lower BMI, and Caucasian race, yet these did not reach statistical significance. It is particularly noteworthy that lower BMI, and lack of obesity, were associated with incomplete CE, as obesity is correlated with gastroparesis. Larger studies are necessary to further evaluate these characteristics.
The lack of association between mode of capsule administration and completion rate is noteworthy, as delayed gastric transit time has been associated with incomplete CE [10,15]. As such, one may hypothesize that bypassing the stomach by administering the capsule endoscopically into the duodenum may lead to higher rates of complete CE. This may have perhaps been mediated by the use of 12 h capsules. Larger trials are necessary to evaluate this hypothesis further.
Our findings of positive CE fall within the range of reported positive CE in outpatient studies (38% and 83%) [17][18][19][20][21][22]. Among the few inpatient studies, a recent retrospective study of 204 patients (103 inpatients) found the diagnostic yield of identifying a bleeding source by CE was 39% among inpatient compared to 34% outpatient (findings not statistically significant) [23]. When performed for obscure bleeding with reported overt blood loss, they found a significant difference (Diagnostic yield: 49% inpatient vs 23% outpatient; OR 3.32, 95% CI 1. 25-8.84, p = 0.02). Other retrospective studies have found that advanced age was associated with increased diagnostic yield of CE [24][25][26]. We observed a trend towards elderly age being associated with a positive result on capsule endoscopy which did not reach statistical significance. We propose our high rates of positive CE may relate to proper selection of patients, as the majority of patients had previous endoscopy and/or colonoscopy prior to CE. Utilizing CE in inpatient settings for its diagnostic potential is imperative. By establishing a diagnosis, patients are spared from unnecessary repeat invasive procedures on subsequent hospitalizations, thereby increasing patient safety and decreasing hospital costs.
Of note, prolonged small bowel transit time, which may lead to incomplete CE, has been associated with higher diagnostic yield [27][28][29] As such, the endpoints of complete CE and positive CE may have an inverse relationship.
Identifying the role of inpatient CE in guiding further management remains a major factor preventing its widespread use. We identified that a large proportion of our patients underwent a subsequent inpatient procedure following CE for the same indication. Within the limitations of a retrospective study, we assume that the selection of which subsequent procedure was directly influenced by the CE results, and the high rate of congruence between CE findings and invasive procedure findings attests to this. As such, inpatient CE not only provides diagnosis, but can also aid in therapeutic intervention. Utilizing inpatient CE results to perform subsequent procedures in a timely fashion during the same hospitalization provides therapeutic intervention, can potentially decrease length of stay and hospital readmissions, and can certainly decrease delays that would take place in the outpatient setting. In particular, the current study benefited from being performed in a tertiary academic center where double balloon enteroscopy is routinely performed and a GI hospitalist is available on-site to accommodate the procedure in a timely manner. It is possible that patients in similar hospital systems will see the greatest benefit from inpatient CE, as the majority of subsequent procedures performed in the current study were double balloon enteroscopy. Patients with obscure gastrointestinal bleeding have frequent bleeding events, often requiring hospitalization. Our study is the first to our knowledge to analyze readmission after inpatient CE. Our rates of 30-day readmission is higher than other studies assessing admission rates for obscure gastrointestinal bleeding following double balloon enteroscopy [30]. We anticipated that CE would affect hospital readmission in two opposite manners. First, several investigators have noted negative CE predicts low rebleeding risk, though this conclusion is contested by other studies [31,32]. Alternatively, specific treatment after positive CE is associated with decreased rebleeding [32]. By identifying culprit lesions and providing therapeutic interventions, we anticipated positive CE findings would be associated with decreased hospital readmission. However, positive CE was not associated with decreased 30-day readmission. We suspect that 30-day follow up is too short an interval to assess for decreased hospitalizations, as previous studies have noted median time to rebleeding is 15 months [33]. Therefore, longer follow-up time is necessary to assess whether inpatient CE can decrease hospital admissions.
The strengths of the current study include the large cohort of inpatient CE. Additionally, it is the first study to correlate CE with subsequent endoscopic procedures, as well as assess readmission after inpatient CE. There are several limitations of our study. The retrospective nature precludes controlling for all cofounders, and relies on information documented in the electronic medical record. We also recognize the potential lack of generalizability as the cohort was recruited from a tertiary academic center. Lastly, several of our findings trended towards significance, and larger trials are necessary to further elucidate associations with positive CE, complete CE, and hospital readmission.
In conclusion, in this retrospective study of inpatient CE, we found high rates of complete CE, positive CE, and 30-day hospital readmission. Additionally, we correlated CE findings with subsequent procedures. Clinicians may utilize inpatient CE to guide further inpatient management in obscure gastrointestinal bleeding. | 2022-05-12T23:59:29.114Z | 2022-05-12T00:00:00.000 | {
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3424838 | pes2o/s2orc | v3-fos-license | Hemolytic anemia in alcoholic liver disease: Zieve syndrome
Abstract Rationale: Zieve syndrome, a rarely reported disease resulting from alcohol abuse, consists of a triad of symptoms: hemolytic anemia, cholestatic jaundice, and transient hyperlipidemia. It is largely under-recognized and under-reported, possibly because of unawareness of the condition by physicians. Here, we report a case of Zieve syndrome managed at the Jilin University First Bethune Hospital. Patient concerns: A 30-year-old Chinese woman presented with a 4-month history of fatigue, yellowish discoloration of the eyes, and tea-colored urine. She had been a heavy drinker for 2 years prior to onset of the disease with an average daily alcohol intake of 60 g/d and more than 80 g/d for the previous 6 months. Diagnosis: The diagnosis of Zieve syndrome was confirmed based on hemolysis and cholestatic jaundice secondary to alcoholic liver disease and heavy drinking. Bone marrow biopsy and liver biopsy both supported the diagnosis. Interventions: We treated her with abstinence from alcohol and supportive therapy. Outcomes: The patient was discharged 14 days after admission with an improvement in symptoms, which continued to subside during the 2-month follow-up period. Lessons: Doctors confronted with hemolysis in a patient with alcoholic liver disease should be aware of the under-reported Zieve syndrome. Recognition of this syndrome could help doctors avoid unnecessary invasive procedures and emphasize the importance of alcohol abstinence as the mainstay of management. Glucocorticoids may not be useful in treating hemolytic anemia in Zieve syndrome.
Introduction
Zieve syndrome was first reported by Leslie Zieve in 1958 based on a retrospective study of patients with similar clinical features. [1] The syndrome was not widely accepted by the medical community until Balcerzak published a prospective study and corroborated Zieve's report in 1968. Zieve syndrome is considered to be rare, with about 200 case reports published in the literature since 1958. [1] The pathogenesis of Zieve syndrome remains obscure. Abstinence from alcohol remains the most effective way of treating Zieve syndrome, and most patients will recover within 4 to 6 weeks after alcohol withdrawal.
Case presentation
A 30-year-old Chinese woman sought medical attention at the Jilin University First Bethune Hospital with complaints of yellowish discoloration of the eyes, dark urine, and fatigue for 4 months prior to admission. The patient had been a heavy drinker for 2 years prior to admission, with an average alcohol intake of 60 g/d and more than 80 g/d over the previous 6 months. The symptoms had progressively worsened since onset of the illness, and she had a history of 2 previous local hospitalizations before being seen in our facility.
First hospitalization
During the patient's first hospitalization, she was diagnosed with hemolytic anemia after some routine tests and examination of her bone marrow. The bone marrow biopsy at her local hospital had demonstrated an obviously active bone marrow proliferation.
Profligate, active, nucleated red cells could be seen in a blood smear, and there were signs of hyperplastic anemia, indicating that the anemia was due to hemolysis. At the time, the patient was prescribed a 2-month course of oral prednisolone, but her symptoms worsened after 2 months.
Second hospitalization
The patient's heavy drinking habit persisted even after diagnosis of the illness and her first hospitalization. A second hospitalization occurred 1 month prior to admission at our facility, and it was during this time that the patient was diagnosed with severe acute pancreatitis. A lipid panel also showed serum triglycerides of 311.87 mg/dL. Fortunately, she recovered after treatment at the local hospital. The fatigue and jaundice persisted and worsened nevertheless.
Admission to Jilin University First Bethune Hospital
The patient was then transferred to our hospital for further treatment. She was first admitted to the hematology department of our hospital where a bone marrow biopsy was performed again, the result of which supported the diagnosis of hemolytic anemia but could not explain the hepatic damage and elevated conjugated bilirubin. She was then transferred to the hepatology department.
Physical examination revealed that the patient had scleral icterus. There was facial and conjunctival edema, moon face, and central obesity evident, likely due to the steroid therapy received during her first hospitalization. Her liver could be palpated 6 cm below the right costal margin, and her spleen was palpable 7 cm below the left thoracic cage. There was also mild edema of the lower limbs.
Notable laboratory studies on admission are summarized in Table 1. A peripheral blood smear was compatible with hemolysis and revealed anisocytosis, and an occasional target cell was seen. Hemoglobin electrophoresis was normal, ruling out thalassaemia. An erythrocyte incubation osmotic fragility test was negative, ruling out hereditary spherocytosis. A bone marrow biopsy revealed active bone marrow proliferation of mainly erythroid hyperplasia (Fig. 1). The proportion of granulocytes was normal. The patient's lipid levels tested in the normal or below normal range, in contrast to the elevated triglyceride levels seen during her second hospitalization. Hyperlipidemia in Zieve syndrome can be transient and the patient at that point had been sick for 4 months.
An abdominal ultrasound revealed hepatic steatosis. The inferior border of the liver was 5.8 cm below the right costal margin, and the inferior border of the spleen was 7.1 cm below the left thoracic cage. The oblique diameter of the right lobe of the liver was 16.3 cm, and the spleen pachydiameter was 7 cm. The diameters of the portal vein and the splenic vein were 13 and 11 mm, respectively.
Pulmonary computed tomography scanning showed evidence of mild pneumonitis. A liver biopsy revealed mild fatty degeneration, Mallory bodies, and cholestasis (Figs. [2][3][4][5]. Viral hepatitis, nonhepatotropic viral infection, autoimmune liver disease, and metabolic liver disease were ruled out. We are of the opinion that this patient had Zieve syndrome based on the combined clinical features of cholestasis, hemolytic anemia, and alcoholic liver disease. Treatment consisted of alcohol abstinence, blood transfusion, and administration of glutathione, diuretics, ursodeoxycholic acid, vitamin supple-ments, and cefoperazone sodium/tazobactam sodium. Her condition gradually improved, and she was discharged 25 days after admission with orders to completely withdraw from consumption of alcohol and to take oral glutathione, ursodeoxycholic acid, and polysaccharide iron complex for a period of time. Two months after discharge, her liver size, portal vein diameter, and levels of blood glucose, hemoglobin, bilirubin, alkaline phosphatase (ALP), and gamma-glutamyltransferase (g-GT) were almost normalized (Tables 2-4; Fig. 6) at her followup. Her spleen pachydiameter dropped from 70 to 56 mm, and the patient's general condition was found to be well.
Literature review
A search of the PubMed database uncovered 120 publications that were related to "Zieve's syndrome" or "Zieve syndrome" after removing duplications and unrelated literature. Among the 120 papers, mostly published by Europeans, 96 were published in non-English languages, and 24 were written in English. The publications were in the form of case reports (47), reviews (5), comparative studies (2), English abstracts (21), letters (6), journal articles (114), and a clinical conference (1). The time periods of publication were as follows:1958to1978(79),1978to1998(30),and1998to2017(11).
Discussion
The pathogenesis of Zieve syndrome remains obscure but is defined by the triad of jaundice, hyperlipidemia, and hemolytic anemia. The hyperbilirubinemia seen in patients with Zieve syndrome is often profound given the dual contributory mechanisms of hepatocyte injury and hemolysis. [2] Jaundice of this triad is caused by direct bilirubin elevation, which suggests cholestasis caused by liver damage in alcoholism as the main reason instead of hemolytic anemia. Hyperlipidemia in Zieve syndrome can be transient, as seen in our patient. Suggested mechanisms include an episode of massive mobilization of fat to or from the fatty liver [3] and dysregulated blood lipids due to damaged pancreatic a cells, [4] as alcohol also damages the pancreas.
Zieve suspected that the hemolysis is due to hyperlipidemia and that an abnormal lipid, possibly lysolecithin, may also be present. [5] Some triggering factors, such as lysolecithin and lysocephalin, could markedly aggravate the hemolysis. In addition, alcohol-induced vitamin E deficiency, which reduces polyunsaturated fatty acid levels and causes oxidation of reduced erythrocyte glutathione, leads to enzyme instability and erythrocyte hemolysis. [6] Moreover, Coombs test-negative hemolysis is a salient feature in this triad, which may indicate it is less likely to be an autoimmune hemolytic anemia and would be insensitive to glucocorticoid therapy. Overtreatment of Zieve syndrome with steroids, like our patient experienced during her first hospitalization, may lead to many unnecessary complications. [7] Our patient's diagnosis was established based on a history of heavy drinking, the clinical triad, and other pertinent physical and lab examinations. The patient had a history of alcohol abuse and displayed the triad of jaundice, hyperlipidemia, and hemolytic anemia. Her laboratory examination also supported our diagnosis. Direct bilirubin, ALP, and g-GT were elevated more markedly than aspartate aminotransferase/alanine aminotransferase. The liver biopsy showed that cholestasis existed in the hepatocyte and bile canaliculi (Fig. 4), suggesting cholestatic jaundice. The liver biopsy also demonstrated steatosis, perisinusoidal fibrosis (Fig. 5), and Mallory corpuscles (Fig. 3), which are features of alcohol liver disease. The peripheral blood smear and bone marrow biopsy demonstrated reticulocytosis, and a negative Coombs test supported the diagnosis of Coombs testnegative hemolytic anemia. The patient also experienced transient hyperlipidemia. Subsequent to her abstinence from alcohol and the initiation of other supportive treatment, the patient recovered gradually. Based on the above findings, the diagnosis of Zieve syndrome was established. Interestingly, the patient also suffered from severe acute pancreatitis, which may be related to Zieve syndrome. The patient had resumed drinking alcohol after her first hospitalization and diagnosis of hemolytic anemia, and afterward the anemia and jaundice became more serious. She then developed acute pancreatitis. Some reports suggest that pancreatitis is an essential feature of Zieve syndrome, [8,9] with possible mechanisms including an increase in lysophosphatide, which could lead to pancreatitis.
Zieve syndrome is a special type of alcoholic hepatitis and is usually recognized by its associated features: the clinical triad of hemolytic anemia, alcoholic fatty liver, and unexplained jaundice. Unfortunately, this association may not be recognized by most physicians. When diagnosed with Zieve syndrome, most patients will recover 4 to 6 weeks after alcohol withdrawal. Continuing to drink alcohol, however, may aggravate the illness and potentially prove to be fatal.
The critical feature that distinguishes Zieve syndrome from acute alcoholic hepatitis is hemolytic anemia, the hallmark of Zieve syndrome. The differential diagnosis between alcoholrelated anemia and Zieve syndrome can be seen in Table 5. The prognosis of the 2 disorders is different: 40% of patients with acute alcoholic hepatitis may progress to liver failure and even death, while in Zieve syndrome, abstaining from alcohol would result in a resolution of the symptoms. [10] Patients with Zieve syndrome may have a higher alcoholic hepatitis score, and this could potentially lead to overtreatment with steroids, inducing immunosuppression and possibly leading to further complications. [7] Early recognition and diagnosis of Zieve syndrome could avoid unnecessary invasive examinations [11] and treatment. Patients with Zieve syndrome will often exhibit right upper quadrant pain, without acute cholecystitis or bile duct obstruction. Articles have reported that patients with severe jaundice and abdominal discomfort were planned for surgery, but with the eventual recognition of Zieve syndrome as the primary cause of their illnesses, the invasive procedures were avoided. [12,13] For Zieve syndrome, abstinence from alcohol is the most effective treatment. Glucocorticoids, most useful for autoimmune hemolytic anemia, may not be useful for treating Zieve syndrome and may in fact lead to further complications. [7]
Conclusions
Zieve syndrome should be more readily recognized by physicians and the medical community. Best accomplished by increasing medical education, this would enable providers to make the correct diagnosis and perform proper treatment. We anticipate that with the growing consumption of alcoholic beverages in China, there will be more patients afflicted with Zieve syndrome in the future. The early diagnosis and treatment of such patients is of paramount importance to their management. There is a real need to report and increase the body of published English language medical literature describing Zieve syndrome, as most of the present literature is written in non-English languages. Such a spread of English-based medical reporting would more widely circulate necessary knowledge of the disorder and aid in the progress of treating and ameliorating this disease. | 2018-04-03T03:29:28.651Z | 2017-11-01T00:00:00.000 | {
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228975873 | pes2o/s2orc | v3-fos-license | Researching cycling innovations The contested nature of understanding and shaping smart cycling futures
With this commentary, we share our re fl ections at the end of a fi ve-year interdisciplinary research project (from 2016 to 2020) on cycling innovations in livinglabsacrossthe Netherlands. Thecommentaryis the product of a collective writing effort of the researchers. It combines re fl ections on both the content of our research project (cycling through the lens of innovations)fromvariousdisciplinaryperspectives – includingsocio-technicaltransitions,mobilities,urbandesign,trans-portplanningandhistory – andre fl ectionsonthetransdisciplinaryapproach(livinglabs)underpinningourresearch.We hope that our re fl ections can bene fi t other researchers concerned with similar topics and approaches. Our research project, Smart Cycling Futures (SCF), began as a proposal within the Smart Urban Regions of the Future (SURF) program, funded by the Dutch Council for Academic Research (NWO). This call of € 16.5 million was co- fi nanced by the Dutch Ministries of Infrastructure & Environment and Internal Affairs. As one of the fi ve winning consortia – the other consortia focused on self-driving cars, in fl uencing travel behaviour, demand-oriented public transport, and small-scale experiments – our group of mobility-oriented researchers played an active role in shaping the evolving landscape
With this commentary, we share our reflections at the end of a five-year interdisciplinary research project (from 2016 to 2020) on cycling innovations in living labs across the Netherlands. The commentary is the product of a collective writing effort of the researchers. It combines reflections on both the content of our research project (cycling through the lens of innovations) from various disciplinary perspectivesincluding socio-technical transitions, mobilities, urban design, transport planning and historyand reflections on the transdisciplinary approach (living labs) underpinning our research. We hope that our reflections can benefit other researchers concerned with similar topics and approaches. Our research project, Smart Cycling Futures (SCF), began as a proposal within the Smart Urban Regions of the Future (SURF) program, funded by the Dutch Council for Academic Research (NWO). This call of €16.5 million was co-financed by the Dutch Ministries of Infrastructure & Environment and Internal Affairs. As one of the five winning consortiathe other consortia focused on self-driving cars, influencing travel behaviour, demand-oriented public transport, and small-scale experimentsour group of mobility-oriented researchers played an active role in shaping the evolving landscape of smart mobility innovation in the Netherlands.
With this commentary, we share our reflections at the end of a five-year interdisciplinary research project (from 2016 to 2020) on cycling innovations in living labs across the Netherlands. The commentary is the product of a collective writing effort of the researchers. It combines reflections on both the content of our research project (cycling through the lens of innovations) from various disciplinary perspectivesincluding socio-technical transitions, mobilities, urban design, transport planning and historyand reflections on the transdisciplinary approach (living labs) underpinning our research. We hope that our reflections can benefit other researchers concerned with similar topics and approaches.
Our research project, Smart Cycling Futures (SCF), began as a proposal within the Smart Urban Regions of the Future (SURF) program, funded by the Dutch Council for Academic Research (NWO). This call of €16.5 million was co-financed by the Dutch Ministries of Infrastructure & Environment and Internal Affairs. As one of the five winning consortiathe other consortia focused on self-driving cars, influencing travel behaviour, demand-oriented public transport, and small-scale experimentsour group of mobility-oriented researchers played an active role in shaping the evolving landscape of smart mobility innovation in the Netherlands.
Our research originated from the growing consensus that cycling has a positive impact on social and ecological sustainability. In recent years, many cities and urban regions have articulated ambitious cycling policies to encourage growth in cycling rates. Likewise, academic attention for cycling by transport planners, geographers, and historians has boomed in recent years. While many studies have studied long-term effects of cycling policies, the social and geographical determinants of cycling, and the environmental impacts of cycling (Heinen et al., 2010;Pucher et al., 2010), the effect of smart innovations on the larger cycling system, urban resilience and liveability has yet to be studied systematically.
Our project explored how cycling innovations may help build the foundations for vital, more resilient and liveable urban regions. Cycling innovations promise to deliver substantial benefits to urban areas in terms of accessibility, social equality, health, liveability, and greenhouse gas emission reductions. However, a host of smart cycling innovations has not been fully realized because they are not yet embedded in existing urban environments, infrastructures and institutions. We lack an understanding of to
Contents lists available at ScienceDirect
Transportation Research Interdisciplinary Perspectives j o u r n a l h o m e p a g e : h t t p s : / / w w w . j o u r n a l s . e l s e v i e r . c o m / t r a n s p o r t a t i o n -r e s e a r c hi n t e r d i s c i p l i n a r y -p e r s p e c t i v e s what extent these innovations have either positive or negative social, economic, and environmental impacts. Cycling can be conceptualized as a "socio-technical system in transition" (Rotmans et al., 2001;Geels, 2002;Shove, 2012;Gössling, 2013). Such a concept focuses on the transformative change in urban mobility structures by looking beyond the bicycle as a technological artefact and its supporting physical infrastructures. Instead, it highlights cycling practices in everyday life as embedded in spatial, historical, social, cultural, economic, and political structures. These socially embedded structures provide stability to cycling as a "system". At the same time, such structures may also impede efforts towards more radical change and substantial improvements to cycling practices. Much of the societal debate on mobility and, relatedly, the way that urban design is approached, remains car-centric by default, with cycling, walking and other ways of moving through space or dwelling being marginalised (see Prytherch, 2018;Verkade and Te Brömmelstroet, 2020). From innovation studies, we know that novelties paving the way towards alternative futures that depart from the current car-centric mobility regime are generally first introduced on a smaller scale as pilot projects in an experimental environment (Hoogma et al., 2002;Brown et al., 2003;Bulkeley et al., 2015). To engage with the process of transition through experimentation, our project emphasized the role of so-called urban 'living labs'. Through transdisciplinary collaboration in various urban living labs, we studied how these processes of innovation to the socio-technical system of cycling can be accelerated.
Given the call's requirements, we worked closely together with four Dutch cities and their corresponding regions. These cities included Amsterdam, Eindhoven, Utrecht, and Zwolle which also participated in co-funding the project. 1 Although this set of cities (and surrounding regions) differ considerably in terms of size, composition, location, and design, they share a common belief in the potential of cycling to deal with various contemporary urban challenges. Participating in the SCF project enabled these cities to obtain policy-relevant knowledge in relation to how smart technologies and changing mobility patterns may shape the future of cycling.
To this end, four living labs were co-created in these four Dutch cities, in which practitioners (i.e. local/regional authorities and innovators/entrepreneurs) and researchers engaged with each other over the entire duration of the project. This bottom-up approach resulted in a variety of living labs across the four cities (see Table 1). Living labs were loosely understood both as a form of collaboration, and as actionable research projects. The positioning of 'living labs' as a key concept forged a sense of shared ambition and approach across researchers with diverse disciplinary backgrounds, whilst also enabling collaboration between researchers, policy actors, business, entrepreneurs and civil society actors. For policy actors, on the other hand, the living lab concept generated expectations regarding 'useful cycling research with impact' that moved beyond conventional research-policy interfaces.
Monitoring and evaluating these living labs and the ways in which they unfolded generated valuable lessons about future cycling innovations, and on the potential and limitations of the living lab approach. In this commentary, we aim to share these lessons with the wider research community, based on a collective brainstorm session in which four distinct themes emerged. These four themes are used below to structure our commentary: 1. Challenges and tensions of a living lab approach in relation to academic research. 2. Questions about the position of existing cyclists in relation to cycling innovations 3. Realisation of the limited understanding of the specificness of cycling in transportation thinking 4. Awareness about the political nature of doing critical research on cycling innovations while also accelerating their introduction through living labs. Reflection 1 focuses on the living lab process itself, while Reflections 2 and 3 discuss current approaches to cycling innovation; Reflection 4, finally brings the discussions on the process and the contents of living labs together. For the reader who may be interested in diving deeper in a particular subject we provide a full overview of publications that came out of the project thus far in the Appendix 1.
Reflection 1: challenges of living labs
Why did we adopt a living lab approach in this research project about cycling innovations? We observed that experimentation has become a contemporary and diverse phenomenon in governing urban transformations, and that it is key to understanding, designing and navigating processes of urban innovation (Evans et al., 2016). Drawing on a long tradition of scholarly work on socio-technical experimentation (Sengers et al., 2019), we know that successful innovation depends to a large degree on the early involvement, enrolment and collaboration of a variety of stakeholders such as researchers, users, businesses and regulators. On first sight, this seems much harder than just introducing a new product or service. However, testing an innovation in a real-world living lab environment allows for learning by doing, fostering new partnerships and collaborations, testing and verifying assumptions across technical, economic, policy or cultural dimensions, generating a sense of collective direction and future possibilities, and as such generating and broadening the potential for innovations that are more socially robust and acceptable.
Nevertheless, the living lab approach also raised a number of challenges. Living lab experimentation is not only about learning about an innovation (such as the desirability and impact of cycling innovations), but also about understanding the processes of experimentation. How should living labs be set up, managed and improved to provide the conditions for joint learning and reflection? An important success factor of living labs is to align levels of knowledge and expectations about experimentation processes across organizations. In our project, instead of aligning this upfront we practiced learning by doing through a joint process of co-creation, in which throughout the first 18 months we collectively explored which questions, processes, mechanisms and resources were needed and available in each urban context.
We experienced that the resulting challenges can be overcome by making them explicit. For instance, we quickly learned that the phasing and development of a PhD research project is fundamentally different from the implementation process of a living lab, in which the dynamics of multiple actors and interests create high levels of uncertainty. Making this explicit led to an alignment of learning goals, expectations, activities and roles. In some cases, PhD research became more deeply linked to actual living lab projects, whereas in other cases PhD research acted as a sounding board for reflection on the basis of fundamental research. In Amsterdam, for example, the living lab aiming to accommodate and further encourage the growth in bike-train commuting took a long time to take shape, so it was not possible for the PhD student to wait for the "exchange bike" system to be launched. 2 Instead, he participated in the meetings leading up to the experiment, designed the questionnaire addressed to users of the system, and conducted a large-scale survey of people using the bike-train combination for commuting in the Netherlands.
This process of "learning by doing" raised questions about how to organize leadership and management of a living lab. In the start-up phase, our living labs were mainly platforms of interaction with (sometimes too) broad visions and varying expectations. All parties (academics, innovators, policy makers) were expected to be involved in the living lab based on their own motivation to learn, and had an equal weight in the decision-making and development of the process. This is typical for a living lab approach, but also led to difficulties in arriving at more specific decisions about which cycling innovations to experiment with. When some of the living labs decided to appoint a dedicated local manager at a later stage, this led to immediate progress. Imposing such a management structure took the co-creation phase into a more traditional project form, but fast-tracked progress in terms of living lab implementation. The role of these managers was to navigate between the academic and practical sides of the living lab and to take responsibility for its progress by providing clarity, direction and action to the living labs. Additionally, they also provided the linkages with formal governance structures.
Having partners from different backgrounds, roles and disciplines also posed challenges of understanding each other's language and logic. Practitioners tended to struggle with learning from academics, as the latter were working on more fundamental questions that may be less relevant for the daily practice of local governments and vice versa. What limits the learning potential is that stakeholders are grounded in different contexts representing different professional 'worlds', with different languages and professional jargon (English vs Dutch; abstract vs practical), different incentive structures, different outputs (policy & concrete plans vs. scientific articles), and different timeframes (long vs short term). We believe that the continuous contact during living lab experimentation contributed to overcoming some of these differences and in any case to learning about them and building connections across these two worlds for future collaborations.
Reflection 2: including existing cyclists
One central tenet in the living lab approach is a user-driven, citizencentric approach to innovation (Bergvall-Kåreborn and Ståhlbröst, 2009). Envisioned users can be included in the design process through lobby groups, institutions or direct representation. Users can also be included in evaluating/improving the innovation on the ground. Our project mainly took this second route. Discussing the different cycling innovations, however, raised fundamental questions about who the envisioned user should be.
Current cycling policies, planning and innovations have a strong tendency to focus primarily on increasing the attractiveness of cycling for people who do not currently cycle (Bruno and Nikolaeva, 2020). Typically, policies and innovations that try to convince people to get out of their cars and on a bicycle come with the unstated premise that people who drive will change their behaviour when they see new infrastructure, new monetary incentives, or new studies on the health benefits of cycling.
While such policies may have an important role to play, in our living labs we became increasingly convinced that such a focus can constitute a distraction from paying attention to the critical role of people who already cycle in sustaining and promoting a transition towards a more sustainable mobility system. Building on previous Dutch research, we argue that a cycling culture is shaped and defined by existing cyclists, and that a transition to a sustainable transportation system starts with understanding, supporting, and investing in the practices of those people. As illustrated in our research (Nello-Deakin and Harms, 2019; Nello-Deakin and , the existence of a critical mass of existing cyclists constitutes a form of "human infrastructure" which plays a critical role in sustaining and encouraging urban cycling for all. Developing policies that improve the experience of existing cyclists helps advance a mode shift through the process of social feedback (Macmillan and Woodcock, 2017;Skov-Petersen et al., 2017), but it also furthers the transition to sustainable mobility by investing in the people most likely to lead that transition. It works to ensure that people who currently cycle keep cycling in the future, or more specifically, after they go through important 'life events' (Delbosc and Nakanishi, 2017;Jones et al., 2014;Zhang and Van Acker, 2017).
In practice, this involves rethinking the working definition of mode substitution. When considering which car trips could be potentially substituted with bicycle trips, it is easy to forget about all the car trips that are already not occurring because of existing cycling trips. In this latter form of mode substitution, persons who never drive have substituted all their driving for cycling (Piatkowski et al., 2015). Current cost-benefit analyses for cycling in the Netherlands, however, only look at the benefits that would result in people moving from driving to cycling and do not consider the costs of people moving from cycling to driving. This is remarkable, as a 21 year longitudinal study of mode shift in the Netherlands found that people were nearly three times as likely to shift from riding a bicycle to another mode than they were to shift from another mode to riding a bicycle (Oakil et al., 2016).
Rather than focusing exclusively on people who drive and assuming that any cycling investment that might lead to a change in their behaviour would be equally appreciated by existing cyclists, an approach that focuses on existing cyclists takes seriously the needs of people who have already made the choice to travel sustainably by creating policies that support them directly (Bruno and Nikolaeva, 2020). In the Smart Cycling Futures project, this emphasis on existing cyclists was reflected in the living lab in Amsterdam, which sought to provide a better solution for bike parking in the vicinity of train stations primarily for the benefit of existing cyclists.
Reflection 3: the conceptual curb
In Reflection 1, we discuss how living lab partners have to find a common language to understand each other's roles. During our active engagement with cycling innovations, however it became evident that the same was true for the topic of cycling itself.
Metaphorically speaking, we found that the cyclist travels along a boundary that is a ubiquitous feature of every street: the curb. The curb is the clear physical boundary that divides the sidewalk from the street; the people who walk from the people who drive. It represents an invisible boundary between the logic of walking and the logic of driving a car (Hamilton-Baillie, 2004). We see the bicycle as a boundary object through which logics on either side of the curb can be explored; in matters of design, this exploration is both literal and conceptual. In a literal sense, the curb separates the motorized from the human-powered, the people in enclosed vehicles from the people exposed to their environment, and the fast from the slow. Conceptually, we observe that the rules and behaviour that govern these realms are also different. Slower speeds and eye contact leave more time for negotiation; in the absence of lane markings and traffic signs, informal and subtle rules work to allow people to "dance" around each other on the sidewalk. In short, walking is about slow pace and negotiated relations with fellow pedestrians while driving is faster, more anonymous and guided by strict rules (Te Brömmelstroet et al., 2017).
Where does cycling fit in between these two realms? Sometimes the bike is admitted to the sidewalk, but more often, the cyclist has to manoeuvre between the cars. Cycling occupies a variable position within these spaces, and the non-conforming behaviour of cyclists reveals the obduracy of the infrastructure that cyclists often operate within (Latham and Wood, 2015). Where dedicated cycling infrastructure is added to the street, we see an additional modal boundary within the streetscape, thus complicating matters of equity in road space distribution (Nello-Deakin, 2019). Transport planners and engineers may have clear ideas about how to guide cars through the city in a fast, safe, and smooth way, yet cyclists have their own way of relating to the environment and to fellow travelers, and the specifics of this experience has been hitherto largely neglected. In exploring the characteristics of the cyclist, Forsyth and Krizek (2010) ask, "Would urban design considerations and practices be different if the experience of bicycling was given a more central place?". Our research shows that the cyclists' perspective is gradually making its way into the publication of new bicycle infrastructure design manuals that guide the work of practitioners. For academics, there is an opportunity to "mount the curb" by incorporating the research and insights of urban designers in creating environments to enhance the richness of the cycling experience (Liu et al., 2018). While we found that practitioners themselves are aware of the limitations of the 'traffic mode' of thinking, their ability to design bicycle infrastructure as social spaces is limited by requirements of institutions and funding mechanisms that they operate in. Taking the bicycle highway for example, aspects such as speed, directness, and efficiency dominate even when designers are explicitly asked about how cycling infrastructure can enhance user experiences (Liu et al., 2019). This shows us that even in the case of dedicated cycling infrastructure with no requirement to accommodate cars, adherence to strict vehicular parameters is still reflected existing design practices. When considering smart innovations, we should keep in mind that cyclists, like pedestrians, are already capable of smartly negotiating among themselves and that technology should complement, and not detract, from our existing innate ability to interact with others.
Reflection 4: the politics of cycling innovation
What exactly are smart cycling futures? What does it mean to make cycling "smarter", as promised by different cycling innovations? In our work, we analysed descriptions of smart cycling innovations in order to understand what kind of futures are lurking behind promises of making cycling faster, safer, easier and more fun Nikolaeva and Nello-Deakin, 2019;van Waes et al., 2018;Van Waes et al., 2020a). We concluded that smart cycling futures are multiple and contested, and may potentially bring about radically different types of transformations of urban mobility. For instance, while some innovators see smart technology as a way to make mobility even more efficient and the bicycle as a perfect car of sorts (with cyclists being little more than non-car drivers), others call for displacing the dominant narrative of mobility that prioritizes efficiency and speed, and replacing it with an appreciation of the sensory and social dimensions of velomobility (Nikolaeva and Nello-Deakin, 2019;Popan, 2019). Our analyses of a spectrum of cycling innovations and possible "smart cycling futures" served as a mapping of the territory well beyond the focus of our own living labs. These analyses were a way of orienting ourselves in the world of cycling innovations and thinking about their consequences. In conversations related to our living labs in Utrecht and Amsterdam, in particular, we raised questions that followed from our analyses and related to issues such as the transformation of the culture of bike ownership, the way cycling data is treated, and our understanding of the motivations of cyclists. Thus, we drew attention to the politics of smart cycling futures, which open some opportunities but also foreclose others. As academics, we should keep giving attention to that political nature of smart cycling futures and our own role within these. Choices for implementing particular smart cycling innovations in the present inevitably leads us to transformation of cycling practices, subjectivities and meanings of cyclingand we call for more reflexivity on the consequences of those transformations.
We have developed a critical perspective on the necessity, jeopardy and perversity of smart cycling innovations (see Te Brömmelstroet, 2014), recognizing the importance of the convivial simplicity of the existing bicycle. Nevertheless, our consortium was also a vehicle that accelerated local niches of cycling innovations and gave them prominence, thereby contributing to a specific politics of how to deal with niches (Savini and Bertolini, 2019). We were forced to confront the possibility that we had contributed to the dominant view of the bicycle as just another mode in the fleet of smart mobility modes. Could such pragmatic research strategies partly foreclose a future where the humble bicycle acts as a true alternative to predominant discourses around "smart mobility" for our urban mobility futures?
One example of this tension between criticizing and supporting cycling innovations is in our living lab in Utrecht. After a long period of searching for relevant and desired cycling innovations, the city of Utrecht and the researchers chose to develop a living lab around dockless bike-sharing. The academic relevance of this was clear for our team: studying whether a new generation of bike-sharing systems can play a role in a mature cycling context. For the municipality, the main question was whether bike-sharing could help to combat the overabundance of parked bicycles in public spaces across the city. Collaborating on researching these questions with an innovator (Donkey Republic was chosen after a market consultation) allowed the team to research the potential of bike-sharing in a hands-on manner. As a part of this, we supported the innovator by introducing their product on the streets of a popular student city (and global poster child for cycling cities) and by improving the product, studying what works and what does not. However, it also allowed us to study a more critical question: how does commercial bikesharing affect the conviviality of the personal bicycle? What does it mean if people lose a direct sense of ownership of the vehicle that they use? And how might this influence how citizens relate to each other and their surroundings? 3 There is no one-size-fits-all approach to engaging in transdisciplinary research on innovation in a way that strengthens its critical potential. Learning from our own path, we see that each collaboration requires designing one's own way of combining observation and intervention, giving over the lead to other societal parties and taking a clear stance on the importance of particular academic questions. One of the ways that the Smart Cycling Futures project attempted to amplify its contribution to making the politics of cycling innovation explicitly is through regular dissemination of results via events organised for a diverse knowledge community of practitioners in the sphere of mobility and cycling in the Netherlands (from relevant ministries and teams in municipalities to an array of private actors and independent consultants). Furthermore, continuous individual and collective reflection on the role of the researcher in the project as a facilitator and (explicitly or implicitly) an agenda-setter in transdisciplinary projects is key for leveraging the value of such projects both for academic research and for society.
Living labs on cycling innovations: overall reflections
We have shared four reflections from our experiences in the Smart Cycling Futures research project. These reflections are based on the experiences and insights of the academic researchers in five years of living lab research around cycling innovations. With this commentary, we aim to provide guidance for ongoing and future research around cycling innovations. As we see it, a living lab approach is an important prerequisite for responsible innovation (Stilgoe et al., 2013), a way to improve the design of innovation and improve the sensitivity for potential perverse effects. Instead of launching a pilot, a living lab promises a shared, multidisciplinary learning process that profits innovators, policy makers, academics and the general population. An important prerequisite is this willingness for shared learning. As our reflections show, it is also crucial that all parties are willing to reflect on deeper, political aspects of what cycling innovations mean and how they should be approached.
Reflections 2 and 3 centred on the perspectives that are used to approach cycling innovations. Currently, innovations often assume that by improving cycling conditions, non-cyclists will switch to using the bicycle. In Reflection 2, we have argued that it is also important to also give more consideration to existing cyclists in designing and evaluating cycling innovations. In Reflection 3, we argue that cycling falls in between the car-based logic of the street and the pedestrian logic of the sidewalk. To develop cycling innovations that benefit cyclists, there is a need for a more specific cycling-oriented logic.
Reflections 1 and 4 focus on the living lab process itself. This is an increasingly popular way of doing research on innovations and we support the importance of this for the quality of innovations and the quality of academic research. In our experience, the living lab approach brought us numerous advantages. The living lab: -facilitated reciprocal learning and collaborations between the policy makers, the innovators and researchers about cycling innovations. -allowed all partners to anticipate unforeseen consequences, for example with regards to parking and data handling. -allowed us to test, learn and fail.
-made the 'responsible' institution (province and/or municipality) aware of more dimensions than just time and budget restrictions of a project and as such contributed to 'better' decisions. However, the living lab method also raises some fundamental questions about the political nature of the work that academics then find themselves in. For us, this represented a dilemma: either staying outside of the innovation process for the sake of objectiveness and academic legitimacy or take an active seat at the table and have a richer, actionoriented understanding of the research object. As we see it, the benefits of a living lab approach mean that adopting an active role is a risk worth taking, but it is vital for researchers to develop a responsible and reflective stance in the process. Researchers need to be aware that the choice to be part of a living lab is in itself a political action, and one that will make certain futures more possible than others. | 2020-11-05T09:11:13.112Z | 2020-11-01T00:00:00.000 | {
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269145048 | pes2o/s2orc | v3-fos-license | Satisfaction rates, function, and return to activity following young adult total hip arthroplasty
Aims Young adults undergoing total hip arthroplasty (THA) largely have different indications for surgery, preoperative function, and postoperative goals compared to a standard patient group. The aim of our study was to describe young adult THA preoperative function and quality of life, and to assess postoperative satisfaction and compare this with functional outcome measures. Methods A retrospective cohort analysis of young adults (aged < 50 years) undergoing THA between May 2018 and May 2023 in a single tertiary centre was undertaken. Median follow-up was 31 months (12 to 61). Oxford Hip Score (OHS) and focus group-designed questionnaires were distributed. Searches identified 244 cases in 225 patients. Those aged aged under 30 years represented 22.7% of the cohort. Developmental dysplasia of the hip (50; 45.5%) and Perthes’ disease (15; 13.6%) were the commonest indications for THA. Results Preoperatively, of 110 patients, 19 (17.2%) were unable to work before THA, 57 (52%) required opioid analgesia, 51 (46.4%) were reliant upon walking aids, and 70 (63.6%) had sexual activity limited by their pathology. One patient required revision due to instability. Mean OHS was 39 (9 to 48). There was a significant difference between the OHS of cases where THA met expectation, compared with the OHS when it did not (satisfied: 86 (78.2%), OHS: 41.2 (36.1%) vs non-satisfied: 24 (21%), OHS: 31.6; p ≤ 0.001). Only one of the 83 patients (75.5%) who returned to premorbid levels of activity did so after 12 months. Conclusion Satisfaction rates of THA in young adults is high, albeit lower than commonly quoted figures. Young adults awaiting THA have poor function with high requirements for mobility aids, analgesia, and difficulties in working and undertaking leisure activities. The OHS provided a useful insight into patient function and was predictive of satisfaction rates, although it did not address the specific demands of young adults undertaking THA. Function at one year postoperatively is a good indication of overall outcomes. Cite this article: Bone Jt Open 2024;5(4):304–311.
Take home message
• Satisfaction rates of total hip arthroplasty (THA) in young adults are high, albeit lower than commonly quoted figures.• Young adults awaiting THA have poor function with high requirements for mobility aids, analgesia, and difficulties in working and undertaking leisure activities.• The Oxford Hip Score provided a useful insight into patient function and was predictive of satisfaction rates, although it did not address the specific demands of young adults undergoing THA.
Introduction
Total hip arthroplasty (THA) has revolutionized the management of debilitating hip arthritis. 1,21][12] Indication for surgery in patients aged under 50 years differs vastly from an older cohort.The 20th annual report from the National Joint Registry (NJR) of England, Wales, Northern Ireland, the Isle of Man and Guernsey recognizes primary osteoarthritis (OA) as the most common indication for surgery across all age groups; however, developmental dysplasia of the hip, Perthes' disease, and avascular necrosis are more prevalent in younger patients. 135][16] The Oxford Hip Score (OHS) 17 is regularly used, yet focuses on the demands of a typical THA cohort, hence it is questionable whether the OHS is generalizable to a young, active population.Previous studies have described mixed results with regard to the ceiling effect of the OHS, and a young patient cohort may be prone to this. 18herefore, the primary aims of the present study are to describe pre-morbid function and impact on QoL factors, and assess analgesic requirements associated with young adult THA.The secondary aims of this study are to assess postoperative satisfaction levels, functional outcomes, and assess how these functional outcomes compare to the OHS.We hypothesize that preoperative disease burden is significant, satisfaction with THA is high, and commonly used patient-reported outcome measures do not assess the demands specific to a young adult.
Study design
We conducted a retrospective cohort analysis of young adults undergoing THA between May 2018 and May 2023 at a tertiary orthopaedic arthroplasty centre (Royal National Orthopaedic Hospital, London, UK).
Inclusion and exclusion criteria
Young adults were defined as patients under the age of 50 years.We included all patients requiring THA during the defined period.For the purposes of this study, six teenagers (aged 14 to 19 years) were included under the definition of young adults.Patients receiving proximal femoral endoprosthesis, or THA secondary to malignancy, were removed from our study.Patients with incomplete datasets, long-term follow-up outside of our institution, and irretrievable records were also excluded.
Data collection
Prior to commencing data collection, our study was reviewed by the local research and ethics committee.Based on the HRA "Defining Research" leaflet, 19 our project was classed as service evaluation and did not require further approval from an institutional review board.Patient consent was obtained prior to questionnaire completion.Cases were identified from the institution's (Royal National Orthopaedic Hospital) prospectively populated surgical database using International Classification of Diseases 10th Revision (ICD-10) codes, 20 and manual review by the senior author (RM).Demographic and clinical data was extracted from selected patients' electronic medical records (EMRs).
Patients were contacted via email and telephone to complete the Oxford Hip Score (OHS) and Young Adult Hip Questionnaire (YAHQ) (see Supplementary Material).The OHS was selected due to its proven validity and utility in previous publications on the subject.The YAHQ was developed by orthopaedic surgeons (RM, JD, JS), in collaboration with a focus group of patients who have previously undergone THA.The questionnaire was evaluated for its content validity by the wider authorship group (RG, KM, RM).It was pilot tested within a focus group of young adult THA patients prior to its distribution.
Outcome evaluation
Demographic and clinical data included age, sex, and American Society of Anesthesiologists (ASA) grade. 21Comorbidities were graded utilizing the Charlson Comorbidity Index (CCI). 22HA procedure date, indication, and laterality were recorded.Perioperative outcomes included complications (intra-and postoperatively), length of hospital stay, and reoperation rate.Longer-term outcomes included OHS, YAHQ, and revision rates.The YAHQ evaluated premorbid functionality, hospital stay, and postoperative functionality, including assessment of satisfaction levels.Ambulation, occupation, analgesic requirements, ability to exercise and drive, and take part in sexual activity were documented pre-and post-THA.The questionnaire facilitated free text answers, from which the authors derived and collated major paraphrased themes.Responses to these questions were analyzed using a reflexive thematic analysis by the authorship group.Both questionnaires were obtained at an average of 31 months following primary arthroplasty, with included cases a minimum of 12 months post-surgery.All data were independently verified by a detailed review of hospital operative reports, anaesthesia records, and clinical records.
Operative protocol
Preoperatively, patients are discussed in the multidisciplinary team setting.Patients are encouraged to engage in serviceled or independent pre-habilitation to strengthen secondary stabilizers of the hip prior to arthroplasty.Patients are discharged following postoperative radiographs, independent mobilization, and once pain is well controlled.Upon discharge, patients are reviewed in orthopaedic clinic after six to eight weeks, typically by a clinical nurse specialist.
Statistical analysis
Statistical analysis was performed on Excel (Microsoft, USA) and using independent-samples t-test for continuous variables, and chi-squared test for categorical variables.Statistical significance was determined as p < 0.05 a priori.
Demographics
We performed a total of 325 THAs in 303 young adults, 22 of these representing bilateral cases, during the study period (Figure 1).Six of these patients were teenagers.No patients were lost to follow-up.Table I describes the baseline patient demographics and clinical data at the time of arthroplasty procedure between questionnaire responders and non-responders.There were no statistically significant differences in baseline demographics or comorbidity.
Results
Table II describes participants' pre-THA status.Before THA, 19 patients (17.2%) were unable to work, 51 (46.4%) required some form of mobility aid, 101 (91.8%) used regular analgesia of varying strengths, 70 (63.6%)were unable to exercise, and 70 (63.6%)felt their sexual activity was limited by their hip pathology.Of nine patients, 35% of those aged under 30 years required a mobility aid and 18 (72%) were unable to exercise.Of 47 patients, 39 (83%) of those aged between 40 and 49 years remained in full-time work, while six (12.8%) used a wheelchair to mobilize preoperatively.The highest proportion of those unable to work were aged between 30 and 39 years.Patients using narcotics post0peratively had a mean OHS of 38 (9 to 48), with THA meeting expectation in 79% (45/57) of cases.Those not using postoperative narcotics had a higher mean OHS of 41 (9 to 48) (p = 0.105), with THA also meeting expectation in 79% (42/53) of cases.
Table III delineates perioperative and short-term patient-reported outcomes.Mean length of stay was 3.8 days (1 to 27).One case required revision, indicated secondary to prosthesis instability, and was within the aged 40 to 49 years cohort.THA met expectation in 78.2% of cases (86/110).In all, 76 patients (69.1%) returned to their previous level of work, all within a 12-month period.In addition, 76 patients (69.1%) returned to their pre-morbid level of sexual activity, and 83 (75.5%) returned to their pre-morbid exercise levels; all but one patient returned to pre-morbid exercise levels within 12 months.A higher proportion of those aged less than 30 years returned to their previous level of work (18/25; 72%), resumed normal sexual activity (19/25; 76%), and returned to pre-morbid exercise levels (20/25; 80%) than their elder counterparts.
Qualitative comments demonstrated several themes, which are summarized in Table VI.Direct quotes included: "Before my operation, questions asked felt more relevant to an older individual, such as do I have difficulty climbing the stairs?","The OHS does not assess the most relevant challenges in my day-to-day life", and "I am more challenged with fear of picking up and playing with my children".Others expressed frustration that clinicians, family, and friends overestimated their ability to recover and rehabilitate from surgery: "Family and friends must be patient with relatives undergoing surgery and understand their frustration at their lack of independence post operatively".Patients described exercise and sexual activity being limited not by the hip itself, but by the fear of pushing their new implant too far.While exercise with their native hip was often very painful, this was sometimes anxiety-free.
Discussion
This paper demonstrates the preoperative functional burden of young patients undergoing THA and their postoperative outcomes using a mixed qualitative methodology.We report good outcomes for THA in young patients, but with significant differences compared to a 'standard' THA population.
In our cohort, functional recovery at 12 months appeared predictive of maximum function post-THA.Additionally, qualitative data analysis demonstrated themes of frustration from patients towards clinicians, family, and friends regarding the often-unrealistic expectation for rapid recovery due to their young age.The mean length of stay in the study Satisfaction rates, function, and return to activity following young adult total hip arthroplasty R. Galloway, K. Monnington, R. Moss, J. Donaldson, J. Skinner, R. McCulloch group was 3.8 days, which is in fact higher than the average for the department as a whole.It is important to note that these patients are often more surgically complex; therefore, despite being a young cohort, these patients often need a prolonged hospital stay before a safe discharge.Of note, younger adults in our study did not return to work significantly faster than their elder counterparts.4][25] THA is major surgery and those involved in pre-and postoperative care must recognize and validate the impact it has on patient QoL.This study's results can guide clinicians and patients regarding defining preoperative expectations for rehabilitation.There was a significant difference between the OHS of cases where THA met expectation, compared with the OHS when it did not.Nevertheless, qualitative themes reflected concern that the OHS does not assess the specific demands of young adults undertaking THA, such as commuting to work, performing sports, and engaging in sexual activity.The OHS has been criticized due to a potential ceiling effect, lack of clarity, and restrictive or irrelevant questions. 26,27The authors conclude that the OHS provides a useful insight into patient function and may be predictive of overall satisfaction rates; however, it may also be susceptible to the previously described ceiling effect.The necessity of further qualitative research in this field is evident, as without exploring young adults' priorities, expectations, and experiences, the THA, total hip arthroplasty.
literature may be presenting what researchers merely assume is important to this patient cohort. 25ur results provide unique insight into preoperative morbidity, analgesic requirements, and function.An unemployment rate of 17% was similar to previously quoted numbers; however, these were based on a "typical" THA population, highlighting the QoL impact preoperatively on a young and active population. 28Over 50% of our cohort required opioid analgesia preoperatively, vastly higher than previously reported rates. 29In our study, these patients had near identical satisfaction with THA than those not using narcotics preoperatively, and lower average OHS, without statistical significance.0][31] Considering the toxicity, dependence, link with poorer outcomes, and societal impact associated with chronic opioid usage, clinicians should counsel patients regarding their usage and consider alternative regimes where possible. 32A previous study analyzing sexual function in young adults pre-THA demonstrated difficulty in 64% of patients, almost equal to our findings. 33exual activity has a large bearing on QoL, and correlates with mental wellbeing and physical health; however, further commentary on this goes beyond the scope of this paper.Despite low rates of revision and complications, the relatively high proportion of patients not returning to pre-morbid function indicates the complexity of operating in this cohort. 34Length of stay was notably prolonged compared to our centre's median length of stay post-THA of 2.2 days, drawing attention to the associated resource requirement. 35early 80% of patients felt their THA met personal expectation, despite a lesser percentage returning to their previous level of work, sexual activity, and exercise levels.These results provide patients and clinicians with useful data to inform preand postoperative counselling.There is a paucity of research exploring the expectations of young adults undergoing THA, with some evidence emphasizing the importance of managing preoperative expectations. 36atients with poorer preoperative mobility had a worse OHS, but higher satisfaction rate with THA than those mobilizing independently preoperatively.This suggests that high OHS is not an independent predictor of patient satisfaction, and re-emphasizes the importance of patient selection and defining expectation preoperatively.OHS was highest in the aged under 30 years cohort, whereas those aged 30 to 39 years appeared least satisfied; the group also had the lowest rates of return to work, exercise, and sexual activity.DDH was the indication for surgery in over 50% of this sub-group.There are mixed results in the literature with regard to the effects of age on post-THA satisfaction, with some evidence suggesting that younger patients have less satisfaction. 37,38This could be related to differences in patient expectations compared to a typical arthroplasty population.
Thematic analysis revealed that young adults often wanted to know the limits of their rehabilitation and prosthesis.Patients often advocated early surgery.Our cohort had a maximum of five years' follow-up; therefore, a prospective cohort analysis may be useful to analyze how these patient perspectives change over a longer-term follow-up period.
Our study has limitations.Looking at the number of respondents, the response rate is less than 40%.This only increases to 45% when the sarcoma cases and patients who declined or were unable to take part were excluded.Additionally, this is a heterogenous group in terms of case mix, implant manufacturer, and indication for procedure.However, we would argue that there were no significant differences in demographic variables between responders and non-responders, therefore adding to the applicability of our findings.Surgical technology and technique have naturally progressed throughout our study period, potentially confounding our results.The OHS does not require clinician input, and measures patients' perceptions, yet has its own limitations.The authors note the relatively short study period, but this is essential to ensure accurate recall from the patients and their experience.Our study period encompassed the COVID-19 pandemic and thus, considering the increase in hybrid office work, the return to the previous level of work may have been underestimated, as many did not return secondary to a change in work circumstances.
In conclusion, this article informs both clinician and patient on expectations and outcomes of THA in the young adult, while highlighting the under-reported comorbidity in this group.Satisfaction rates of THA in young adults is high, albeit lower than commonly quoted figures.Young adults awaiting THA have poor function, with high requirements for mobility aids and analgesia, and difficulties in working and undertaking leisure activities.The OHS provides a useful insight into patient function and was predictive of satisfaction rates, although it did not address the specific demands of young adults undertaking THA.Function at one year postoperatively is a good indication of overall outcomes.
Table I .
Demographics of responders and non-responders.
Table II .
Participant status three months prior to operation.
Table III .
Outcomes, Oxford Hip Score, satisfaction, and return to activity postoperatively.
Table IV .
Associated Oxford Hip Scores.
Table V .
Preoperative mobility versus Oxford Hip Score and satisfaction.
Table VI .
Major paraphrased themes.
Satisfaction rates, function, and return to activity following young adult total hip arthroplasty R. Galloway, K. Monnington, R. Moss, J. Donaldson, J. Skinner, R. McCulloch | 2024-04-16T05:06:05.857Z | 2024-04-01T00:00:00.000 | {
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